JVPT-2012 Vol11

Review Article
G-PROTEIN COUPLED RECEPTORS: PAST AND PRESENT STATUS

S.P. SINGH*, A.H. AHMAD, WASIF AHMAD AND N.K. PANKAJ
Department of Veterinary Pharmacology and Toxicology C.V.A.Sc, G.B.P.U.A & T, Pantnagar-263145. * Corresponding author : E-mail: sppharma@rediffmail.com
ABSTRACT
Among membrane-bound receptors, the G protein-coupled receptors (GPCRs) are of most diverse type of receptors which might have originated during evolution and are capable of transducing messages through different photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins. A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. This is an attempt to update and visualize overall concepts regarding GPCR including their isoforms, database information, agonist desensitization, coupling mechanism etc. Key words: Database, GPCR, isoforms, metabotropic, signaling pathways, transduction.
INTRODUCTION "G-proteincoupled receptors (GPCRs) form a remarkable modular system that allows transmission of a wide variety of signals over the cell membrane, between cells and over long distances in the body. Today, we understand the molecular mechanism of how these receptors work in intricate detail" . Kobilka and Lefkowitz, Nobel Laureates for Chemistry (2012). GPCRs have been recognised in the genomes of unicellular organisms like bacteria and yeast to multicellular like plants, nematodes and other invertebrate groups. This hints towards an early evolutionary origin of this group of molecules. The diversity of GPCRs is dictated both by the multiplicity of stimuli to which they respond, as well as by the variety of intracellular signaling pathways they activate. These include light, neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones, nucleotides, chemokines and, undoubtedly, many others. In addition, there are at least 18 different human G proteins to which GPCRs can couple. These G proteins form heterotrimeric complexes with G subunits, which have at least 5 types, and G subunits, which have at least 11 types (Hermans, 2003; Wong, 2003). STRUCTURE OF CPCRs In a recent analysis of the GPCRs in the human genome, more than 800 GPCRs have been listed out of which 701 belong to rhodopsin family (type A) and 241 to non-olfactory type (Fredriksson et al., 2003). According to this analysis, there are approximately 460 type A olfactory receptors, although estimates range from 322 (Glusman et al., 2001; Takeda et al., 2002) to 900 (Venter et al., 2001) out of which 347 have been cloned (Zozulya et al., 2001). This large number of olfactory receptors accounts for the ability of humans to detect a wide variety of exogenous olfactory ligands. In an another report, 367 human endoGPCRs and 392 mouse endoGPCRs
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(Vassilatis et al., 2003) were listed. The term endoGPCR refers to GPCRs for endogenous (non-olfactory) ligands. In view of the known existence of alternatively spliced variants and editing isoforms of GPCRs, it is likely that the true number of GPCRs might never be known and is much higher than predicted. The GPCRs, also known as metabotropic receptors or 7-transmembrane heptahelical receptors that couple to intracellular effector systems via a G-protein. They constitute the largest family, and include receptors for many hormones and slow transmitters, viz the muscarinic acetylcholine receptor (mAChR), adrenergic receptors and chemokine receptors (Milligan, 2006). Gproteins comprise a family of membrane-resident proteins whose function is to recognize activated GPCRs and pass on the message to the effector systems that generate a cellular response and represent the interlocutor in the executive setup, intercepting between the receptors and on the effector enzymes or ion channels. They are called G-proteins because of their interaction with the guanine nucleotides i.e. GTP and GDP (Offermanns, 2002). Gproteins consist of three subunits: , and . Guanine nucleotides bind to the subunit, which has enzymic activity, catalyzing the conversion of GTP to GDP. The and subunits remain together as a complex (Kostenis, 2006). All three subunits are anchored to the membrane through a fatty acid chain, coupled to the G-protein through a reaction known as prenylation. G-proteins appear to be freely diffusible in the plane of the membrane, so that single pool of G-protein in a cell can interact with several different receptors and effectors. In the resting state, the G-protein exists as an unattached trimer, with GDP occupying the site on the subunit. After a GPCR is activated by an agonist molecule, a conformational change occurs in cytoplasmic domain of the receptor causing it to acquire high affinity for . Association of with the receptor causes the bound GDP to dissociate and to be replaced
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with GTP (GDP-GTP exchange), which in turn causes dissociation of the G-protein trimer releasing -GTP and subunits; the active forms of the G-protein which diffuse in the membrane and can associate with various enzymes and ion channels causing activation of the target. It was originally thought that only the subunit has a signaling function and complex serving merely as a chaperone to keep subunits out of range of the various effector proteins to prevent it to get otherwise excited. However, the complexes actually make assignations of their own and control effectors in much the same way as the subunits (Clapham et al., 1997). In general, it appears that higher concentrations of complex than of subunits are needed. Thus, -mediated effects occur at higher levels of receptor occupancy than -mediated effects. Association of subunits with target enzymes can cause either activation or inhibition depending on the involving G protein. ISOFORMS OF THE GPCR RECEPTORS Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 (GPCR) subtype were obtained from alternative RNA splicing. The two isoforms differ in the sequence of the putative cytoplasmic carboxylterminal tail and their hydrophobicity; one isoform, named EP3, has a hydrophilic tail, and the other, named EP3, has a hydrophobic tail. On expression, the two receptors displayed identical ligand binding properties but differed in responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 EP3 and decreased that of EP3. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 than EP3 for 50% inhibition of cAMP formation. A similar difference in agonist potency was also observed for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails showed variation in coupling to the Gi protein leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins (Sugimoto, 1993). PHARMACODYNAMICS GPCRs are present on the plasma membrane as a bundle of 7--helices. The biology of GPCRs is highly complex, their ultimate importance is depicted by the fact that at least one third (Robas et al., 2003) and perhaps as many as half (Flower, 1999) of currently marketed drugs target GPCRs, although only 10% of GPCRs are known drug targets (Vassilatis et al., 2003). Agonists bind to a
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cleft within the extracellular face of the bundle or to a globular ligand-binding domain sometimes found at the amino terminus. G proteins bind to the cytoplasmic face of the receptors. Agonist binding is followed by a change in the conformation of the receptor that may involve disruption of a strong ionic interaction between the third and sixth transmembrane helices (Ballesteros et al., 2001; Shapiro et al., 2002) which facilitates activation of the G-protein heterotrimer. Depending on the coupling of the receptor with type of G proteins, a variety of downstream signaling pathways can be activated (Marinissen and Gutkind, 2001; Neves et al., 2002). Signaling is then attenuated by GPCR internalization facilitated by arrestin binding. Signalling, desensitization and eventual resensitization are regulated by complex interactions of various intracellular domains of the GPCRs with numerous intracellular proteins (Bockaert et al., 2003). Receptors in this family respond to agonists by promoting the binding of GTP to the G protein subunit. GTP activates the G protein and allows it, in turn, to activate the effector protein. The G protein remains active until it hydrolyzes the bound GTP to GDP and becomes inactive. G proteins are composed of a GTP-binding subunit which confers specific recognition by receptor and effector and an associated dimer of and subunits that confer both membrane localization of the G protein e.g., via myristoylation and direct signaling such as activation of inward rectifier K+ (GIRK) channels and binding sites for G protein receptor kinases (GRKs). Activation of the G subunit by GTP allows it to both regulate an effector protein and drive the release of G subunits which in addition to regulating their own group of effectors, re-associate with GDP-liganded G, returning the system to the basal state. The major effect of many GPCRs is to release calcium (Ca2+) from intracellular stores. For example, receptors for norepinephrine activate Gq specific for the activation of phospholipase C b . Phospholipase C b (PLC b ) is a membrane-bound enzyme that hydrolyzes phosphatidylinositol-4,5-bisphosphate, a membrane phospholipid, to generate inositol-1,4,5-trisphosphate (IP3) and the lipid, diacylglycerol (DAG). IP3 binds to receptors on Ca2+ release channels in the IP3-sensitive Ca2+ stores of the endoplasmic reticulum, triggering the release of Ca2+ and rapidly raising [Ca2+]i from approximately 100 nM to the micromolar range. When Ca2+ levels rise following release from intracellular stores, the elevation of Ca2+ is transient due to reuptake of Ca2+ into these stores. Ca2+ can bind to and directly regulate ion channels e.g., large conductance Ca2+-activated K+ channels, or Ca2+ can bind to calmodulin and the resulting Ca2+-calmodulin complex then can bind ion channels e.g., small conductance Ca2+activated K+ channels or to intracellular enzymes such as Ca2+-calmodulin-dependent protein kinases e.g., CaMKII, MLCK, and phosphorylase kinase. While the complexity
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of receptor signaling through G proteins is clear from the abundance of GPCRs present on a single cell, receptorligand interactions alone do not regulate all GPCR signaling. It is now clear that GPCRs undergo both homoand heterodimerization and possibly oligomerization (Milligan, 2003). TERNARY COMPLEX AND DRUG DESIGN Kobilka and his coworkers revealed the threedimensional (3D) structure of a fully functional ternary complex at high resolution: the complex of 2-adrenergic receptor (AR) with agonist and G-protein. The ternary complex structure and a range of other structures provide a basis for pharmacologic development of drugs with high specificity, efficacy and few side effects. The biochemical strategies developed by Kobilka for AR make it possible to produce crystals of other 7TM receptors as well (Rasmussen et al., 2011). Membrane protein crystallization technology and fragment screening lead to many new X-ray structures of GPCRs which provide more accurate homology models across the Family A class of GPCRs which are helpful in structure-based drug discovery (SBDB) to GPCRs. In silico screening will be useful to define the three dimensional structure and the extent of binding sites of the GPCR receptors. Hot spot within the binding sites of GPCRs to ligands can be used to select virtual hits. Thus homology models may be derived from structural information available from closely related proteins (Cavasotto and Phatak, 2009). These approaches have been used successfully to identify antagonists and will be useful in future as well in reducing the attrition rate of drugs targeted at GPCRs for identification of agonists. Thus, combining the new approaches like structure-based discovery and in silico screening with advancements in the molecular biology and genetic engineering of GPCRs will open new vistas of GPCR targeted drug discovery (Congreve et al., 2011). PARTIAL AGONISTS AND GPCR DESENSITIZATION Weak or partial agonists desensitize GPCRs more efficiently than strong agonists (Clark, 1999). Receptors that mediate their actions by stimulating GTP binding regulatory G proteins share structural as well as functional similarities. Genetic analysis of the -adrenergic receptor (-AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein (Strader,1989). A model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the -AR and the G protein Gs has
been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the -AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur (Dixon, 1989). Structural similarities among G proteinlinked receptors suggest that the information gained from the genetic analysis of the -AR should help define functionally important regions of other receptors of this class (Sigal,1989). PROBABLE ARRANGEMENT OF THE HELICES IN GPCRs The probable arrangement of the seven helices, in all receptors, has been deduced. The location, relative to the centre of the lipid bilayer of each of the seven helical sequence segments and their probable lengths and orientation are deduced from sequence analysis relative to the centre of the helix bundle of each helical segment around its axis. The packing of the helices in the model is revealed by the density in a three-dimensional map of frog rhodopsin determined by electron cryo-microscopy (Baldwin,1997). A model proposed for the interaction between G proteins and G protein-coupled receptors, is based on the fact that this interaction shows little specificity and conserved parts of the G proteins interact with conserved parts of the receptors. These parts are a conserved negative residue in the G protein, a fully conserved arginine in the receptor and a series of residues that are not conserved but always hydrophobic like the hydrophobic side of the C-terminal helix of the G protein and the hydrophobic side of a helix in the C-terminal domain of the receptor. Other, mainly cytosolic, factors determine the specificity and regulation of this interaction (Oliveira,1999). MOLECULAR MEDIATORS OF GPCRs G protein-coupled receptors (GPCRs) are certainly the most diverse among membrane-bound receptors and are capable of transducing messages through different nucleotides, nucleosides, photons, organic odorants, peptides, proteins and lipids. Twodimensional crystallization of rhodopsin have led to a useful model of a common central core, composed of seven transmembrane helical domains and its structural modifications during activation. There are at least six families of GPCRs showing no sequence similarity (Bockaert, 1999). Some GPCRs have been found to form either homo- or heterodimers with a structurally different GPCRs but also with membrane-bound proteins having one transmembrane domain such as nina-A, odr-4 or RAMP, the latter being involved in their targeting, function and pharmacology (Pin, 1999). Certain findings provided new insight into Gprotein signaling during mammalian development and
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helped to explain how color of hair and skin can be controlled both coordinately and independently (Van Raamsdonk, 2004). A new class of dominant dark skin (Dsk) mutations was discovered after screening of ~30,000 mice with increased dermal melanin. Three out of four such mutations as hypermorphic alleles of Gnaq and Gna11, which encode widely expressed Gq subunits and require Ednrb in an additive and quantitative manner (Fuchs et al., 2004).The G-protein-coupled receptors are hijacked by viruses and harness their activated intracellular signaling pathways during the development of the disease (Sodhi et al., 2004). REGULATORS OF G PROTEIN SIGNALING (RGS) PROTEINS RGS proteins in drug discovery RGS proteins play an important role in signal regulation via GPCRs and have shown potential clinical importance in CNS and CVS disorders, cancer and diabetes which thus need to be targeted in drug discovery (Sjogren, 2011). During recent years GPCR signaling has been greatly explored and targeted for drug discovery. Various drugs have been developed that target these receptors, and a lot of research has been done to understand their physiological and pathophysiological function. Now the pathways are well understood that GPCRs signaling occurs through heterotrimeric G proteins consisting of an and a -subunit. After receptor activation, the subunit is exchanged from GDP to GTP resulting in its dissociation from heterotrimer. The signal is turned off upon hydrolysis of GTP to GDP reforming heterotrimeric complex. This process in vitro occurs at fairly slow rate in minutes whereas at a fast rate in vivo in seconds which is due to the missing component identified during early 1990s as regulator of G protein signaling (RGS) proteins. RGS proteins accelerate GTPase activity at active (GTP-bound) G subunits causing reduced amplitude and duration of GPCR-mediated signaling (Hollinger and Hepler, 2002). RGS proteins have a common domain called the RGS or RH (RGS homology) domain responsible for the catalytic activity toward G proteins. It is now understood that RGS proteins are involved in regulating GPCR signaling as well as non GTPase-activating (non-GAP) proteins mechanisms suggesting a potential role for RGS proteins as drug targets. Since RGS discovery over 15 years ago, RGS proteins have been characterized in their ability to regulate GPCR signaling, structure, tissue distribution, and physiological function. Albeit much to be worked out, it is evident that RGS proteins are emerging as novel drug targets in several pathophysiological states (Sjgren et al., 2010; Tesmer, 2009). Apart from the canonical function as GAPs, many RGS proteins also have other properties which have been
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a research area of great interest in recent years. Several RGS proteins contain additional domains to their RGS domain that can play a role in these functions. However, the presence of additional functional domains is not always necessary for an RGS protein to have functions that are not directly related to the GAP activity. The ability to participate in proteinprotein interactions has been well described for members of the R7 family of RGS proteins. Inhibition of RGS protein function Inhibition of RGS proteins enhance signaling via certain GPCRs. Many RGS proteins have a very limited expression (Gold et al.,1997) and this could increase tissue specificity and enable lower dosage of an endogenous or exogenous agonist (Blazer and Neubig, 2009). RGS proteins enhance 2a adrenergic suppression of hippocampal CA3 epileptiform bursts, for their role in the treatment of epilepsy (Goldenstein et al., 2009). The models using RGS-insensitive G proteins can be used for understanding the potential of RGS protein inhibitors in the treatment of CNS disorders. Augmentation of RGS protein function Enhancer of RGS protein function mediated through RGS-insensitive G proteins could be beneficial in the treatment of various hormones and neurotransmitters, such as noradrenaline, angiotensin II, 5-HT, endothelin, and acetylcholine associated cardiovascular disease e.g., hypertension, heart failure, and arrhythmias by reducing signaling via muscarinic receptors that couple to Gi2 (Fu et al., 2006; 2007). Identifying the RGS protein(s) responsible for the phenotype will be crucial in developing novel modulators of RGS protein function. RGS4 could enhance the muscarinic responses in the RGS-insensitive G i2 expression as evident in RGS4 knockout mice exhibiting increased responses to agonists at the M2 muscarinic receptor, decreased GIRK channel desensitization (Bender et al., 2008) and altered kinetics of acetylcholine-activated K currents (Cifelli et al., 2008) in the heart which suggested the potential for RGS4 enhancement in the regulation of cardiac automaticity. Beneficial roles of enhancers of RGS protein function was also evident from the phenotype of the RGS2 knockout mice, which were hypertensive and prone to early heart failure (Heximer et al., 2003; Oliveira-Dos-Santos et al., 2000) and is related to increased signaling through several receptors responsible vasoconstriction such as PAR- 1 (Tang et al., 2003), noradrenaline, angiotensin II, vasopressin, and endothelin receptors in vascular smooth muscle cells (VMSCs). RGS2 regulates blood pressure likely through its actions in the kidney (Gurley et al., 2010). RGS9 enhancers could be beneficial in the treatment of drug addiction and side effects of L-DOPA treatment in Parkinsons disease (PD). G-protein-coupled receptors (GPCRs) have been
GPCRs
recently credited as crucial players in tumour growth and metastasis. Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, invade their surrounding tissues and disseminate to other organs. Interfering with GPCRs might provide unique opportunities for cancer prevention and treatment. DATABASE ON GPCR A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Data on the GPCRs mutants were collected from the literature to develop a database of mutants and their effects. The GPCR, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of over 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned and mutation data may be retrieved from this alignment. Alternatively, a detailed specification of mutants may be searched, for example, search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://wwwgrap.fagmed.uit.no/GRAP/+ +homepage.html) using the World-Wide Web system. (Kristiansen, 1996). CONCLUSION The GPCRs, also called metabotropic receptors, have been reported in the genomes of unicellular organisms like bacteria and yeast to multicellular like plants, nematodes and other invertebrate groups and are comprised of seven membrane-spanning -helices often linked as dimeric structures. One of the intracellular loops is larger than the others and interacts with the G-protein. The G-protein, a trimeric membrane protein comprising three subunits (, , ) with the subunit possessing GTPase activity, binds to antagonist-occupied receptor, the subunit dissociates and activate an effector i.e., a membrane enzyme or ion channel. RGS proteins play an important role in signal regulation via GPCRs and have shown potential clinical importance in CNS and CVS disorders, cancer and diabetes which thus need to be targeted in drug discovery. There are several types of Gprotein, which interact with different receptors and control different effectors through second messengers such as cAMP, IP3, DAG etc. Various isoforms have been identified in different species. The GPCRs, which is the largest family of cell-surface molecules involved in signal transmission, have recently credited as crucial players in tumor growth
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Tang, K. M., Wang, G. R., Lu, P., Karas, R. H., Aronovitz, M., Heximer, S. P., et al. (2003). Regulator of Gprotein signaling-2 mediates vascular smooth muscle relaxation and BP. Nature Med. 9: 1506 1512. Tesmer, J. J. G. (2009). Structure and function of regulator of G protein signaling homology domains. In R. A. Fisher (Ed.), Mol. Biol. of RGS Proteins (pp. 75113). Van Raamsdonk, C., Fitch, K., Fuchs, H., de Angelis, M. and Barsh, G. (2004) Effects of G-protein mutations on skin color Nature Genetics. 36:961 - 968 Vassilatis, D. K., Hohmann, J. G., Zeng, H., Li, F., Ranchalis, J. E., Mortrud, M. T., Brown, A., Rodriguez, S. S., Weller, J. R., Wright, A. C., Bergmann, J. E. and Gaitanaris, G. A. (2003). The G protein-coupled receptor repertoires of human and mouse. Proc. Natl. Acad. Sci. USA. 100: 4903-4908. Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A. et al. (2001). The sequence of the human genome. Science. 291: 1304-1351. Wong, S. K. F. (2003). G protein selectivity is regulated by multiple intracellular regions of GPCRs. Neurosignals. 12: 1-12. Zozulya, S., Echeverri, F. and Nguyen, T. (2001). The human olfactory receptor repertoire. Genome. Biol. 2: RESEARCH0018.1-0018.12.
Received on:16-08-2012 Accepted on:26-12-2012
Research Article
CNS DEPRESSANT AND ANTI-CONVULSANT ACTIVITY OF HYDROETHANOL EXTRACT OF DRYMARIA CORDATA WILLD
C. C. BARUA1*, J. D ROY1, B. BURAGOHAIN1, A. TALUKDAR1, A. G. BARUA2, P. BORAH3, MANGALA LAHKAR4
1
Department of Pharmacology & Toxicology, 2Department of Veterinary Public Health, 3Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati- 781022, Assam 4 Department of Pharmacology, Guwahati Medical College, Bhangagarh, Guwahati- 781001, Assam * Corresponding author: E-mail: chanacin@satyam.net.in ; chanacin@gmail.com.
ABSTRACT The present study evaluated the CNS depressant and anticonvulsant effects of hydroethanol extract of Drymaria cordata (DCHE) in various behavioral models viz. spontaneous motor activity using actophotometer, anticonvulsant activity (electroconvulsion and chemoconvulsion test) and muscle relaxant activity ( rotarod test). In spontaneous motor activity study, DCHE showed significant reduction in spontaneous motor activity in dose dependent manner and the maximum effect was observed at 200 mg.kg-1 p o was better than the standard drug. Administration of DCHE could partially block tonic and clonic convulsions in dose dependent manner in maximal electroconvulsion and chemoshock convulsion tests in mice. But the extract was devoid of muscle relaxant activity. Phytochemical screening of the extract showed presence of steroids, terpenoids, tannin and diterpenes which might be responsible for the neuropharmacological activity of DCHE. Key words: Anti-convulsant effects, CNS depressant, Drymaria cordata, hydroethanol.
INTRODUCTION Neuropsychiatric and neurological disorders viz. depression, schizophrenia, epilepsy, parkinsonism etc are important clinical conditions that require serious attention. Drugs currently used in treatment of these disorders are either refractory, have massive side effects or posses unfavourable drug-drug/drug-food interactions (Danjuma et al., 2009). Moreover, these synthetic drugs are very expensive to develop (Rakh and Chaudhari, 2010). It is therefore essential that efforts should be made to introduce new drug with fewer side effects and to develop cheaper drugs. Medicinal herbs constitute the basis of traditional medicinal practice worldwide (Amos et al., 2001). The World Health Organization (WHO) has shown that, over 80% of the population in traditional medicinal system depends on these medicinal plants. Medicinal plants have also been used in the development of new drugs and continue to play an invaluable role in the drug discovery process (Cragg et al., 1997, Farnsworth, 1994). Drymaria cordata Willd (Caryophyllaceae) locally known as Laijabori is one of the traditionally used plants of North East India used for its analgesic, wound healing, anti-inflammatory activity and is also used as antidote, appetizer, depurative, emollient, febrifuge, laxative and stimulant in both human and animals. The pounded leaf is applied to snake bites (Saklani and Jain, 1994). Literature survey revealed that D. cordata have been reported for its antitussive (Mukherjee et al., 1997), antibacterial (Mukherjee et al., 1998) and antiinflammatory properties (Mukherjee et al., 1998). Our previous studies showed that methanol extract of D. cordata possessed analgesic
8
(Barua et al., 2009) and antiinflammatory activities (Barua et al., 2010) and, in addition, hydroethanol extract exhibited anxiolytic (Barua et al., 2009) and antinociceptive activities (Barua et al., 2011). As its effect on CNS has not been studied so far, the present study was undertaken for evaluation of hydroethanol extract in D. cordata in various neuropharmacological behavioral models in laboratory animals. MATERIALS AND METHODS Experimental animals Swiss Albino mice (20-30 g) and Wistar rats (150200 g) were used for the study. The animals had free access to food and water. They were fasted overnight before the experiment. They were housed in animal room, with alternating light-dark cycle of 12 hours each. The animals were acclimatized to the laboratory conditions for at least five days prior to the experiments. All the experiments were conducted between 0900 h 1800 h. The study was conducted after obtaining the approval of the Institutional Animal Ethics Committee. Acute toxicity study Acute toxicity study was carried out according to the Organization of Economic Corporation Development (OECD) guidelines No. 425. DCHE was administered orally in doses of 100, 200, 400, 800, 1000 and 2000 mg.kg1 to groups of mice (n=3) and percentage mortality was recorded for a period of 24 hours (Vogel, 2002). Based on the above toxicity study, direct limit test was done. Initially, a particular dose on the basis of the above study was administered to single female rat and the rat was observed for 48 hours with close
Activity of Drymaria cordata
surveillance up to initial 4 hours (same as in case of first rat) after 48 hours (of the second administration), same dose was administered in 2 more female rats and the observation was done same as for previous rats. The rats were observed for 14 days. The weight of the animals was recorded on 7th and 14th day. Preparation of plant extract The leaves of Drymaria cordata were collected during the month of July-Sept, 2010 from the medicinal garden of the department and identified by Botanical Survey of India, Shillong, Meghalaya. A voucher specimen (No AAU/CVSC/PHT/ 07-08/ 03) has been deposited in the Herbarium of Botanical Survey of India, Shillong, Meghalaya. Fresh leaves of Drymaria cordata were cleaned and under shade dried in clean dust free environment, grinded and stored in air-tight container. They were (250 g) soaked in 1000 ml of hydroethanol (50:50) for 72 hours in separate beakers. It was stirred every 18 hours using a sterile glass rod. The solvent was filtered every 3rd day using muslin cloth and What mans filter paper No 1. The filtrate obtained was concentrated in rotary evaporator (Equitron, Roteva) at 50-60C under reduced pressure leaving a dark brown residue. The Drymaria cordata hydroethanol extract (DCHE), thus obtained was transferred to a Petri dish and kept over water bath (50C-) until the solvent gets completely evaporated. It was stored at 4C for future use. Recovery was 18.06% (w/w).Freshly prepared DCHE was subjected to standard phytochemical screening tests for various constituents by standard methods (Harborne, 1991). Spontaneous motor activity The animals were administered with control, DCHE at the doses 50, 100 and 200 mg.kg-1 p o and standard drug diazepam (1 mg.kg-1 p o). After 30 minutes of pretreatment, the mice were placed individually in the activity cage for 10 minutes and activity score of each animal was recorded. The locomotor activity was measured using an actophotometer (Rolex), which operates on photoelectric cells and are connected in circuit with a counter (Finney, 1952). Percent reduction/ increase in motor activity in test groups was calculated and compared with the control and the standard group. Anti- convulsant activity Electro-convulsion test In this test, the animals were pre-treated with control, DCHE at the doses 50, 100 and 200 mg.kg-1 p o and standard drug diazepam (1 mg.kg-1 po). After 30 minutes, two electrodes were placed on both the ears and 150 mA current was applied for 0.2 sec and different phase of convulsions were noted (Swinyard et al., 1952). Reduction/ increase in time or abolition of tonic extensor phase in test groups was recorded and compared with the control and the standard group using electroconvulsiometer (Rolex).
Chemo-convulsion test In this test, the animals were fed with control, DCHE at the doses 50, 100 and 200 mg.kg-1 p o and standard drug diazepam (1 mg.kg-1 po). After 30 min of pre-treatment, pentylenetetrazole (80 mg.kg-1 ip) was injected and onset and severity of convulsion in test groups was recorded and compared with the control and the standard group (Swinyard et al., 1952). Rota-rod test The animals were placed one by one on the rotating rod and the fall-off time was recorded when the mouse fell from the rotating rod. The animals were administered with control, DCHE at the doses 50, 100 and 200 mg.kg-1 p o. and standard drug diazepam (1 mg.kg-1 p o). After 30 minutes of pre-treatment with the test compound or standard drug, the mice were again placed over the rotating rod and fall-off time was recorded. Percent reduction/ increase in motor activity in test groups was calculated and compared with the control and the standard group (Kinnard and Carr, 1957).
RESULTS Oral administration of DCHE up to 2 g/kg did not produce any toxic effects in the normal behavior of the mice. No mortality was observed and the extract was found to be safe at the given dose. The phytochemical screening of DCHE showed the presence of tannins by ferric chloride and gelatin test; diterpenes and triterpenes by Salkowskis and Liberman Buchardts test and steroids by Salkowskis and Liberman Buchardts test. The locomotor activity is an index of alertness and a decrease indicates sedative effect. Administration of DCHE caused significant reduction (P<0.01) in locomotor activity from 13.30 to 41.02 % at the dose 50 to 200 mg.kg-1 po. The effect of DCHE at the dose 200 mg.kg1 po was better than the standard drug diazepam which showed a percent reduction of 34.36% (Table 1). DCHE exhibited a significant reduction (P<0.01) in the duration of tonic phase from 22.13 to 40.96 % at the doses 50 to 200 mg.kg-1 p o. The standard drug phenytoin on the other hand, showed 96.87% protection (Table 2). DCHE treated group shows significant percent inhibition (P<0.01) in seizures from 38.04 to 95.25% at the doses 50 to 200 mg.kg-1 po in a dose dependent manner. In the chemo-convulsion also, the standard drug diazepam showed 100% protection. DCHE was found to be devoid of muscle relaxant property as none of the animals fell off the rotarod whereas animals treated with diazepam fell off within 3-5 seconds (Table 3). DISCUSSION The present study was undertaken to study the effect of hydroethanol extract of Drymaria cordata
Barua et al.
on central nervous system. DCHE showed CNS depressant property as there was reduction in spontaneous motor activity. It showed anticonvulsant activity by exhibiting significant protection against electrical and chemical induced seizures but was devoid of muscle relaxant activity. The locomotor activity is an index of alertness and a decrease indicates sedative effect. Decrease in locomotion reveals depressant effect of CNS drugs (Goloubkova et al., 1998). The increase in motor activity gives an indication of the level of excitability of the CNS and decrease may be related to sedation resulting from depression of CNS (Leewanich et al., 1996). DCHE showed significant reduction in spontaneous motor activity which might be closely related to sedation resulting from CNS depressant activity. The maximal electro convulsion test is considered to be a predictor of likely therapeutic efficacy against generalized tonic-clonic seizures (Vijayalakshmi et al., 2011). The MES induced convulsion in animals represent grandmal type of epilepsy. In electro-convulsion test, DCHE was able to reduce the duration of tonic phase. The reduction in time to recover from electrically induced convulsions by DCHE indicates anticonvulsant effect of the plant extract. This activity might be due to the inhibition
TABLE 1: Effect of DCHE on spontaneous motor activity. Treatments Control DCHE Dose(mg/kg p.o) 50 100 200 1
of excitatory mechanisms in the CNS (Radhakrishnan et al., 2001) Maximal electro shock (MES) causes significant increase in the level of norepinephrine, dopamine and acetylcholine activity in the brain (Shafiuddin et al., 2009). It has been also reported that there may be an increase in the turnover of norepinephrine by MES induced seizures. An excitatory effect on the cerebellum, to activate inhibitory pathways that extend to the cerebral cortex, has been suggested to contribute to the anticonvulsant effects of phenytoin ( Syed et al., 2010) The anticonvulsant effect of DCHE might be similar to the phenytoin. Hence it can be said that DCHE might follow the similar mechanism of anticonvulsant activity as that of phenytoin and might be effective in grandmal type of epilepsy. Pentylenetetrazole (PTZ) induced clonic convulsion test is a valuable model for studying the effect of putative anticonvulsant drugs on the propagation of seizure activity (Goodman et al., 1953). The PTZ induced clonic convulsion resemble petit mal type of convulsions in man. It has been demonstrated that a neuronal pathway sub-serving clonic PTZ convulsion is located in the forebrain while the brainstem is involved in the network of tonic PTZ convulsion (Browning et al., 1981; Browning and Nelson, 1986). Since DCHE specifically suppresses clonic PTZ
Before treatment 154.660.49 142.161.49* 138.500.67* 148.161.49* 185.661.25
Aftertreatment 147.500.56* 123.161.30 95.001.50 87.330.49 121.830.87
% reduction 4.62 13.30 31.30 41.02 34.36
Diazepam
Values are given as Mean S.E.M.; n=6; *P< 0.01 as compared to control. TABLE 2: Effect of DCHE on maximal electro convulsion. Treatments Control DCHE Dose(mg/kg p.o). 50 100 200 25 Duration of tonic phase (seconds) 27.881.29 21.710.26* 18.430.04* 16.460.10* 0.870.278 % inhibition 0.00 22.13 33.89 40.96 96.87
Phenytoin (i.p.)
Values are given as Mean S.E.M.; n=6; *P< 0.01 as compared to control. TABLE 3: Effect of DCHE on chemoshock convulsion test. Treatments Control DCHE Dose (mg/kg p.o). 50 100 200 1 Reaction time (seconds) 17.48 0.47 10.83 0.30* 5.66 0.49 0.83 0.04 0.000.00 % inhibition 0.00 38.04 67.62 95.25 100.00
Diazepam
Values are given as Mean S.E.M.; n=6; *P< 0.01 as compared to control.
10 Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/8-12
Activity of Drymaria cordata
convulsion, thus it can be suggested that DCHE contains active compound(s) that inhibit the propagation of convulsive seizure activity in the forebrain implicated by the inhibition of clonic PTZ convulsion. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter has shown to attenuate the convulsion. PTZ might be exerting its convulsive effect by inhibiting the activity of Gamma aminobutyric acid at the GABAA receptors, the major inhibitory neurotransmitter which is implicated in epilepsy (Vijayalakshmi et al., 2011). Diazepam, a benzodiazepine is reported to antagonize PTZ induced seizure by enhancing GABA neurotransmission (Thomas et al., 1994). In our study, DCHE could prevent PTZ induced seizure was significantly higher at the dose 100 (67.62 %) and 200 mg/kg p.o. (95.25 %) than electro shock induced convulsion Hence, diazepam was employed as a standard drug in PTZ model. It has been indicated that PTZ induced seizures can be prevented by drugs that reduce T-type Ca2+ currents such as ethosuximide and also by drugs that enhance GABAA receptor mediated inhibitory neurotransmission such as benzodiazepines (De Sarro et al., 2003). Hence, it might be possible that DCHE might prevent PTZ induced seizures by decreasing T-type Ca2+ currents and/ or by enhancing GABA A receptor mediated inhibitory neurotransmission and might be effective in petit mal type of convulsions. The study clearly demonstrated that the DCHE is devoid of muscle relaxant activity as indicated by negative results obtained in the rotarod test. Phytochemical analysis of the extract showed the presence of steroids, terpenoids, tannin and diterpenes..Various phytoconstituents such as terpenoids particularly triterpenoids are reported for anticonvulsant activity (Medina et al., 1990, Avallone et al., 2000). Similar anticonvulsant activity was observed in the rhizome of the Smilax china Linn. in mice. The neuropharmacological effects of DCHE might be attributed to these compounds present therein. These findings revealed CNS depressant and anticonvulsion activities of DCHE in addition to its anxiolytic (Barua et al., 2009) and analgesic activity (Barua et al., 2011) which might be due to various mechanisms such as decreased serotonergic and dopaminergic transmission and increased cholinergic transmission and vice versa. From the present findings, it can be concluded that the hydroethanol extract of DCHE possessed CNS depressant and anticonvulsant activity. ACKNOWLEDGEMENT The authors are grateful to National Medicinal Plant Board, Govt. of India, New Delhi for providing financial assistance to carry out this work. Physical facility provided by the Director of Research (Vety), C.V.Sc is also gratefully acknowledged.
REFERENCES Amos, S., Kolawole, E., Akah, P., Wambebe. C. and Gamaniel, K. (2001). Behavourial effects of the aqueous extract of Guiera senegalenesis in mice and rats. Phytomed. 8: 356-361. Avallone, R., Zanoli, P. and Puia, G., (2000) Pharmacological profile of apigenin, a flavonoid isolated from Matricaria chamomilla. Biochem. Pharmacol. 1: 1387-1394 Barua, C.C., Barua, A.G, Roy, J.D., Buragohain, B., and Borah P. (2010). Anti-inflammatory properties on Drymaria cordata Willd. Indian J. Ani. Sci. 80: 1268-1270. Barua, C.C., Barua, A.G., Roy, J.D., Buragohain, B., and Borah, P. (2011). Analgesic and antinociceptive activity of hydroethanolic extract of Drymaria cordata Willd. Indian J. Pharm. 43: 121-125 Barua, C.C., Pal, S.K., Barua, A.G., Roy, J.D., Buragohain, B., Bora. R.S. and Lahon, L.C. (2009). Analgesic activity of methanolic extract of Drymaria cordata Willd Cayophyllaceae. Phamacologyonline. 2: 470-476. Barua, C.C., Roy, J.D., Buragohain, B., Barua, A.G., Borah, P. and Lahkar, M. (2009). Anxiolytic activity of hydroethanolic extract of Drymaria cordata Willd. Indian J. Exp Biol .47: 969-97. Browning, R.A. and Nelson, O.K. (1986). Modification of electroshock and pentylenetetrazol seizure patterns in rats after precouicular transection. Exp. Neuro 93: 546-556. Browning, R.A., Simonton, R.L. and Turner, F.J. (1981). Antagonism of experimentally-induced tonic seizures following a lesion of the midbrain tegmentum. Epilepsia. 22: 595-601. Cragg, G.M., Newman, D.J. and Snader, K.M. (1997). Natural products in drug discovery and development. J. Nat. Pro. 60: 52-60. Danjuma, N. M., Abdu-Aguye, I., Anuka, J. A., Hussaini, I., Zezi, A, U., Yaro, A. H., Maiha, B. B. and Dabo, I. (2009). Central nervous system depressant effect of the hydroalcoholic extracts of leaves, stem and root barks of Randia nilotica Stapf. (Rubiaceae). Eur. J. Sci. Res. 25: 353361. De Sarro, A., Cechetto, V., Fravolini, V., Naccari, F. Tabarrini, O., De Sarro, G. (2003). Effects of novel a- desfluroquinolines and classic quinolines on pentylenetetrazole induced seizures in mice. Antimicrob Agents Chemother. 43: 1729-1736. Farnsworth, N.R. (1994). Ethnopharmacology and drug development. In Prance, G.T. and Marsh. J. Ethnobotany and The Search for New Drugs, Ciba Foundation Symposium 185, pp. 42-59 John Wiley and Sons, Chichester.
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Finney, D.J. (1952). Probit analysis. Cambridge University Press, Cambridge. Goloubkova, T.D., Heckler, E., Rates, S.M.K., Henriques, J.A.P. and Henriques, A.T., (1998). Inhibition of cytochorme P450 dependent monooxygenases by and alkaloid fraction from Helietta apiculata markedly potentiate the hypnotic action of pentobarbital. J. Ethnopharmacol. 60: 141-148. Goodman, L.S., Grcwal, M.S., Brown, W.C. and Swinyard, E.A (1953) Comparison of maximal seizures evoked by penetylenetetrazol (metrazol) and electroshock in mice, and their modification by anticonvulsants. J. Pharmacol. Exptl. Therap. 108: 168-176. Harborne, J.B. (1991). Phytochemical Screening Guide to modern techniques of plant analysis. pp. 653. Chapman and Hall, NewYork. Kinnard, W.J. and Carr, C. J. (1957). A preliminary procedure for the evaluation of CNS depressant. J. Pharmacol. Exptl. Therap. 121: 354. Leewanich, P., Tohda, M., Matsumoto, K., Subhadhir sakul, S., Takayama, H.,Watanbe, H. (1996). Behavioural studies on alkaloids extracted from leaves of Hunteria zeylanica. Biol. Pharmaceut. Bull. 19: 394399. Medina, J.H., Paladini, A.C., Wolfman, C., Levi de Stein, M., Calvo, D. and Diaz, L.E. (1990). Chyrsin (5,7di-flavone, a naturally occuring ligand of the benzodiapeines receptors with anticonvulsant properties. Biochem Pharmacol. 40: 2227-2231. Mukherjee, P.K., Bhattacharya, S., Saha, K., Giri, S.N., Pal, M. and Saha, B. P. (1997). Studies on antitussive activity of Drymaria cordata Willd (Caryophylaceae). J. Ethnopharmacol. 56: 74-77. Mukherjee, P.K., Mukherjee, K., Bhattacharya, S., Saha,B.P. and Pal, M. ( 1998). Studies on the anti- inflammatory effects of Drymaria cordata Willd. Nat. Prod. Sci. 4:.91-94.
Mukherjee, P.K., Saha, K., Bhattacharya, S., Giri, S.N., Pal, M., Saha, B.P.(1998) Studies on anti bacterial activity of Drymaria cordata Willd (Caryophylaceae). Phytother. Res. 11: 249-250. Radhakrishnan R., Zakaria M.N.M., Islam M.W., Chem H.B., Kamil M., chan K., Al-Attas A., (2001). Neuropharmacological actions of Portulaca oleraceae L.V. Sativa (Hawk). J. Ethnopharmacol. 76: 171-176. Rakh, M. S. and Chaudhuri, S. R. (2010). Evaluation of CNS depressant activity of Momordica dioica Willd fruit pulp. Int. J. Pharm. Pharmaceu. Sci. 2: 124126. Saklani, A. and Jain, S.K. (1994). Cross cultural ethno botany of North East India. pp. 97. Deep publishers, New Delhi. Shafiuddin, M.D., Liyakat Ahmed, M.D., Taranalli, A.D and Khaja Pasha (2009). Influence of cyclohexanoyl thiosemicarbazide and some anticonvulsant drugs on neurotransmitter levels in rat-brain. Int.J. Chem. Sci., 7: 264-272. Swinyard, E.A, Brown, W.C. and Goodman, I.S. (1952). Comparative Assays of antiepileptic drugs in mice and rat. J. Pharmacol. Exptl. Therap. 319: 166. Syed, K. M., Liyakha,T., Ahmed, M.D. and Paramjyothi, S. (2010). Neuropharmacological Effects of Ethanolic Extract of Portulaca quadrifida Linn. In Mice. Int. J. Pharm. Tech. Res. 2: 1386-1390, Thomas, C. P. and Kevin, D. A. (1994). Medical Neuroscience, USA Rochester: Mayo Foundation; p.307-312. Vijayalakshmi, A., Ravichandran, V., Anbu, J., Veraj, M. and Jayakumari, S. (2011). Anticonvulsant and neurotoxicity profile of the rhizome of Smilax china Linn. in mice 43: 27-30. Vogel, H.G. (2002). Drug discovery and evaluation: Pharmacological Assay. Berlin Heidelberg, New York, 385.
Received on: 21-1-2012 Accepted on: 23-4-2012
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Research Article
HOMOLOGY MODELING OF CANINE PROSTAGLANDIN G/H SYNTHASE-2

BHASKAR GANGULY*, SUDHIR KUMAR, TANUJ AMBWANI AND S.P. SINGH
Department of Veterinary Physiology and Biochemistry, Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences; G. B. Pant University of Agriculture & Technology, Pantnagar, PIN: 263145, INDIA. *Corresponding author; E-mail: vetbhaskar@gmail.com.
ABSTRACT Prostaglandin G/H synthase-2 is an oxidoreductase involved in the synthesis of several inflammatory mediators. It is an important target for many anti-inflammatory agents. Our study describes an in silico derived 3D model for the canine prostaglandin G/H synthase-2. Structure-activity features of the enzyme were characterized, and active site was identified. Also, its interactions with inhibitor molecules and involvement in other biochemical pathways were elucidated. The study shall facilitate the designing of novel anti-inflammatory agents for canids. Key words: COX-2, Diclofenac, Homology modeling, inhibition, Prostaglandin G/H synthase-2; PTGS-2.
INTRODUCTION Prostaglandin G/H synthase-2 (PTGS-2) is a member of the oxidoreductase family (EC 1.14.99.1). Also known as cyclooxygenase-2 (COX-2), prostaglandinendoperoxide synthase-2 (PES-2), Prostaglandin H2 Synthase 2 (PGHS-2/PHS II), glucocorticoid-regulated inflammatory cyclooxygenase (GRIPGHS), TIS10 protein or macrophage activation-associated marker protein P71/ 73, it catalyzes in a two-step reaction the cyclization of arachidonate to prostaglandin H2, the parent substance for the prostaglandins, prostacyclins, and thromboxanes, using up O2 during the process (Koolman and Roehm, 2005) making it an important target for various mediators. Futuristic drug design and development rely heavily on the availability of molecular models of the enzyme. Our study describes an in silico derived 3D model for the canine PTGS-2; to the best of the knowledge of the authors, it is the first ever molecular model for this enzyme obtained from a canine source. In addition, studies on the structureactivity features, including active site identification and interactions with inhibitors, were also performed. MATERIALS AND METHODS Sequence retrieval and 3D modeling of PTGS-2 The amino acid sequence of the complete canine PTGS-2 enzyme was retrieved from GenBank database at NCBI (Accession ADO27661.1; Benson et al., 2007). The amino acid sequence of the complete canine PTGS-2 enzyme, retrieved from GenBank, was used as a query in PSI-BLAST (Altschul et al., 1997) to find a suitable template for homology modeling. Default parameters were used in PSI-BLAST and the search was performed against the Protein Data Bank (PDB) database. The template, hence identified, was used for homology modeling using the modeling package MODELLER9v10 (Sali et al., 1995).
Model optimization, quality assessment and visualization Hydrogen addition, and clash reduction was performed in Swiss-Pdb Viewer 4.0.4 (Guex and Peitsch, 1997). Energy minimization was also performed using GROMOS96 (van Gunsteren et al., 1996) force field in SwissPdb Viewer. The errors in the model were, further, fixed using the tools at What IF Web Interface (Vriend, 1990). For structural evaluation and analysis of stereo-chemical quality, the 3D model was submitted to PDBsum (Laskowski et al., 1997) and RAMPAGE (Lovell et al., 2002). Overall quality of the structure was determined by ERRAT (Colovos and Yeates, 1993). Protein dimerization, visualization of 3D structures, and superposition, alignment and RMSD determination of query and template structure were performed in YASARA view (Krieger et al., 2002). For structural alignment of model and template, MUSTANG implementation (Konagurthu et al., 2006) of YASARA view was used. Protein structure-activity and accession number The final 3D structure of canine PTGS-2 was submitted to the Protein Model Database (PMDB; Castrignan et al., 2006). The structure-function characteristics of the enzyme were inferred from the NCBICDD output. NCBI-CDD is a protein annotation resource consisting of a collection of well-annotated multiple sequence alignment models for ancient domains and fulllength proteins (Marchler-Bauer et al., 2011). To determine the involvement of the enzyme in the bio-chemical pathways, KEGG automatic annotation server (KAAS) was used (Moriya et al., 2007).
RESULTS AND DISCUSSION The present study focused on the structural and functional analysis of canine PTGS-2. The complete canine PTGS-2 enzyme was found to be a homo-dimeric
Ganguly et al.
protein made up of two identical chains A and B. The sequence corresponding to one monomeric chain, made up of 604 amino acids, was identified and used for comparative modeling. The 3D model of a protein provides us invaluable insights into the structural basis of its function. Comparative or homology modeling is the most common structure prediction method. Numerous online servers and tools are available for homology modeling of proteins. Upon a PSI-BLAST search against the Protein Data Bank (PDB), 1PXX_A was identified as the best template (100% sequence coverage and 90% sequence identity) available for the homology modeling of the canine PTGS-2. 1PXX_A is the X-ray diffraction model of chain A of murine COX-2 at a resolution of 2.9 . The query sequence and template structure were then provided as inputs in MODELLER9v10 to generate the 3D model of canine PTGS-2. The homo-dimeric structure was obtained by dimerization of the output from MODELLER in YASARA view (Fig. 1).
Fig. 2: Ramachandran plot of Canine Prostaglandin G/H synthase2; no outlier residue is present (Generated from RAMPAGE).
Fig. 1: 3D model of Canine Prostaglandin G/H synthase-2; A helix rich structure is evident (Visualized in YASARA View).
The model generated by MODELLER was subjected to energy minimization and assessed for geometric and energy aspects using Swiss-Pdb Viewer and refined using What If Web Interface. The corrected model showed a quality factor of 93.601 % in ERRAT. The positioning of secondary structural elements was generated from PDBsum. 4 sheets, 4 hairpins, 8 strands, 32 helices, 56 helix-helix interactions, 38 turns, 8 turns and 5 disulphide linkages were predicted in the 3D structure of each chain. Several structure assessment methods including Ramachandran plots and RMSD were used to check the reliability of the predicted 3D model. Ramachandran plots were obtained from RAMPAGE server for quality assessment. No residues were present in the disallowed region whereas 52 (4.7 %) residues were present in the generously allowed regions. All other (95.3 %) residues were in the most favored regions (Fig. 2). This indicated that the backbone dihedral angles, phi and psi, in the 3D
model, were reasonably accurate. The RMSD value indicates the degree to which two 3D structures are similar; the lower the value, the more similar the structures. Both template and query structures were superimposed for the calculation of RMSD. The RMSD value obtained from superimposition of our model and 1PXX in YASARA View was found to be 0.067 over a total of 1102 aligned residues. The overall quality factor, Ramachandran plot characteristics and RMSD values confirm the quality of the homology model of the Canine Prostaglandin G/H synthase-2. Structural alignment of query and template also allowed the docking of diclofenac, pre-docked to the template, with the query structure (Fig. 3). The final protein structure was deposited in PMDB and is available under the Accession ID: PM0078233. The structure of a protein determines its function. The complete sequence of monomeric canine PTGS-2 is 604 amino acids long and consists of three functional domains and one dimerization interface. The first domain is the calcium binding domain spanning from residue 20 to 34. Both the terminal residues of this domain, i.e., 20
Fig. 3: Docking of Diclofenac in the active site of Canine Prostaglandin G/H synthase-2.
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Homology modeling of canine PTGS-2
and 34 comprise the functional site of the domain. The second domain in the canine PTGS-2 is the substrate binding site spanning from residue 103 to residue 520. The functional residues of this site were identified as 103, 191, 331, 334, 335, 339-41, 367, 504, 508, 512, 513, 516, 517 and 520. The third domain in the canine PTGS-2 is the heme binding site spanning from residue 134 to residue 436. The functional residues of this site were identified as 134, 185, 189, 193, 196-98, 281, 368, 371-74, 377, 394, 430, 432, 433 and 436. The dimerization interface was formed of residues 111, 113-15, 122, 123-26, 128, 215, 305-09, 312, 313, 316, 319, 320, 323, 353-60, 367, 371, 373, 523, 524, 527-31, 533-35, 537 and 538. Based on a KEGG search to identify the involvement of the enzyme in other biological processes, performed via KAAS, canine PTGS-2 was identified to also participate in biological pathways involving vascular endothelial growth factor (VEGF) signaling, NF-kappa B signaling, serotonergic synapse, retrograde endocannabinoid signaling, and in the pathogenesis of Leishmania and cancers. In the VEGF signaling pathway PTGS-2 increases PGI2 production, promoting angiogenesis this is of importance in the neo-vascularization of neoplasms. At serotonergic synapses, it exerts neuroprotective role by inhibiting adenylate cyclase and decreasing amyloid beta protein. In retrograde endocannabinoid signaling, it favours PGG synthesis from 2-arachydonoil glycerol (2AG), thereby reducing 2 AG levels. The family of endocannabinoids includes at least five derivatives of arachidonic acid; the two best characterized are arachydonoyl ethanolamide (anandamide, AEA) and 2arachydonoil glycerol (2AG). They are released from postsynaptic neurons upon postsynaptic depolarization and/ or receptor activation. The released endocannabinoids then activate the CB1 receptors (CB1R) at presynaptic terminals and suppress the release of inhibitory transmitter GABA. Therefore, PTGS-2 indirectly promotes the release of GABA. In leishmaniasis, PTGS-2 increases PGE2 production under the influence of the parasite glycoproteins. In this study, comparative modeling approach has been used to propose probably the first 3D structure for the Canine Prostaglandin G/H synthase-2. With the assistance of a well-defined structure and annotations, we can predict protein functional and binding sites, which can help in understanding the mechanism of bio-molecular functioning. REFERENCES Altschul, S.F., Madden, T.L., Schaeffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell,
J. and Wheeler D.L. (2007). GenBank. Nucleic. Acids Res. 35:21-25. Castrignan, T., De Meo, P.D., Cozzetto, D., Talamo, I.G. and Tramontano, A. (2006). The PMDB Protein Model Database. Nucleic. Acids Res. 34 (Database issue): D306-309. Colovos, C. and Yeates, T.O. (1993). Verification of protein structures: patterns of nonbonded atomic interactions. Protein. Sci. 2:1511-1519. Guex, N. and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis. 18: 2714-2723. Konagurthu, A.S., Whisstock, J.C., Stuckey, P.J. and Lesk, A.M. (2006). MUSTANG: A multiple structural alignment algorithm. Proteins. 64: 559-574. Koolman, J. and Roehm, K.H. (2005). Color Atlas of Biochemistry. 2nd edn. Georg Thieme Verlag, Stuttgart. Krieger, E., Koraimann, G. and Vriend, G. (2002). Increasing the precision of comparative models with YASARA NOVA - a self-parameterizing force field. Proteins 47: 393-402. Laskowski, R.A., Hutchinson, E.G., Michie, A.D., Wallace, A.C., Jones, M.L. and Thornton, J.M. (1997). PDBsum: a Web-based database of summaries and analyses of all PDB structures. Trends Biochem. Sci. 22: 488-490. Lovell, S.C., Davis, I.W., Arendall III, W.B., de Bakker, P.I.W., Word, J.M., Prisant, M.G., Richardson J.S. and Richardson, D.C. (2002). Structure validation by C alpha geometry: phi, psi and C beta deviation. Proteins: Structure, Function & Genetics. 50: 437-450. Marchler-Bauer, A. et al. (2011). CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 39(Database issue):D225-229. Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A.C. and Kanehisa, M. (2007). KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic. Acids. Res. 35(Web Server issue): W182-185. Sali, A., Potterton, L., Yuan, F., van Vlijmen, H. and Karplus, M. (1995). Evaluation of comparative protein modeling by MODELLER. Proteins. 23: 318-326. van Gunsteren W.F. et al. (1996). In Biomolecular simulation: the GROMOS96 manual and user guide. Vdf Hochschulverlag ETHZ. Vriend, G. (1990). WHAT IF: A molecular modeling and drug design program. J. Mol. Graph. 8: 2-56.
Research Article
EFFECT OF DOCOSAHEXAENOIC ACID ON CONCENTRATIONRESPONSE RELATIONSHIP OF 5-HT AND REVERSAL OF 5-HT CONTRACTION IN SHEEP CORONARY ARTERY
THAKUR UTTAM SINGH1, SUBHASHREE PARIDA1, SATISH KUMAR GARG2, SANTOSH KUMAR MISHRA3
Scientist, 3Head, Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar-243122, Uttar Pradesh, India 2 Dean, College of Veterinary Science & Animal Husbandry, Pandit Deen Dayal Upadhyaya Veterinary University & Go-Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh, India 1 Corresponding author E-mail: tusingh80@gmail.com
ABSTRACT
1
Present study was done to investigate the effect of docosahexaenoic acid (DHA) on concentration-response relationship of 5-HT, reversal of 5-HT contraction and basal tone in sheep coronary artery. Pretreatment of tissues with DHA (100 M) for 30 minutes caused significant (p<0.05) inhibition of contractions elicited with 5-HT (n=5). To assess the vasodilator response of DHA on coronary artery, endothelium-denuded arterial rings were constricted with 5-HT. At the plateau of 5-HT contraction, DHA was added in a cumulative manner at 0.5 log unit. DHA (0.1-100M) produced concentration-dependent relaxation. The Emax was 38.527.03% at 100 M concentration of the DHA. DHA (107-104M) added cumulatively at an increment of 0.5 log unit caused a concentration-dependent contraction of coronary arterial rings with an Emax of 20.423.76 (n=4). Key words: Coronary artery, Docosahexaenoic acid, 5-HT, Sheep.
INTRODUCTION N-3 Polyunsaturated fatty acids (PUFAs) are known to protect the cardiovascular system and improve blood pressure (Wang et al., 2011). 17S-HDHA, an endothelium-derived docosahexaenoic acid (DHA) product via lipooxygenase, activates BK(Ca) channels in coronary arterial smooth muscle cells, leading to coronary vasodilation, which may represent an important mechanism mediating the beneficial actions of DHA in coronary circulation (Li et al., 2011). Vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK(Ca) channels (Lai et al., 2009). DHA and eicosapentaenoic acid (EPA) are the major n-3 fatty acids in fish oils. Dietary supplements with fish oils have been considered as indirectly cardioprotective because of their antithrombotic, antiatherosclerotic, and antihypertensive properties. Some studies show that DHA may be the active agent in dietary fish oils responsible for lowering systemic blood pressure. Daily ingestion of fish oil was shown to reduce the blood pressure of patients with essential hypertension (Knapp and FitzGerald, 1989). Diets enriched in DHA, a major n-3 fatty acid in fish oil, have hypotensive properties (Ye et al., 2002). Omega-3 fatty acids are classed as essential fatty acids and common omega-3 fatty acids in the human body are DHA and EPA (Lees and Karel, 1998). However, there is no report on the effect of DHA on 5-HT-induced contraction, reversal of 5-HT contraction and basal tone in sheep coronary artery. Therefore, the first objective of the present study was to evaluate the effect of DHA on 5-HT-induced contraction in
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sheep coronary artery. The second and third question was that whether DHA would affect reversal of 5-HT contraction and basal tone in this vessel. MATERIALS AND METHODS Chemicals Acetylcholine chloride and 5-Hydroxytryptamine hydrochloride (Sigma Chemicals, St Louis, MO) and DHA (Cayman Chemicals, Ann Arbor, MI, USA) were used in this study. DHA was dissolved in absolute alcohol. All other drugs were dissolved in distilled water. Tissue Collection Heart from freshly slaughtered sheep was collected from local slaughterhouse in cold (4-6 oC) oxygenated modified Krebs-Henseleit solution (MKHS), pH 7.4. Coronary artery was dissected from heart, cleared of connective tissue, and cut into rings of about 2-3 mm length. Tension Experiments Arterial rings of 2-3 mm were prepared from sheep coronary artery. Rings were mounted between two Lshaped stainless steel hooks under a resting tension of 1.5 g in a thermostatically controlled (37.00.5 C) organ bath of 10 ml capacity containing MKHS and continuously aerated with carbogen (95% O2+5% CO2). Experiments were done in endothelium-denuded coronary arterial rings. Endothelium was removed by mechanical rubbing with a cotton swab. Endothelium removal was confirmed by demonstrating the absence of relaxation to ACh (10 M). The arterial rings were equilibrated for 90 min with a
Effect of DHA on sheep coronary artery
repeated replacement of bath solution every 15-20 min. A high sensitivity force displacement transducer (Model MLT0202/D; Power Lab, Castle Hill, Australia) was used to measure the change in tension, and the data were recorded in a PC using chart version 4.1.2 software program (Power Lab). Effect of DHA on the concentration-response relationship of 5-HT in sheep coronary artery To study the effects of DHA on the vasoconstrictor responses of 5-HT, coronary artery was used. After equilibration for 90 min, the coronary arterial rings were constricted with 5-HT (1 M), and when the contraction reached the plateau, ACh (10 M) was added to examine the endothelial integrity. Once the tissue viability was confirmed, the tissue was washed off drugs and allowed to relax for a period of 30 min. Then, a concentrationresponse curve (109-105M) to 5-HT agonist was elicited. The same tissue was used to plot a second concentrationresponse curve following pre-treatment with DHA for 30 min. Effect of DHA on reversal of 5-HT contraction in sheep coronary artery To assess the vasodilator response of DHA on coronary artery, endothelium-denuded arterial rings were constricted with 5-HT (5.0 M; mean absolute tension 0.290.08 g (n=5). At the plateau of 5-HT contraction, DHA was added in a cumulative manner at 0.5 log unit. Direct effect of DHA on basal tone in sheep coronary artery To find out the direct effect of DHA on basal tone in the coronary artery, DHA (107-104M) were added cumulatively at an increment of 0.5 log unit to induce a concentration-dependent contraction of coronary arterial rings.
Fig. 2. Effect of DHA on reversal of 5-HT-induced contraction in sheep coronary artery.
Fig. 3. Effect of DHA on basal tone in sheep coronary artery.
Statistical analysis Results are expressed as MeansS.E.M and multiple comparisons were done using two-way ANOVA followed by Bonferroni post hoc test. P<0.05 was considered statistically significant. Emax was determined using non linear regression analysis of Graphpad Prism. pD2 is expressed as log EC50 of the agonist.
RESULTS Cumulatively added 5-HT (109-105M) at 0.5 log interval elicited concentration-dependent contractions in sheep coronary artery. In control arterial rings, the maximum contraction achieved was 80.5818.29% of 80mM KCl-induced contraction and pD2 value was 4.500.62. Pretreatment of tissues with DHA (100 M) caused significant (p<0.05) inhibition of contractions
Fig. 1. Effect of DHA on the concentration-response relationship of 5-HT in sheep coronary artery .
Singh et al.
elicited with 5-HT (n=4-5). The Emax and pD2 values of 5HT were 26.104.53% and 3.060.41 in presence of DHA, respectively (Fig. 1). Figure 2 shows that DHA (0.1-100M) produced concentration-dependent relaxation. The Emax was 38.527.03% at 100 M concentration of the DHA. DHA (107-104M) added cumulatively at an increment of 0.5 log unit caused a concentration-dependent contraction of sheep coronary arterial rings with an Emax of 20.423.76 (n=4) (Fig. 3). DISCUSSION Present study shows that DHA inhibited 5-HTinduced concentration-dependent contraction in coronary artery. DHA caused a significant relaxation in 5-HTcontracted coronary arterial rings and also caused a marginal increase in basal tone. The relaxant effects of omega-3 fatty acids may contribute to hypotensive effects by modulating vascular tone (Engler et al., 1990). To gain an understanding of the omega-3 fatty acids and their vascular relaxant properties, the current investigation was performed to examine the vascular effects of this fatty acid in ovine coronary artery. Cumulative concentrationresponse curves was generated for DHA in vessels precontracted with 5-HT. A significant relaxant effect was observed for DHA in coronary artery (38%) at 100 M concentration. Our findings are strengthened by the report of Engler (1992) who showed relaxant effect of this fatty acid in rat aorta in a concentration range of 1-255 M. At 31 M concentration, the maximal relaxation observed in this tissue was 20% for DHA. Increasing the concentration to 255 M, a corresponding increase in the magnitude of relaxation was evident with DHA (63%). DHA also antagonized concentration-dependent contractions elicited by 5-HT in coronary artery. The relaxant effect of DHA was endothelium-independent. Similar observation has been made in aorta where removal of endothelium did not alter DHA-induced relaxations (Engler et al., 1987). EPA, however, caused endothelium-dependent relaxation of porcine coronary artery (Shimokawa and Vanhoutte, 1989). Another possible mechanism of action of omega-3 fatty acids such as epoxydocosapentaenoates (EDPs)-induced relaxation in coronary microvessels is due to activation of BKCa channels directly (Ye et al., 2002). Thus the observation made in the present study demonstrating DHAinduced relaxation of coroonary artery may be relevant to its beneficial effect in hypertension.
REFERENCES Engler, M.B., Karanian, J.W. and Salem, N. Jr. (1987). Doco-sahexaenoic acid (22:6w3) induces a concentration-dependent relaxation of rat aortic rings. Eur. J. Pharmacol. 185: 223-226. Engler, M.B., Karanian, J.W. and Salem, N. Jr. (1990). Docosahexaenoic acid (22:6n3)-induced relaxation of the rat aorta. Eur. J. Pharmacol. 185: 223-226. Engler, M.B. (1992). Effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone. Eur. J. Pharmacol. 215: 325-328. Knapp, H.R. and FitzGerald, G.A. (1989). The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension. N. Engl. J. Med. 320: 10371043. Lees, R.S. and Karel, M.D. (1998). Health effects of omega-3 fatty acids in health and disease edt. Lees, R.S. and Karel, M.D. Part I Printed in USA. Li, X., Hong, S., Li, P.L. and Zhang, Y. (2011). Docosahexanoic acid-induced coronary arterial dilation: actions of 17S-hydroxy docosahexanoic acid on K+ channel activity. J. Pharmacol. Exp. Therap. 336(3): 891-899. Lai, L.H., Wang, R.X., Jiang, W.P., Yang, X.J., Song, J.P., Li, X.R. and Tao, G. (2009). Effects of docosahexaenoic acid on large-conductance Ca2+activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells. Acta Pharmacol. Sin. 30(3): 314-320. Shimokawa, H. and Vanhoutte, P.M. (1989). Dietary omega 3 fatty acids and endothelium-dependent relaxations in porcine coronary arteries. Am. J. Physiol. 256: H968-973. Wang, R.X., Chai, Q., Lu, T. and Lee, H.C. (2011). Activation of vascular BK channels by docosahexaenoic acid is dependent on cytochrome P450 epoxygenase activity. Cardiovasc. Res. 90(2): 344-352. Ye, D., Zhang, D., Oltman, C., Dellsperger, K., Lee, H.C. and Rollins, M. (2002). Cytochrome P-450 Epoxygenase Metabolites of Docosahexaenoate Potently Dilate Coronary Arterioles by Activating Large-Conductance Calcium-Activated Potassium Channels. J. Pharmacol. Exp. Therp. 303(2): 76876.
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Research Article
PROGESTERONE PROFILE AND CONCEPTION RATE FOLLOWING INSULIN TREATMENT DURING LUTEAL PHASE OF ESTROUS CYCLE IN BUFFALOES
Q. I. HUQ1, H.P. GUPTA2, SHIV PRASAD3, V.S. RAJORA4 AND ASAD HUSSAIN5
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MVSc Scholar, 2Professor and Head, Department of Veterinary Gynecology and Obstetrics, 3Professor, Professor and Head, Veterinary Medicine, and 5PhD scholar, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar - 263 145 2 Corresponding author: E-mail: hpguptavgo@gmail.com
ABSTRACT The present study was conducted to evaluate the response of insulin treatment (Human Mixtard, @ 0.25 I.U/kg body weight) during luteal phase of estrous cycle on serum progesterone and conception rate in buffalo (Bubalus bubalis). Estrus was induced using 2 ml. (263 g/ml) Cloprostenol Sodium (i/m) and the animals were bred naturally. The buffaloes (n=20) were grouped as control (n=10, PBS) and treatment group (n= 10). Insulin was given on day 12 of estrous cycle in treatment group. The conception rate was 50% in both groups . The serum progesterone concentration was significantly (p<0.05) higher 4.7100.342, 3.249 0.541 and 3.7550.576 ng/ml on days 14, 18 and 30 in treatment group vs 4.5800.249, 2.9040.681 and 3.4710.420 ng/ml, respectively, in control group. Comparison of data on progesterone profile between pregnant and non-pregnant (within group) animals of treatment and control groups showed higher levels of serum progesterone in pregnant animals from day 14 to 30 in treatment group. Present study concluded that insulin administration during luteal phase of estrous cycle increased progesterone secretion without any effect on conception rate in buffaloes. Key words: Bubalus bubalis, conception rate, insulin, progesterone
INTRODUCTION Numerous causes of pregnancy loss in bovine include body condition score, disease, high milk yield, heat stress, oocyte quality, insemination protocol, synchronization method, concentration of progesterone and the uterine environment (Santos et al., 2004). Lalthazuali et al. (2010) suggested that luteal deficiencies following AI may be a major cause of embryonic mortality in buffaloes. High proportion of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both (Lemley et al., 2008). Lemley et al. (2007, 2008) demonstrated that, in the cow, either providing a gluconeogenic feed stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism. Thus, the present study was under taken to study the effect of insulin on serum progesterone profile and conception rate in buffalo. MATERIALS AND METHODS Normal cyclic buffaloes (n=20) were selected on the basis of their previous health records and per-rectal examination of their genital organs. The experimental buffaloes aged 4-7 years were divided into two groups (Treatment group, n=10 and control group, n=10).The animals having mature corpus luteum (CL) were given 2 ml intramuscular injection of Cloprostenol sodium (Cyclix, Intervet India Pvt. Ltd., India) to synchronize the
estrus. The estrus was detected at every morning and evening by close observation for external signs, such as bellowing, mucus discharge from vulva, mounting, frequent micturition, swollen and edematous vulva and later on confirmed by per-rectal palpation. All the animals of treatment and control groups detected in heat were bred by bull. Insulin (Human Mixtard , Torrent Pharmaceutiscals Ltd., India) was given @ 0.25 IU / kg body weight by subcutaneous route to the animals of the treatment group on day 12, and PBS was given subcutaneously to the animals of the control group. Human Mixtard is a mixture of dissolved insulin (30 %) and isophane insulin (70 %). It contains both fast acting (Insulin) and long acting insulin (Isophane insulin). Blood samples were taken by jugular venipuncture as per the schedule on day 0, 12, 14, 18 and 30 in all groups. About 4-5 ml blood without anticoagulant was collected in sterilized glass tubes and kept at room temperature as a slant for 6-8 hours for separation of blood serum. Blood serum was separated, centrifuged at 3000 rpm for 15 minutes and was transferred into sterilized serum vials. All samples were stored at -20C till analysis. Progesterone was estimated in blood serum by radioimmunoassay (RIA) using RIA kits (Immunotech, France). Pregnancy was checked by per-rectal examination of the genital organs after 60 days of service. The data obtained in the present study was analyzed statistically and compared for significant difference using t-test (Snedecor and Cochran, 1994).
Huq et al.
TABLE 1: The effect of single insulin treatment @ 0.25 IU/ kg b.wt., im, on day 12 of estrous cycle on concentration of serum progesterone (ng/ml) in buffaloes. S. No. 1. 2. 3. 4. 5.
*
Days of estrous cycle Day-0 Day-12 Day-14 Day-18 Day -30
Mean SE of progesterone (ng/ml) Mean SE of progesterone (ng/ml; within group) Control, (n=10) Treatment (n=10) Pregnant (n=05) Non-pregnant (n=05) 0.2960.048 4.2200.324 4.5800.249 2.9040.681 3.4710.420 0.2730.046 4.079 0.199 4.7100.342 3.249 0.541 3.7550.576 0.2380.062 4.2160.786 4.9680.646 4.5580.646* 4.5580.661 0.3120.053 3.862.0283 4.5520.332 1.9400.218 2.9220.843
Significant at 5% level of significance within group.
RESULTS AND DISCUSSION In the present study the serum progesterone concentration remained higher 4.7100.342, 3.249 0.541 and 3.7550.576 ng/ml on days 14, 18 and 30 in treatment group vs control group 4.5800.249, 2.9040.681 and 3.4710.420 ng/ml respectively, irrespective of pregnancy status of animals (Table 1). However, the differences were not significant. Comparison of data on progesterone profile between pregnant and non-pregnant animals of treatment group showed higher levels of serum progesterone in pregnant animals from day14 to 30 in treatment group (Table 2). But, difference was significant only on day 18 (P< 0.05). The significant difference on day 18 might be due to effect of luteolysis (Ahmad, 2001). The higher serum progesterone on different days after insulin administration in different treatment groups may be possibly due to effect of insulin on progesterone metabolism, or progesterone release from corpus luteum or due to effect of insulin on steroidogenesis, however, in this study slight increase in progesterone concentration may be because of giving single insulin injection. Lemley et al. (2009) found that, cows consuming a high starch diet had elevated insulin concentrations, lower hepatic cytochrome P450 activity and a longer progesterone half-life compared to cows consuming a high fiber diet. Lemley et al. (2007, 2008) demonstrated that, in the cow, either providing a gluconeogenic feed stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism. Lalthazuali et al. (2010) reported higher (P<0.05) plasma progesterone concentration in insulin-treated buffaloes on day 12, 15, and 21 post-AI. Suguna et al. (2009) observed significantly increased progesterone concentrations following insulin administration in insulin treated goats than control during gestation indicating its favourable effect on steroidogenesis. Increase in progesterone might be due to direct effect of insulin on CL or by increasing the ovulation rate. Selvaraju et al. (2002) recorded significantly higher plasma progesterone concentrations in insulin treated repeat breeding cows than control. Sarath (2005) also reported similar findings in acyclic goats. Lucy et al. (1993)
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also reported the direct effect of insulin and/or IGF-I on the follicles and corpus luteum resulting in increased peripheral concentration of progesterone. In vitro studies utilizing bovine luteal cell culture also recorded insulininduced increase in progesterone secretion (Saurwein et al., 1992). Lalthazuali et al. (2010) found that there was no improvement in corpus luteum diameter in insulin treated buffaloes as compared to control and suggested that increase in conception rate in treatment group may be due effect of insulin on progesterone synthesis/release from corpus luteum, which resulted in increase in progesterone concentration. The conception rate in control and treatment group was 50 %. Thus, there was no difference in conception rate between treatment group and control. Kharche et al. (2003) reported that single subcutaneous injection of long acting purified bovine insulin @ 0.2 IU/kg body weight on day 10 had no beneficial effect on conception rate. However, Lalthazuali et al. (2010) injected long acting purified bovine insulin @ 0.2 IU/kg body weight subcutaneously during post-insemination mid-luteal phase (days 8-12) in buffaloes and found significantly (P<0.05) higher first service conception rate (58% vs. 20%) in treatment group as compared to control group. In this study lack of difference may be due to use of single injection (Kharche et al. 2003) or due to small sample size (Lalthazuali et al., 2010) or due to injecting insulin late in luteal phase. Thus, it may be concluded that insulin administration on day 12 resulted in the increase in serum progesterone concentration, however, conception rate remained unaffected. ACKNOWLEDGEMENT Financial help from the National Fund for Basic and Strategic Research in Agriculture (NFBSRA), ICAR, New Delhi, under the project Antiluteolytic strategies- a novel approach to enhance fertility in buffalo is thankfully acknowledged. REFERENCES Ahmad, N. (2001). Reproduction in buffaloes. In: Noakes, D.E., Parkinson, T.J. and England, G.C.W.
Progesterone in insulin treated buffaloes
Arthurs Veterinary Reproduction and Obstetrics. 8th edn. New Delhi, India, Harcourt Publishers limited. pp. 789-799. Kharche, S.D., Majumdar, A.C and Srivastava, S.K. (2003). Influence of exogenous insulin on conception rate in crossbred cattle. Indian J. Animal Sc. 73(8): 890-891. Lalthazuali, Singh, J., Ghuman, S.P.S., Pandey, A.K. and Dhaliwal, G.S. (2010). Impact of insulin treatment during post-insemination mid-luteal phase on luteal profile and conception rate in buffalo. Indian J. Anim. Sc. 80 (9): 854-856. Lemley, C.O., Butler, S.T., Butler, W.R. and Wilson, M.E. (2007). Insulin reduces the mRNA abundance of the progesterone catabolic enzymes cytochrome P450 2C and 3A in the dairy cow. Biol. Repr. 77:146. Lemley, C.O., Butler, S.T., Butler, W.R. and Wilson, M.E. (2008). Insulin alters hepatic progesterone catabolic enzymes Cytochrome P450 2C and 3A in dairy cows. J. Dairy Sci. 91: 641-645. Lemley, C.O., Koch, J.M., Blemings, K.P. and Wilson, M.E. (2009). Alteration of progesterone catabolic enzymes, CYP2C and CYP3A, in hepatocytes challenged with Insulin and Glucagon. J. Anim. Vet. Adv. 8(1): 39-46 Lucy, M.C., De La Sota, R.L., Staples, C.R. and Thatcher, W.W. (1993). Ovarian follicular populations in lactating dairy cows treated with recombinant bovine somatotrophin or saline and fed diets differing in fat content and energy. J. Dairy Sc.
76: 1014-1027. Santos, J.E.P., Thatcher, W.W., Chebel, R.C., Cerri, R.L.A. and Galvao, K.N. (2004). The effect of embryonic death rates in cattle on the efficacy of oestrous synchronization programs. Anim. Repr. Sc. 82: 513-535. Sarath, T. (2005). Follicular dynamics, endocrine and nitric oxide profiles in cyclic and insulin administered acyclic goats. Thesis, IVRI, Izatnagar (U.P). Sauerwein, H., Miyamoto, J., Gunther, J., Meyer, H.H.D. and Schams, D. (1992). Binding and action of insulin like growth factors and insulin in bovine luteal tissue during the oestrous cycle. J. Repr. Fert. 96: 103-15. Selvaraju, S., Agarwal, S.K., Karche, S.D., Srivastava, S.K., Majumdar, A. C. and Shanker, U. (2002). Fertility responses and hormonal profiles in repeat breeding cows treated with insulin. Anim. Repr. Sc. 73: 141-49. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods, 8th Edn. Ames, Iowa State University Press. p 503. Suguna, K., Mehrotra, S., Agarwal, S.K., Hoque, M., Shanker, U., Singh, S. K. and Varshney, V.P. (2009). Effect of exogenous insulin administration on ovarian function, embryo/fetal development during pregnancy in goats. Anim. Repr. Sc. 111: 202-213.
Research Article
PHARMACOKINETICS OF CEFEPIME IN FEBRILE BUFFALO CALVES

SHWETA JAIN1, NEETU RAJPUT AND Y. P. SAHNI
Department of Pharmacology and Toxicology College of Veterinary Science and Animal Husbandry,J. N.K.V.V., Jabalpur- 482001, India 1 Corresponding author: E-mail: drshwetavety@yahoo.co.in
ABSTRACT The present study was planned to investigate the effect of Brewers yeast-induced fever on the pharmacokinetics and dosage regimen of cefepime in buffalo calves (n = 4) following a single intravenous dose of 10 mg.kg-1 body weight. Fever was induced by subcutaneous administration of Brewers yeast at a dose of 20 mg.kg-1 body weight. The drug concentration in plasma was estimated by microbiological assay. At 1 min, the peak plasma concentration of cefepime was 42.9 0.40 g.ml-1 and the drug was detected upto 24 h. The apparent volume of distribution and area under the concentrationtime curve were 0.63 0.006 L.kg-1 and 70.1 0.59 g.ml-1.h, respectively. The elimination half-life was 3.05 0.01 h and total body clearance was 0.143 0.001 L.kg-1.h-1. Based on the results a satisfactory dosage regimen of cefepime in febrile buffalo calves was calculated as 9.55 mg.kg-1 followed by 8.92 mg.kg-1 at 12 h intervals. Keywords: Buffalo calves, cefepime, dosage, fever, pharmacokinetics.
INTRODUCTION Cefepime, a novel fourth generation cephalosporin, has potent bactericidal activity against a wide range of gram-negative and gram-positive micro-organisms including Pseudomonas aeruginosa and methicillinsusceptible Staphylococcus spp. It is highly active against Pneumococci, Streptococcus pneumoniae, Enterobactor cloacae and many members of the family Enterobacteriacea. It has several advantages over earlier generation cephalosporins and is highly stable to hydrolysis by -lactamases. Pharmacokinetics of cefepime has been investigated in rats (Brindley et al., 1991), dogs (Stampley et al., 1992), monkeys (Forgue et al., 1987), neonatal foals (Gardner and Papich 2001), horses (Guglick et al., 1998), ewes (Ismail, 2005), goats (Patani et al., 2006), cow calves (Pawar and Sharma, 2008) and human beings (Barbhaiya et al., 1990). Febrile conditions are known to markedly alter the pharmacokinetics of antimicrobials (Burrows, 1985). Fever is one of the most common manifestations of many infectious diseases and is reported to induce biochemical and physiological alterations in cells (Lohuis et al., 1988). In view of the paucity of such pharmacokinetic data, the present study was undertaken to determine the pharmacokinetics of cefepime in febrile buffalo calves. MATERIALS AND METHODS Four male buffalo calves of 6-12 months age and weighing between 70-100 kg, were housed in an animal shed with a concrete floor and adequate ventilation. A constant supply of water was maintained in the shed. The animals were healthy at the time of experiment. All the animals were acclimatized to the animal shed under
uniform conditions and were maintained on green fodder, wheat straw and water ad libitum. Fever was induced by subcutaneous injection of Brewers yeast (Sisco Laboratories, Mumbai, India) at the dose rate of 20 mg.kg1 body weight. Rise in body temperature was monitored by frequent recording of rectal temperatures. Cefepime (Sefdin, Unichem Laboratories, Mumbai, India) was administered as a single intravenous injection into the jugular vein of all the animals at the dose rate of 10 mg.kg-1 body weight when there was a 2oC rise in the rectal temperature of the animals. Blood samples (5 ml) were withdrawn from contralateral jugular vein into heparinized glass centrifuge tubes before and at 1, 2.5, 5, 7.5, 10, 15, 30, 45 min and 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24 and 36 h after cefepime administration. Plasma was separated by centrifugation at 3000 rpm for 15 min at room temperature and kept at 20C until analysis, which usually took place on the day after collection and was used for estimation of the drug concentrations. The concentration of cefepime in plasma was estimated by a standard microbiological assay technique (Arret et al., 1971) using Escherichia coli (MTCC 739) as test organism. Based on diameter of zone of inhibition of the reference concentrations, the plasma levels of cefepime were calculated and expressed as g.ml -1 . Pharmacokinetic parameters were calculated manually by the least square regression technique (Gibaldi and Perrier, 1982). Using the values of and Vdarea obtained after intravenous injection in febrile calves, the maintenance dose of cefepime was calculated using the formula D/ = Cp (min).Vd(area). (e 1). Where, Cp (min) is the minimum inhibitory concentration of cefepime. Initial dose
22
Kinetics of cefepime in calves
was calculated by deleting 1 from the above equation. RESULTS AND DISCUSSION In all the animals there was significant rise in body temperature within 20-21 of administration of Brewers yeast. The elevated body temperature attained its peak value of 102.6 0.07oF (2-3oF above normal) at 24 h of injection of Brewers yeast. The body temperature of animals then declined slowly but remained above the normal range upto 26-28 h and thereafter became normal. Evaluation of the data revealed that the pharmacokinetics of cefepime was best described by a two-compartment open model. Initially there was a rapid fall in plasma concentration during the distribution phase and then a gradual fall during the elimination phase. The various pharmacokinetic parameters of cefepime in febrile buffalo calves are given in Table 1. At 1 min following single intravenous administration, the peak plasma levels of cefepime in febrile animals was 42.9 0.40 g.ml-1, which rapidly declined to 9.740.16 g.ml-1 at 1 h. The levels of cefepime above the therapeutic plasma concentration were maintained from 1 min to 10 h of administration. Similar to the present findings, the plasma concentration of cefepime at 1 min following intravenous administration in febrile crossbred calves was reported as 50.0 0.48 g.ml-1 (Pawar and Sharma, 2008).
The pattern of disappearance of drug from plasma of febrile buffalo calves followed two-compartment open model. Similarly, the disposition pattern of cefepime in febrile cross-bred calves (Pawar and Sharma, 2008) and cefpirome in buffalo calves (Rajput et al., 2008) also followed two-compartment open model. The high value of distribution rate constant (9.50 0.14 h-1) indicated that cefepime was rapidly distributed into various body fluids and tissue compartments of febrile buffalo calves. This correlated well with the rapid decline of drug in plasma in the early phase. The high value of K12/ K21 ratio (1.71 0.02) also indicated the rapid transfer of drug from central to peripheral compartments in febrile buffalo calves. The high value of AUC (70.1 0.59 g.ml1 .h) and AUMC (295.8 2.90 g.ml-1-.h2) showed that vast area of the body was covered by cefepime concentrations. In contrast to our findings much higher value of AUC (101.0 g.ml-1-.h) and AUMC (414.2 2.90 g.ml-1-.h2) for cefepime in febrile calves (Joshi, 2005) has been reported. Extensive tissue distribution was reflected by the high value of Vd(area) (0.63 0.006 L.kg-1) and P/C ratio (1.84 0.03) established in the present study indicated that cefepime is present in greater concentration in peripheral than the central compartments in febrile buffalo calves. The elimination half-life of 3.05 0.01 h and ClB of 0.143 0.001 L.kg-1.h-1 in febrile buffalo calves, reflected
TABLE 1: Pharmacokinetic parameters of cefepime in febrile (20 % Brewers yeast @ 20 mg.kg-1, subcutaneous) buffalo claves after a single intravenous dose of 10 mg.kg-1 body weight (n = 4). Parameter A B t t K12 K21 K12/K21 AUC AUMC Vd(area) Cl B Kel MRT P/C Td Unit g.ml-1 h-1 g.ml-1 h-1 h h h-1 h-1 ratio g.ml-1.h g.ml-1.h2 L.kg-1 L.kg-1h-1 h-1 h ratio h Mean SE 30.0 0.53 9.50 0.14 15.2 0.15 0.226 0.001 0.07 0.001 3.05 0.01 5.74 0.07 3.35 0.07 1.71 0.02 70.1 0.59 295.8 2.90 0.63 0.006 0.143 0.001 0.64 0.007 4.21 0.02 1.84 0.03 10.1 0.04
A and B, zero-time plasma concentration intercepts of regression lines of distribution and elimination phases, respectively; and , distribution and elimination coefficients, respectively, in the biexponential equation that describes the plasma concentration-versus-time data; t1/2 and t1/2, half-lives of distribution and elimination phases, respectively; K12 and K21, rate constants defined in the two compartment model; AUC, area under the plasma concentration-time-curve; AUMC, area under the first moment of the plasma concentration-time-curve; Vdarea, volume of distribution from AUC; ClB, total body clearance of the drug; Kel, elimination rate constant from the central compartment; MRT, mean residence time of drug in body; T/P, ratio of the drug present in the peripheral to central compartment; td, total duration of pharmacological effect.
Jain et al.
rapid elimination and body clearance of the drug. In agreement to our findings high value elimination half-life (3.0 h) was also reported in calves (Joshi, 2005). Low value of ClB (114.2 ml.kg-1h-1) was also reported in cow calves (Pawar and Sharma, 2008). The high value of elimination half-life (1.97 h) and ClB (327 ml.kg-1.h-1) was also reported for ceftizoxime in febrile cow calves (Chaudhary et al., 2002). The value of Kel, MRT and td of cefepime in febrile buffalo calves were 0.640.007 h-1, 4.21 0.02 h and 10.1 0.04 h, respectively. Comparable values of MRT (4.15 h) have been reported for cefepime in febrile buffalo calves (Joshi, 2005). However, a higher value of Kel (2.58 h-1) was reported for ceftizoxime in febrile cow calves (Chaudhary et al., 2002). Taking a convenient dosage interval of 12 h, the priming and maintenance doses of cefepime to maintain an MIC of 1 g.ml-1- were calculated to be 9.55 and 8.92 mg.kg-1, respectively. However, under field conditions a suitable intravenous dosage regimen of cefepime in febrile buffalo calves would be 9.5 mg.kg-1 to be repeated at 12 h intervals. REFERENCES Arret, B., Johnson, D.P. and Krishbaum, A. (1971). Outline of details for microbiological assay of antibiotics. Res. Vet. Sci., 49: 34-38. Barbhaiya, R. H., Forgue, S. T., Gleason, C. R., Knupp,C. A., Pittman, K. A., Weidler D. J. and Martin, R. R. (1990). Safety, tolerance and pharmacokinetic evaluation of cefepime after administration of single intravenous doses. Antimicrob. Agents Chemother. 34: 1118-1122. Brindley, C. J., Brodie, R. R., Cook, S. C. , Oldfield, P. R., Chasseaud, L. F. and Barbhaiya, R. H. (1991). Dose proportional pharmacokinetice of cefepime in rats. Eur. J. Drug Metab. Pharmacokinet. 3: 9-14. Burrows, G.E. (1985). Effect of experimentally induced P. haemolytica pneumonia on the pharmacokinetics
of erythromycin in calf. Am. J. Vet. Res. 46: 798803. Chaudhary R.K., Srivastava, A.K. and Rampal, S. (2002). Pharmacokinetics and dosage regimen of ceftizoxime in normal and febrile calves. Indian J. Ani. Sci. 72 (2): 133-135. Gardner, S. Y. and Papich, M. G. (2001). Comparison of cefepime pharmacokinetics in neonatal foals and adult dogs. J. Vet. Pharmacol. Ther. 24: 187192. Gibaldi, M., and Perrier, D.(1982) Methods of residuals. In: Pharmacokinetics, 2nd ed. Marcel and Dekker Inc. New York, 433-444. Joshi, B. (2005). Pharmacokinetics of cefepime in healthy and febrile buffalo calves (Babulus bubalis) M.V.Sc. Thesis, Punjab Agriculture University, Ludhiana, India. Lohuis, J.A.C.M., Varheijden, J.H.M., Burvenich, C. and Van Miert, A.S.J.P.A.M.(1988). Pathophysiological effects of endotoxin in ruminants . Vet. Quart. 10: 109-125. Patani, K., Patel, U. , Bhavsar, S., Thaker, A. and Sarvaiya, J.(2006). Single dose pharmacokinetics of cefepime after intravenous and intramuscular administration in goats. Turk. J. Vet. Ani. Sci. 32(3): 159-162. Pawar, Y. G. and Sharma, S. K. (2008). Influence of E. coli lipopolysaccharide induced fever on the plasma kinetics of cefepime in cross-bred calves. Vet. Res.Commun. 32(2): 123-130. Rajput, N., Dumka, V. K. and. Sandhu, H. S. (2008). Influence of experimentally induced fever on the disposition of cefepirome in buffalo calves. Environ. Toxicol. Pharmacol. 26(3): 305-308. Stampley, A. R., Brown, M. P., Gronwell, R. R., Castro, L. and Ston, H. W. (1992). Serum concentration of cefepime (BMY 28142), a broad-spectrum cephalosporin, in dogs. Cornell. Vet. 82: 69-77.
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Research Article
IMPACT OF SUB-CHRONIC ORAL EXPOSURE OF IMIDACLOPRID ON BIOCHEMICAL PROFILE OF CROSSBRED CALVES

BARINDERJIT KAUR, RAJDEEP KAUR* AND H.S. SANDHU
Department of Veterinary Pharmacology and Toxicology College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana - 141 004 * Corresponding author: E-mail: rajksidhu@yahoo.com ABSTRACT In the present investigation, oral administration of imidacloprid, a neonicotinoid insecticide, at a dose rate of 0.5 mg/kg/day for 150 consecutive days did not produce any toxic symptoms but significantly elevated the levels of plasma AST (11.8-22.0%), ALT (22.0-34.0%) and alkaline phosphatase (18.0-27.7%) without affecting the activity of plasma acid phosphatase and cholinesterase enzymes. A significant increase in the levels of serum globulins (15.1%), creatinine( 23.3%), glucose 14.1-17.0% and cholesterolemia (16.9%) were observed, however, it did not change the levels of total serum proteins, albumin and blood urea nitrogen. Thus, on the basis of long-term oral toxicity study, imidacloprid was found to be a low risk insecticide cross bred cows. Keywords: : Biochemical parameters, cow calves, imidacloprid, oral toxicity.
INTRODUCTION In the present world scenario of insect pest management, newer and safer compounds are being developed for various agricultural and veterinary practices. Imidacloprid is a recently introduced insecticide belonging to the group neo-nicotinoids which have outstanding potency and systemic action for crop protection against piercing-sucking pests with high efficacy against flea in cats and dogs (Tomizawa and Casida, 2005). Its insecticidal activity is attributed to its complete and virtually irreversible blockage of post-synaptic nicotinergic acetylcholine receptors in the central nervous system of insects (Phua et al., 2009). Imidacloprid was introduced in India in 1997-98 due to its high efficacy against insectpests of agriculture and animal importance. However, following to its repeated use, it may enter into food chain and cause residual toxicity in man and animals. As there is scarcity of reports on the toxicological aspects of this compound in livestock, the present study was designed to evaluate toxic effects of imidacloprid in crossbred cow calves. MATERIALS AND METHODS Eight healthy male crossbred cow calves (612months; 70-120kg) were randomly divided into two groups of four animals, each. All the animals were dewormed and maintained on seasonal green fodder and water was provided ad libitum. Group I served as control, to which no insecticide was administered. The animals in group II were administered imidacloprid at a dose rate of 0.5 mg/kg/day for 150 consecutive days. The requisite amount of insecticide was suspended in about 50 ml of water and drenched to the animals daily between 9.00
a.m. 10.00 a.m. The animals were weighed weekly and doses of imidacloprid corrected according to the change in body weight. To study the biochemical parameters, blood samples were collected weekly in heparin containing test tubes via jugular vein puncture. Plasma was separated and stored for analysis of biochemical parameters. Heparinized blood was also used for blood glucose estimation (Frankel et al, 1970). Plasma enzymes viz. aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and acid phosphatase (ACP), and blood urea nitrogen (BUN), plasma creatinine and plasma cholesterol levels were estimated using the standard methods as described by Wootton (1964). Plasma cholinesterase activity was estimated according to the method of Voss and Sachsse (1970) as modified by Moroi et al (1976). Level of total serum proteins in blood without any anticoagulant was determined (Reinhold, 1953). Data was analysed employing statistical method as per Snedecor and Cochran (1967). RESULTS AND DISCUSSION Daily oral administration of 0.5 mg/kg/day of imidacloprid in male cow calves did not produce any apparent clinical signs associated with toxicosis. However, long-term administration of imidacloprid produced significant elevation in the levels of both plasma aspartate aminotransferase and plasma alanine aminotransferase (Table 1). The levels of plasma aspartate aminotransferase were significantly enhanced from day 90 (11.8%) through day 135 (22%). Similarly, imidacloprid produced a significant elevation in the levels of plasma alanine aminotransferase to the extent of 22% from day 90th to
26
TABLE 1: Effect of subchronic oral administration of imidacloprid (0.5 mg/kg/day) on plasma enzymes and total serum proteins in cow calves. 15 68.3 2.34 67.5 2.08 66.5 2.16 67.8 1.92 71.3 1.83** 75.4 2.06*a 76.8 1.63*a 78.4 1.67*a 42.7 1.64 40.2 3.01 41.2 2.43 43.1 1.83 60.5 1.41** 62.2 2.30**a 64.3 1.66*a 66.4 2.12*a 50.6 3.00 51.1 1.67 52.3 1.72 53.2 2.06 59.3 1.37** 62.3 1.52*a 65.6 1.72*a 63.7 1.66*a 4.35 0.15 4.98 0.33 4.32 0.33 4.72 0.26a 4.33 0.28 4.56 0.32a 4.33 0.30 4.61 0.44a 30 45 60 75 90 105 120 135 150 68.3 1.81 80.1b 40.5 1.96 68.5b 52.3 1.44 64.8b 4.31 0.26 5.17b
Kaur et al.
Time (days)
186.2 5.15 183.6 5.95 183.3 8.68 184.3 6.21 186.7 5.62 204.5 b 198.0 6.33 200.3 6.01a 199.4 5.76a 201.5 6.19a 5.87 0.15 6.87 0.32 5.82 0.17 6.81 0.29a 5.90 0.33 6.83 0.31a 5.88 0.29 6.81 0.33a 5.94 0.31 6.80b
Aspartate Aminotransferase (nmol pyruvate formed/min/ml) Control 66.1 1.46 69.1 1.72 67.5 2.19 68.4 1.42 67.5 1.84 69.3 1.28 Imidacloprid 64.3 1.60 64.1 1.32 66.0 2.01 67.3 1.72 70.5 2.11 68.6 2.19 Alanine Aminotransferase (nmol pyruvate formed/min/ml) Control 43.5 2.21 42.4 1.95 44.2 1.50 43.2 2.26 40.6 0.99 42.3 1.72 Imidacloprid 49.6 3.17 50.2 1.96 49.2 2.53 50.8 2.61 54.9 1.63 58.3 2.14 Alkaline Phosphatase (nmol penol liberated/min/ml) Control 52.6 2.73 50.1 2.52 49.7 1.92 49.4 2.36 51.7 2.14 50.3 2.60 Imidacloprid 49.9 1.83 50.1 2.25 51.6 2.20 52.3 1.97 54.6 1.86 58.8 0.98** Acid Phosphatase (nmol phenol liberated/min/ml) Control 4.56 0.12 4.52 0.15 4.43 0.24 4.29 0.22 4.33 0.17 4.29 0.20 Imidacloprid 4.83 0.23 4.81 0.74 4.52 0.08 4.77 0.19 4.67 0.25 4.69 0.32 Plasma Cholinesterase (nmol acetylthiocholine hydrolysed/min/ml) Control 188.7 5.38 192.9 6.24 187.4 4.68 187.6 5.12 187.4 5.43 184.6 3.96 Imidacloprid 195.2 6.43 195.7 6.66 189.4 6.92 191.4 5.99 190.4 6.21 191.9 6.18 Total Serum Proteins (g/dl) Control 6.03 0.06 6.26 0.40 5.99 0.32 6.11 0.07 6.13 0.07 5.99 0.05 Imidacloprid 6.96 0.41 6.94 0.36 6.23 0.43 6.92 0.31 6.63 0.46 6.88 0.42
Values given represent the mean SE of 4 animals unless otherwise stated., aValue is Mean SE of 3 animals, bValue is mean of 2 animals, * P<0.05, **P<0.01
TABLE 2: 15 4.47 0.11 3.92 0.31 2.04 0.31 3.32 0.26 1.89 0.35 3.28 0.27 2.57 0.19 3.32 0.36 2.53 0.21 3.40 0.15 3.86 0.21 4.05 0.33 4.32 0.19 4.02 0.33 4.16 0.32 4.03 0.28 30 45 60 75 3.97 0.42 3.98 0.19 1.91 0.26 3.38 0.21 90 3.99 0.18 3.97 0.26 2.30 0.16 3.52 0.12 105 3.96 0.22 3.97 0.23a 120 4.02 0.19 4.02 0.32a 135 4.04 0.21 4.09 0.41a 2.37 0.26 2.29 0.17 2.32 0.27 3.61 0.14a 3.82 0.12**a 3.78 0.13a 150 4.01 0.23 3.99b 2.33 0.30 3.72b
Effect of subchronic oral administration of imidacloprid (0.5 mg/kg/day) on biochemical parameters in cow calves.
Time (days)
112.4 7.41 108.5 5.98 109.6 5.95 113.3 6.50 114.2 6.72 109.8 5.99 110.4 4.89 109.9 4.72 112.6 6.01 110.0 5.44 139.7b 122.4 4.36 121.4 4.83 124.3 3.89 130.2 3.26** 136.2 3.45** 135.6 4.11** 138.8 4.08*a 140.8 2.10*a 140.3 3.56*a 6.32 0.69 8.42 0.32 0.97 0.17 1.23 0.05 61.2 1.33 56.2 2.36 64.2 2.52 60.7 1.86 0.92 0.21 1.27 0.10 6.17 1.02 8.40 0.26 6.63 0.78 8.17 0.42 0.95 0.19 1.30 0.09 61.2 2.29 57.2 1.63 6.33 0.54 8.23 0.32 0.93 0.20 1.35 0.11 63.9 1.24 58.5 2.42 6.94 0.63 8.22 0.28 0.96 0.16 1.30 0.06 62.9 3.01 62.7 2.42 6.72 0.16 8.58 0.41 0.96 0.12 1.33 0.07 63.7 2.62 66.7 2.06** 6.48 0.32 8.55 0.44a 0.94 0.20 1.38 0.08a 62.2 2.87 63.6 2.14a 6.36 0.31 8.52 0.38a 6.34 0.28 8.60 0.29a 0.91 0.13 0.89 0.15 1.54 0.05*a 1.48 0.06**a 59.2 1.99 60.1 2.32 62.8 2.03a 68.2 1.74**a 6.35 0.32 8.55b 0.90 0.17 1.48b 61.6 2.44 69.4b
Albumin (g/dl) Control 4.24 0.24 Imidacloprid 3.89 0.27 Globulin (g/dl) Control 2.79 0.27 Imidacloprid 3.32 0.11 Cholesterol (mg/dl) Control 104.3 6.21 Imidacloprid 120.0 3.26 Blood Urea Nitrogen (mg/dl) Control 6.27 0.43 Imidacloprid 8.46 0.23 Creatinine (mg/dl) Control 0.97 0.14 Imidacloprid 1.20 0.08 Blood Glucose (mg/dl) Control 64.1 2.49 Imidacloprid 58.5 2.31
Values given represent the Mean SE of 4 animals unless otherwise stated., aValue is mean SE of 3 animals, bValue is mean of 2 animals, * P<0.05, **P<0.01
Impact of imidacloprid in crossbred calves
34% rise till day 135th. These findings are in agreement with results of experiments conducted by USEPA (1998) on rats treated with imidacloprid which showed an elevation in the levels of ALT. Although damage to any particular organ cannot be cited as a cause of increased levels of aminotransferases, the moderate increase in AST and ALT levels in blood in the present study suggests hepatocellular injury. Repeated oral administration of imidacloprid @ 0.5 mg/kg/day for 150 days produced no significant alterations in the plasma levels of acid phosphatase (ACP) enzyme, but a significant elevation to the extent of 27.7% was observed in the levels of plasma alkaline phosphatase (ALP) on 135th day of study (Table 1). An elevation in the plasma levels of ALP in rats upon administration of imidacloprid has also been documented by USEPA (1998). Alkaline phosphatase has high activity in kidneys, intestine, liver, biliary tracts, lungs, etc. in many species (Clampitt and Hart, 1978), howeve, r elevated ALP level is encountered usually where there is hepatobiliary involvement and as documented by USEPA (1998), liver is the principal target organ in imidacloprid toxicity, the elevated ALP levels in the present study could be due to its harmful effect on hepatocytes. Imidacloprid @ 0.5 mg/kg/day for 150 days did not produce any significant alteration in the levels of plasma cholinesterase enzyme (Table1). No change in activity of cholinesterase enzymes by imidacloprid on repeated oral exposure are in agreement with the data generated by USEPA (1998) in experimental adult wistar rats where repeated oral administration of imidacloprid produced no inhibition of cholinesterase activity in brain, plasma or erythrocytes. The effect of repeated oral administration of imidacloprid on total serum proteins, albumin and globulins is depicted in Table 1 and 2. There was no significant alteration in the level of total serum proteins and albumin after repeated drenching of imidacloprid @ 0.5 mg/kg/day for 150 days, however, serum globulins showed a significant elevation to the extent of 15% on 120th day of treatment and then returned to normal. The findings of total serum proteins and albumin are in agreement with work of Premlata (2002) who reported no significant changes in rats. Although liver plays vital role in protein metabolism yet total protein concentration is of little value in assessment of liver functions (Brar et al., 2000). It is difficult to substantiate the temporary rise in serum globulin levels with repeated oral administration of imidacloprid from the available data. No significant alterations in the level of blood urea nitrogen were produced in cow calves exposed to repeated oral administration of imidacloprid, however, it produced a significant hypercreatininemia (Table 2). The plasma creatinine was elevated to the extent of 28% in imidacloprid
treated calves. The present findings of hypercreatininemia are in contrast to the work of Premlata (2002) who reported no change and a non-significant decrease in plasma creatinine levels after i.p. imidacloprid 28 days. The creatinine levels help in evaluating the renal functions especially the glomerular function because after filtration by glomerulli, it is excreted in urine. Increase in creatinine levels indicates nephron damage (Brar et al., 2000). The hypercreatininemia observed in the present study suggested damage to the renal clearance pathways following repeated oral administration of imidacloprid in calves. In addition, imidacloprid produced hyperglycemia and hypercholesterolemia in cow calves (Table 2). The blood glucose levels were elevated to the extent of 17%. Hyperglycemia induced by toxicants has been related to the increased secretion of catecholamines from adrenal medulla following stress conditions and subsequently an increase in circulating glucocorticoids which decrease peripheral glucose (Munck et al., 1984; Clement, 1985). The cholesterol levels were increased to the extent of 17.3%. Alterations in cholesterol levels are not liver specific but elevated level of total cholesterol and its ester can be observed in domestic animals having biliary obstruction (Cornelius, 1989). Therefore, on the basis of the present investigation it can be suggested that imidacloprid is a low-risk insecticide which can be safely used in the recommended concentrations in crossbred cow calves. REFERENCES Brar, R.S, Sandhu, H.S. and Singh, A. (2000). Veterinary Clinical Diagnosis by Laboratory Methods. Kalyani Publishers, Ludhiana-New Delhi. Cornelius, C.E. (1989). Liver function. In: Clinical Biochemistry of Domestic Animals. Kaneko, J J (Ed). 4th edn. Academic Press. San Diego. pp 36497. Clampitt, R.B. and Hart, R.J. (1978). The tissue activities of some diagnostic enzymes in ten mammalian species. J. Com. Pathol. 88: 607-21. Clement, J.G. (1985). Hormonal consequences of organophosphate poisoning. Fund. Appl. Toxicol. 5: 561-77. Frankel, S., Reitman, S. and Sonnerwirtha, A.C. (1970). Gradwhols Clinical Laboratory Methods and Diagnosis. Vol. I. The C.V Mosby Co., St. Louis. pp- 82-83. Moroi, K., Usbiyama, S., Satoh, T. and Kuga, T. (1976). Enzyme induction of repeated administration of tetrachlorvinphos in rats. Revue dElevage et de Medicine Veterinaire des Pays Tropicaure, 43: 431-34. Munck, A., Guyre, M.P. and Holbrook. (1984). Physiological functions of glucocorticoids in stress
Kaur et al.
and their relation to pharmacological action. Endocrine Rev. 5: 25. Phua, D.H., Lin, C.C., Wu, M.L., Deng, J.F. and Yang, C.C. (2009). Neonicotinoid insecticides: an emerging cause of acute pesticide poisoning. Clin. Toxicol. 47(4): 336-41. Premlata. (2002) Pharmacological and Toxicological Studies of Imidacloprid: A Nitroguanidine Insecticide . M.V.Sc. Thesis, CCS Haryana Agricultural University, Hisar. Reinhold, J.G. (1953). Total Proteins: Reiner M (ed). Standard Methods of Clinical Chemistry. Vol 1 pp 88. Academic Press, New York. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods. 6th ed Allied Pacific, Bombay.
Tomizawa, M. and Casida, J.E. (2005). Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu. Rev. Pharmacol. Toxicol. 45:24768. USEPA. (1998) Imidacloprid, Pesticide Tolerance. Federal Register 63(57): 14363-71. Voss, S. and Sachsse, K. (1970). Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetylcholine/DTNB procedures. Toxicol. Appl. Pharmacol. 16: 76472. Wootton, I.D.P. (1964) Microanalysis. In Medical Biochemistry, 4th edn, J and A Churchill Ltd, London.
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28
Research Article
NITRIC OXIDE AND C-GMP-INDEPENDENT 2-ADRENOCEPTORS MEDIATED TOCOLYSIS IN BUFFALOES

SOUMEN CHOUDHURY1*, THAKUR UTTAM SINGH2, SATISH KUMAR GARG3 AND SANTOSH KUMAR MISHRA4
1
Asstt. Prof.,Department of Pharmacology and Toxicology, 3Dean, C.V. Sc. & A. H., U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura - 281 001 3 Scientist, 4Head, Division of Pharmacology and Toxicology, I.V.R.I.Izatnagar-243 122, U.P., India 1 Corresponding author: E-mail-smnpharma@gmail.com
ABSTRACT Control of myometrial excitability employing 2-adrenoceptor agonists has important therapeutic applications in the management of preterm labour and threatened abortion. Present study was undertaken to unravel the possible involvement of nitric oxide and c-GMP and c-AMP as second messengers, if any, in regulating 2-adrenoceptor mediated myometrial relaxation in buffalo uterus. Albuterol produced concentration- dependent relaxation of myometrial spontaneity with pD2 and Rmax value of 8.55 and 101.1 6.3%, respectively. Pre-treatment of myometrial strips with L-NAME (1mM) or ODQ (1mM) failed to alter the potency and efficacy of albuterol compared to control. Exposure of myometrial strips to albuterol (10 nM and 100 nM) although increased tissue c-AMP accumulation to 3.49 0.28 and 4.89 0.58 pmol/mg tissue protein (n=6) respectively, in a dose dependent manner in comparison to basal level of 3.23 0.48 pmol/mg tissue protein but this rise in c-AMP level was not statistically significant. Based on these findings it may be inferred that tocolytic effect of albuterol on buffalo myometrium seems to be nitric oxide and c-GMP-independent. But the involvement of c-AMP in regulating beta2-adrenoceptor mediated relaxation cannot be ruled out. Key words : Albuterol, 2-adrenoceptor agonists, buffalo, cAMP, cGMP, nitric oxide, tocolytic, uterine myometrium.
INTRODUCTION Use of 2-adrenoceptor stimulants as tocolytics is a common practice in veterinary medicine to treat gynaecological and obstetrical disorders like uterine prolapse, delay parturition during odd hours, during foetotomy and caesarean sections in cows, sheep and other domestic animals and also in embryo transfer technology (Salmanoglu et al., 1990). However, the underlying signalling pathways in special reference to the involvement of nitric oxide and second messengers responsible for inducing 2-adrenoceptor agonist-mediated myometrial relaxation in buffalo is not known. Thus the present study was designed to unravel the possible involvement of nitric oxide (NO) and cyclic nucleotides (cGMP and c-AMP), in mediating albuterol-induced tocolysis in buffaloes. MATERIALS AND METHODS Myometrial strips from midcornual region of diestrous stage uterus of cyclic non-descript buffaloes were collected from local abattoir and mounted in a thermostatically controlled (37.0 0.5 C) organ bath (Ugo Basile, Italy) of 10 ml capacity containing continuously aerated (95% O2 + 5% CO2) Ringer Locke solution (RLS, pH 7.4). Isometric tension was recorded under 2g resting tension using Chart V5.4.1 software programme (Powerlab, AD Instruments, Bella Vista, NSW, Australia). In order to evaluate the involvement of NO and c-GMP pathway, the cumulative concentration response of albuterol were recorded alone on spontaneous myometrial contractility
was recorded and presence of L-NAME (L-nitro arginine methyl ester, Sigma, USA), a non-selective nitric oxide synthase (NOS) inhibitor and ODQ (1H [1, 2, 4]oxodiazole[4,3-a] Quinoxaline, Cayman, USA), a soluble guanylate cyclase (sGC) inhibitor, respectively. Tissue cAMP levels in absence (control) and presence of albuterol (10nM and 100nM) were estimated using EIA assay kit (Cayman Chemicals, USA). Rolipram (10M, Santa Cruz, USA) was used as phosphodiesterase (PDE4) inhibitor. Tissue protein was estimated by employing Lowrys method (1951) and concentrations of c-AMP were expressed as pmol/mg tissue protein. Multiple mean values of relaxation response were analysed using twoway ANOVA followed by Bonoferroni post hoc test whereas biochemical parameters were analyzed by one-way ANOVA followed by Tukeys post-hoc test using Graph Pad Prism 4.0 (Graph Pad, La Jolla, USA). pD2 is defined as logEC50 value. RESULTS AND DISCUSSION After an equilibration period of about 2hrs in RLS, myometrial strips exhibited regular pattern of spontaneity with average amplitude and frequency/min of 2.11 0.15 g (n = 25) and 1.72 0.15 (n = 25), respectively. Albuterol (3x10-10 M to 3x10-8 M at 0.5 log dose units) produced concentration-dependent relaxant effect on myometrial spontaneity with pD2 and Rmax value of 8.55 and 101.1 6.3%, respectively. L-NAME (1mM) had no direct effect on myometrial spontaneity and pre-treatment of tissue with L-NAME did not produce any significant effect on albuterol-
Choudhury et al.
induced myometrial relaxation (Fig.1). The pD2 and Rmax values of albuterol (n=6) in the presence of L-NAME were found to be 8.41 and 92.71 5.35%, respectively. Further, pre-incubation of myometrial strips with ODQ (1mM) failed to produce any significant effect either on myometrial spontaneity or relaxation effect of albuterol. The corresponding values of pD2 and Rmax of albuterol in presence of ODQ were recorded as 8.33 and 99.38 0.32%, respectively (n = 6). The DRCs of albuterol in absence and presence of ODQ were depicted in Fig.2 Considering the EC50 value of albuterol, tissue cAMP level was determined in the presence of 10 and 100 nM of albuterol. Fig. 3 illustrates the basal (control) and albuterol-induced c-AMP accumulation in myometrial strips of buffaloes. Treatment with 10 nM albuterol slightly increased the level of c-AMP (3.49 0.28 pmol/mg tissue protein, n=6) while at higher concentration (100 nM) there was marked increase in the tissue concentration of cAMP (4.89 0.58 pmol/mg tissue protein; n=6) in comparison to control (3.23 0.48 pmol/mg tissue protein,
n=6) as shown in fig. 4. However, the changes in c-AMP levels following treatment with albuterol were found to be statistically non-significant. Albuterol, a selective 2 agonist, produced concentration dependent inhibitory effect on myometrial spontaneity, thus, suggesting its potent tocolytic effect on buffalo myometrium. This observation is in conformity with similar reports on ewes (Crankshaw and Ruzycky, 1984), goats and cows (Matsuda et al., 2002) and also from our laboratory on buffalo myometrium contracted with different spasmogens (Garg et al., 2004). Nitric oxide has been reported to be involved in myometrial relaxation both in animals (Kuenzli et al., 1998) and human beings (Bradley et al., 1998) and its use in preterm labour has been suggested by Lees and coworkers (1994). In human myometrium, NO has been reported to induce myometrial relaxation in laboring and non-laboring stage either directly or through second messenger by stimulating guanylyl cyclase (Buxton et
Fig. 1: Spontaneous contractility of isolated myometrial strips of buffaloes.
Fig. 3: Cumulative concentration response curves of albuterol alone and in the presence of ODQ (1 mM) on isolated myometrial strips of buffaloes (n=6). Vertical barsrepresent SEM.
Fig. 2: Cumulative concentration response curves of albuterol alone and in the presence of L-NAME (1mM) on isolated myometrial strips of buffaloes (n=6). Vertical bars represent SEM.
Fig. 4: c-AMP levels in the isolated buffalo myometrium under basal condition (control) and after treatment with albuterol (10 nM and 100 nM).
30
2-adrenoceptors mediated tocolysis in buffaloes
al., 2001). 2-adrenoceptors are reported to mediate relaxation by releasing nitric oxide from epithelial and nonepithelial source of rat trachea (Montalvo et al., 2010) and by promoting eNOS phosphorylation in mouse pulmonary artery (Banquet et al., 2011). Pre-treatment of myometrial strips with L-NAME (1mM) did not produce any significant shift in dose response curve of albuterol in comparison to control. There was no change in the potency (pD2) and efficacy (Rmax) of albuterol following pre-treatment of LNAME compared to control. Thus, it may be inferred that NO does not seem to modulate 2- adrenoceptor agonistinduced myometrial relaxation in non-pregnant buffaloes. Pal (2010) has also observed similar effect of L-NAME on pregnant buffalo myometrium. ODQ did not produce any significant effect on albuterol-induced tocolysis and the dose-response curves of albuterol alone and in the presence of ODQ superimposed over each other and the pD2 value of albuterol alone (8.55) did not differ from the value of albuterol in the presence of ODQ (8.33 ). These observations suggest that blockade of sGC did not influence the tocolytic effect of albuterol thus evidently suggesting that albuterol-induced tocolysis is c-GMP-independent. Effect of albuterol in the present study in buffalo myometrium is similar to that of nitric oxide-induced relaxation in guinea pig (Kuenzli et al., 1996), monkey (Kuenzli et al., 1998) and human myometrium (Bradley et al., 1998) which is reported to be c-GMP independent and possible involvement of ion channels or pump regulation by nitric oxide has been suggested (Buxton et al., 2001). Involvement of KATP and BK Ca channels has also been reported in mediating albuterol-induced tocolysis in non-pregnant buffaloes (Choudhury et al., 2010) Garg and co-workers (2005) have reported a promising potential of papaverine and caffeine as tocolytics in buffaloes which act through the inhibition of phosphodiesterase, thereby resulting in increased intracellular levels of c-AMP and also by causing blockade of Ca2+ and 2-adrenergic receptors are also known to relax smooth muscles by increasing the intracellular levels of c-AMP (Rang et al., 2003). Thus, in the present study an attempt was undertaken to elucidate the possible involvement of c-AMP in regulating albuterol-induced tocolytic effect on buffalo myometrium. Pre-treatment of uterine strips with albuterol although dose dependently increased c-AMP accumulation in myometrial strips but this change was statistically non-significant. Thus, the possible involvement of c-AMP in mediating albuterolinduced tocolysis in buffaloes cannot be ruled out. Observations of this study are in agreement with the results of Hughes et al. (1997). Both the c-AMP-dependent and c-AMP-independent pathways for the action of -adrenergic receptors agonists have been documented (Khac et al., 1996).
In conclusion, albuterol-induced myometrial relaxation in buffaloes is independent of NO and c-GMP and possible involvement of c-AMP in mediating albuterolinduced tocolysis cannot fully be ruled out. REFERENCES Banquet, S., Delannoy, E., Agouni, A., Dessy, C., Lacomme, S., Hubert, F., Richard, V., Muller, B. and Leblais, V. (2011). Role of G(i/o)-Src kinasePI3K/Akt pathway and caveolin-1 in adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery. Cell Signal. 23(7):1136-1143. Bradley, K.K., Buxton, I.L.O., Barber, J.E., McGaw, T. and Bradley, M.E.(1998). Nitric oxide relaxes human myometrium by a cGMP independent mechanism. Am. J. Physiol. 44: C1668-C1673. Buxton, I.L.O., Kaiser, R.A., Malmquist, N.A. and Tichenor, S. (2001). NO-induced relaxation of labouring and non-labouring human myometrium is not mediated by cyclic GMP. . Br.J. Pharmacol. 134: 206-214. Choudhury, S., Garg, S.K., Singh, T.U. and Mishra, S.K. (2010). Cellular coupling of potassium channels with 2 adrenoceptors in mediating myometrial relaxation in buffaloes (Bubalus bubalis). J. Vet. Pharmacol. Therap. 33: 2227. Crankshaw, D.J. and Ruzycky, A.L. (1984) Characterization of putative -adrenoceptors in the myometrium of the pregnant ewes: correlation between the binding of [3H] dihydroalprenolol and the inhibition of myometrial contractility in vitro. Biol. Reprod. 30: 609618. Garg, K.M., Garg, S.K. and Sabir, M. (2005). Pharmacological inhibition of phosphodiesterase inhibitors as potential tocolytic agents in buffaloes (Bubalus bubalis). Ind. J. Anim. Sc. 75: 212-214. Garg, S.K., Garg, K.M. and Sabir, M. (2004). Evaluation of tocolytic efficacy of selective 2 adrenoceptor agonist on buffalo uterus. Ind. J. Exp. Pharmacol. 42: 913-918. Hughes, S.J., Hollingsworth, M. and Elliot, K.R.F. (1997). The role of cAMP-independent pathway in the uterine relaxant action of relaxin in rats. J.Reprod. Fert. 109:289-296. Khac, L.D., Arnaudeau, S., Lepretre, N., Mironneau, J. and Harbon, S. (1996). Adrenergic Receptors Activation Attenuates the Generation of Inositol Phosphates in the Pregnant Rat Mypmetrium. Correlation with Inhibition of Ca++ influx, a cAMPindependent mechanism. J. Pharmacol. Exp. Therap. 276: 130-136. Kuenzli, K.A., Bradley, M.E. and Buxton, I.L.O. (1996). Cyclic GMP independent effects on nitric oxide on guineapig uterine contractility. Br. J.
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Pharmacol. 119: 737-743. Kuenzli, K.A., Buxton, I.L. and Bradley, M.E. (1998). Nitric oxide regulation of monkey myometrial contractility. Br. J. Pharmacol. 124: 63-68. Lees,C., Campbell,S., Jauniaux, E., Brown, R, Ramsay, B., Gibb,D. and Moncada, S. (1994). Arrest of preterm labour of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet. 343: 1325-1326. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, RJ. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275. Matsuda, Y., Kouno, S., Sakamoto, H. and Ikenoue, T. (2002). Effects of meluadrine tartrate and ritodrine hydrochloride on oxytocin-induced uterine contraction, uterine arterial blood flow and maternal cardiovascular function in pregnant goats. Jap. J. Pharmacol. 90: 107113. Montalvo, F., Cantres-Fonseca, O., Santos, G., Vega, M., Torres, I., Carmona, J., Dexter, D. and Santacana,
G. (2010). Nitric oxide is involved in the response of the isolated intact and epithelium-denuded rat trachea to the 2 adrenergic receptor agonist salbutamol. Respiration. 80(5):426-432. Pal, S. (2010). Pharmacological characterization of ATPdependent potassium channels and signaling pathways of terbutaline and forskolin-induced myometrial relaxation in pregnant buffaloes. M.V.Sc. Thesis. U.P. Pt. D.D. Upadhyaya PashuChikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura , India. Rang, H.P., Dale, M.M., Ritter, J.M. and Moore, P.K. (2003). In: Pharmacology. 5th ed. Church Hill Livingstone Publishers. pp. 22-48. Salmanoglu, M.R., Bekyllrek, T. and Kilieoglu, C. (1990). Use of sympathomimetic (ritodine) to produce uterine relaxation in cows, sheep and goats. Veterinary Fakultesi Deraisi Universitesi, Ankara (Turkey). 37: 10-19.
Received on : 14-03-2012 Accepted on : 20-06-2012
32
Research Article
THRESHOLD PLASMA FLUORIDE LEVELS IN GOATS FOR SUBACUTE ORAL TOXICITY OF FLUORIDE AND EFFICACY OF ALUMINIUM SULPHATE AS AN AMELIORATIVE AGENT
VINAY KANT1, A. K. SRIVASTAVA2, R. RAINA3, P.K. VERMA4, N.K. PANKAJ5, P. SINGH6 AND S.K. UPPAL7
1 Ph.D. Scholar, Deptt. of Pharmacology and Toxicology, IVRI, Izatnagar, Bareilly, 2Director, NDRI, Karnal, 3Professor, Division of Pharmacology and Toxicology, F.V.Sc. & A.H., SKUAST-J, R.S. Pura, Jammu-181102, 4Asstant Professor, Division of Pharmacology and Toxicology, F.V.Sc. & A.H., SKUAST-J, R.S. Pura, Jammu-181102, 5 Ph.D scholar, Department of Pharmacology and Toxicology, C.V.&A.Sc, G. B. P. U. A. & T, Pantnagar- 263145, India, 6 M.V.Sc. Scholar, Livestock Product Technology, F.V.Sc. & A.H., SKUAST-J, R.S. Pura, Jammu-181102, 7Associate Professor, Department of Clinic and VEterinary Services, GADVASU, Ludhiana 1 Corrosponding author : E-mail: drvinaykant2001@yahoo.co.in
ABSTRACT Fluorine intoxication is an important global public health concern in humans and animals. In present study, sodium fluoride alone and with aluminium sulphate (ameliorative agent) was administered orally daily for 30 days in healthy goats of group 1 and 2 respectively to access the threshold plasma fluoride levels for appearance of clinical symptoms in subacute toxicity and ameliorative effect of aluminium sulphate. Signs of toxicity, like inappetance, lethargy, weight loss, decreased ruminal motility, decreased milk production, muscle weakness and decreased movement appeared only in group 1 within 7-10 days of exposure. Significant (P < 0.01) increase in plasma and milk fluoride levels was observed in both the groups during the experiment. Finally, it could be concluded that the plasma fluoride level >1.50 0.14 mg/L was associated with the clinical symptoms of subacute toxicity of fluoride in goats and aluminium sulphate may be effectively employed as ameliorative agent. Key words: Aluminium sulphate, goats, milk, sodium fluoride, subacute toxicity.
INTRODUCTION Fluorine is highly reactive and most electronegative element, due to which it combines directly or indirectly with other elements, except oxygen and nitrogen under ordinary temperature and pressure (Banks and Goldwhite, 1966), to form complexes called fluorides. These are released into environment via both human as well as natural activities and exposed to animals and humans mainly via air, drinking water and food. Kidneys are the primary route for the removal of absorbed fluoride from the body and other routes of excretion are sweat, feces, saliva and milk (Mellberg et al., 1983; Whitford, 1996; WHO, 2002). Although, fluorine is one of the essential trace elements required for human or animal body but excess intake produces deleterious effects on the body and causes a disease called Fluorosis; which is one of the important public health problems all over the world. So, the concentration of fluoride in the body is critical and plasma is the biological fluid into which fluoride must pass for its distribution in the body as well as its elimination from the body; therefore, plasma is often referred to as the central compartment of the body (Fejerskov et al., 1996). So, the aim of the present study was to determine the threshold plasma fluoride levels for the appearance of clinical symptoms in subacute toxicity and ameliorative effect of aluminium sulphate in goats.
MATERIALS AND METHODS Experimental design Eight healthy cross bred goats of age 1.5-2 years of 25-30 kg body weight were procured from local farmers of R.S. Pura, Jammu. They were acclimatized for two weeks in the divisional animal shed under standard conditions before the commencement of experiment. The animals were maintained on ad lib feed and water. The experimental protocol was approved by institutional ethics committee. The animals were divided into two groups of 4 each. Group 1 was used to induce the subacute toxicity of F to determine the threshold plasma F levels to induce clinical symptoms and in this group sodium fluoride (NaF, SD Fine- Chem. Ltd.) alone was administered orally by drenching at the dose rate of 20 mg/kg b.wt. (providing 9 mg/kg b.wt. fluorine) daily for 30 days. Where as group 2 was used to study the ameliorative efficacy of aluminium sulphate and in this group same dose of sodium fluoride along with aluminium sulphate [Al2(SO4)3.16H2O, SD FineChem. Ltd.] at the dose rate of 150 mg/kg b.wt. were administered orally by drenching daily for 30 days. Aluminium sulphate was administered 30 minutes before the administration of sodium fluoride. Both the salts were dissolved separately in 100 ml of distilled water and drenched to the animals daily between 9.00 to 10.00 a.m. All the animals were weighed weekly and doses of salts
Kant et al.
were administered accordingly. The body weight and ruminal motility of the goats of both the groups were determined on day 0, 7, 14, 21, 28, 30 of exposure and on day 7, 14 after termination of exposure of fluoride. All animals were closely observed for clinical signs and mortality. Sample collection Blood samples were collected in a thoroughly clean fluoride free glass vial containing heparin (Himedia, Mumbai) @ 5-10 IU/ml blood on day 0, 1, 3, 7, 14, 21, 28, 30 of treatment and on day 3, 7, 14 after termination of treatment by jugular vein puncture. The heparinized blood samples were centrifuged at 3000 rpm (revolution per minute) for 15 min. Plasma samples were separated and stored in polyethylene plastic vials at 20oC until analysis. Milk samples were also collected in polyethylene plastic vials on day 0, 1, 3, 7, 14, 21, 28, 30 of exposure and stored at - 20oC until analysis. Fluoride estimation Plasma fluoride estimation was carried out by Sener et al. (2007) method by using ion selective electrode (ISE) of Orion Fluoride Analyzer (microprocessor based 4 star model) using 96-09 combination fluoride electrode. Equal volume of plasma sample and TISAB III (Total ionic
strength adjustment buffer III) was added in a beaker, well mixed and fluoride was estimated by putting 96-09 combination fluoride electrode in beaker. The electrode was washed with distilled water and cleaned before the estimation of the next sample. Similarly, milk fluoride levels were also estimated. Statistical analysis Mean plasma fluoride levels on different days of exposure and after termination of exposure were compared to pre-exposure (0 day) value of the same group and a probability level of P < 0.05 and P < 0.01 was considered statistically significant (Snedecor and Cochran, 1989). RESULTS The toxic symptoms appeared within 7-10 days of oral administration of fluoride. All the animals of group 1 showed signs of toxicity, like inappetance, lethargy, weight loss (Table 1), decreased ruminal motility (Table 1), decreased milk production, muscle weakness and decreased movement. They sat and stayed at one place during grazing. Two animals out of the four animals in the group showed haemorrhagic diarrhea intermittently after 24 day onwards of the treatment. There was loss of condition and goats on an average lost 7 kgs in body weight
TABLE 1: Effect on body weight and ruminal motility of goats on different days of exposure of sodium fluoride alone (group 1) and along with aluminium sulphate (group 2) (n=4 and values are expressed as mean SE) Parameter Group 0 1 Body weight (Kilograms) Ruminal motility (per 2 minutes) 2 1 2
a b
Treatment days 7 29.75 1.65 32.5 1.04 1.50 b 0.29 2.5 0.29 14 28.5 1.85 33.25 1.03 1.25 b 0.25 2.75 0.25 21 26.75 b 1.84 33.0 0.91 1.50 b 0.29 2.5 0.29 28 24.75 b 1.44 31.75 0.75 1.25 b 0.25 2.75 0.25 30 24.00 b 1.47 31.25 0.48 1.50 b 0.29 2.75 0.25 31.75 1.65 32.0 1.08 2.75 0.25 3.0 0.00
Post Treatment days 7 23.75 b 1.11 31.75 0.75 2.00 b 0.00 2.75 0.25 14 25.00 b 1.08 32.25 0.63 2.25 a 0.25 2.5 0.29
Significantly (P < 0.05) different as compared to pre-exposure (0 day) value of the same group. Significantly (P < 0.01) different as compared to pre-exposure (0 day) value of the same group.
TABLE 2: Fluoride levels (mg/L) in plasma and milk on different days of exposure of sodium fluoride alone (group 1) and along with aluminium sulphate (group 2) (n=4 and values are expressed as mean SE) Parameter Plasma Fluoride levels (mg/L) Group 0 1 2 Milk Fluoride levels (mg/L) 1 2
a b
Treatment days 1 0.541 0.08 0.459 b 0.05 0.447 b 0.07 0.169 b 0.02
b
Post Treatment days 21

b
3 1.191 0.15 0.666 b 0.20 0.507 b 0.09 0.216 b 0.01

b
7 1.500 0.14 0.949 b 0.15 0.711 b 0.19 0.257 b 0.01

b
14 1.595 0.20 1.065 b 0.14 0.774 b 0.16 0.305 b 0.02
28
b
30
b
3
b
7
b
14
b
0.058 0.01 0.03 0.01 0.04 0.01 0.039 0.01
1.493 0.23 0.878 b 0.14 0.829 b 0.13 0.344 b 0.02
1.913 0.30 1.116 b 0.25 0.875 b 0.12 0.379 b 0.03
1.850 0.32 0.759 b 0.27 0.878 b 0.08 0.428 b 0.03
0.513 0.02 0.470 b 0.11 NE NE
0.346 0.02 0.202 a 0.07 NE NE
0.310 b 0.03 0.185 a 0.05 NE NE
Significantly (P < 0.05) different as compared to pre-exposure (0 day) value of the same group. Significantly (P < 0.01) different as compared to pre-exposure (0 day) value of the same group. NE: not estimated
34
Fluoride levels and its amelioration in goats
after 30 days of daily administration. The toxic symptoms were not prominant in animals of group 2. The plasma fluoride levels of goats on different days are presented in Table 2 and the graphical representation is shown in the Figure 1. In group 1, the plasma fluoride (mg/L) increased significantly (P < 0.01) on day 1 of exposure from the pre exposure levels and remained increased significantly (P < 0.01) during the whole experiment. The plasma fluoride level was maximum on day 28 of exposure. Similarly, in group 2, the plasma fluoride levels (mg/L) increased significantly (P < 0.01) on day 1 of exposure and the levels were also maximum on day 28 of exposure. After termination of exposure, the plasma fluoride showed decreasing trend in both the groups, but remained significantly higher than the normal pre exposure levels. The milk fluoride levels on different days are also presented in Table 2 and the graphical representation is shown in the Figure 2. The levels of milk fluoride (mg/L) increased significantly (P < 0.01) on different days of exposure from the pre exposure levels in dose response manner. The maximum milk fluoride levels in both the groups were on day 30 of exposure. DISCUSSION The fluoride toxicity is directly correlated to the
Fig. 1. Plasma fluoride levels (mg/L) on different days in group 1 (only NaF) and 2 [NaF along with Al2(SO4)3].
Fig. 2. Figure 2. Milk fluoride levels (mg/L) of group 1 and 2 on different days of exposure.
level of fluorine in blood and tissues. The tolerance of fluoride varies with the species, concentration of fluoride in water, feed and soil, and solubility of fluoride. The normal safe levels of fluorine in livestock are considered to be less than 0.2 ppm in plasma (Aiello, 1997). The toxic symptoms observed in the present study are in concurrence with the findings in sheep (Tiwary et al., 1979) and buffalo calves (Gujarathi et al., 1991; Marconi, 2000). Poor growth performance of experimental animals is probably due to disturbance of some nutrient utilization, which is in accordance with the findings of Guenter and Hahn (1986) in hens. In present study, the plasma fluoride levels increased significantly on different days of exposure and the appearance of toxic symptoms within 7-10 days revealed that the concentration of >1.50 0.14 mg/L of fluoride in plasma may be taken as threshold for appearance of clinical symptoms of subacute toxicity of fluoride in goats. But Marconi (2000) reported that 121.010.6 ng/ml (0.121 mg/L) may be taken as threshold for appearance of clinical symptoms of subacute toxicity of fluoride in buffalo calves. As the buffaloes are most susceptible and goats are most resistant animals to fluoride toxicity among the ruminants, so, the toxic effects were observed at lower levels in buffalo calves as compared to our study in goats. Also the dose of sodium fluoride used by Marconi in buffalo calves was 6 mg/kg which was about 3.5 times lesser to our used dose in the goats. Kristinsson et al., (1997) also reported that plasma concentration of F > 500 ng/ml (0.5 mg/L) after short term exposure may be considered as potentially toxic in lambs. Our toxic levels are three times higher to the toxic levels observed by the Kristinsson et al. (1997) in lambs and this may be due the age difference, as the young animals are more susceptible to fluoride toxicity as compared to adults. On the other hand, the toxic symptoms were not observed in group 2 and the plasma fluoride levels increased significantly on different days of exposure, but to a lesser extent as compared to group 1. This may be due to the formation of insoluble complexes by aluminium with the fluoride in the gastrointestinal tract and decreases its absorption. Therefore, it could be concluded that the aluminium sulphate has ameliorative effects on the increase in plasma fluoride levels, which is in accordance with the findings in buffalo calves (Marconi, 2000) and cow calves (Mehra, 1981). In both the groups, the plasma fluoride levels showed decline on day 21 and 30 as compared to day 14 and 28 of exposure, respectively, and this may be due to manual error in the sampling time or it may be due to that during those days the body elimination of fluoride was more. But the levels on day 21 and 30 are very much significantly higher as compared to pre-exposure values and they have not showed significant decline on these days as compared to the day 14 and 28, respectively.
Kant et al.
The milk fluoride levels were also increased on different days of exposure from pre-exposure levels in both the groups and the levels were within the ranges reported by Samal and Naik (1995) in cows (0.19 to 1.15 ppm) and in buffaloes (0.31 to 1.21 ppm). Recording of decreased milk yield in present study is in accordance with the findings in cattle (Murray, 1967; Jones, 1972; Sobocinski et al., 1984). This increase of fluoride levels in milk revealed that milk is one of the routes of excretion. On the basis of results, it could be concluded that the plasma fluoride level >1.50 0.14 mg/L was associated with the clinical symptoms of subacute toxicity of fluoride in goats and aluminium sulphate may be effectively employed for alleviation of toxic symptoms of subacute toxicity of fluoride in goats and also milk is one of the routes of excretion of fluoride. ACKNOWLEDGEMENT The authors are thankful to Vice Chancellor, Shere-Kashmir University of Agriculture Sciences and Technology, Jammu for providing necessary facilities. REFERENCES Aiello, S.E. (1997). The Merck Veterinary Manual, pp. 2038-39. 8th edn, Merck and Co. Inc Whitehouse Station, N J, USA. Banks, R.E. and Goldwhite, H. (1966). Fluoride chemistry. In: Smith FA (Ed) Handbook of Experimental Pharmacology. Vol. 20 Part 1, pp. 608. New York Springer-Verlag. Fejerskov, O., Ekstrand, J. and Burt, B.A. (1996). Fluoride in Dentistry. 2nd edn. Copenhagen: Munsksgaard. Guenter, W. and Hahn, P.H.B. (1986). Fluorine toxicity and laying hen performance. Poult. Sci. 65: 769778. Gujarathi, S.R., Singh, B. and Bhikane, A.U. (1991). Effect of acute experimental fluorine poisoning on hematological and biochemical indices in buffalo
calves (Bubalus bubalis). Ind. J. Vet. Med. 11: 80-82. Jones, W.G. (1972). Fluorosis in dairy herd. Vet. Rec. 90: 503-507. Kristinsson, J., Gunnarson, E., Johannesson, P. and Palsson, P.A. (1997). Experimental fluoride poisoning in Icelandic sheep. Buvisindi. 11: 107112. Mehra, U.R. (1981). Effect of ameliorative measures against fluorosis on certain blood constituents of cattle. Ind. J. Nutr .Dietet. 18: 372-374. Marconi, N. (2000). Studies on experimental fluorosis and its amelioration in buffalo M.V.Sc. Thesis, Punjab Agricultural University, Ludhiana, India. Mellberg, J.R., Ripa, L.W. and Leske, G.S. (1983). Fluoride in Preventive Dentistry. Chicago: Quintessence Publishing. Murray, M. (1967). Fluorosis in a herd of cattle in Kenya. Bull. Epizoot. Dis. Afr. 15: 259-262. Samal, U.N. and Naik, B.N. (1995). Fluoride levels in milk and blood serum of cattle. Environ. Ecol. 13: 415417. Senedecor, G.W. and Cochran, W.J. (1989). Statistical Methods, Oxford IBH Co., pp. 61. Bombay. Sener, Y., Tosun, G., Kahvecioglu, F., Gokalp, A. and Koc, H. (2007). Fluoride levels of human plasma and breast milk. Eur. J. Dent. 1: 21-24. Sobocinski, R., Ewy, R. and Ewy, Z. (1984). Ten years study of fluorosis in cattle. Medyeyna Veterynaria. 40: 67-71. Tiwary, S.N., Singh, C.D.N. and Jha, G.J. (1979). Pathology of acute fluorine poisoning in sheep. Ind. Vet. J. 56: 638-640. Whitford, G. (1996). The Metabolism and Toxicity of Fluoride. 2nd ed. Switzerland: Karger. WHO. (2002). Environmental health criteria, pp. 227. Geneva.
36
Research Article
PHARMACOKINETICS OF LEVOFLOXACIN FOLLOWING SUBCUTANEOUS ADMINISTRATION IN CATTLE CALVES

ARVIND KUMAR, ANU RAHAL*, RAM RAGVENDRA, RAJESH MANDIL, ATUL PRAKASH AND SATISH K. GARG
Deptt. of Pharmacology and Toxicology,College of Veterinary Sciences Pt. Deen Dayal Upadhyaya Veterinary Science University (DUVASU), Mathura - 281 001 (Uttar Pradesh). * Corresponding author: E-mail: rahalanu72@gmail.com ABSTRACT Disposition kinetics of levofloxacin following a single subcutaneous administration was determined in cattle calves at a dose rate of 10mg/kg body weight. Plasma concentrations of levofloxacin were determined using HPLC assay and were subjected to compartmental pharmacokinetic analysis using a computer software program Pharmkit. Following SC administration of levofloxacin, the peak plasma concentration of approximately 3g.ml-1 was observed at 1 h. Levofloxacin was detectable in plasma (0.210.05 g.ml-1) up to 12 h following SC administration. The plasma concentration time data of levofloxacin for SC route was best conformed to one-compartment open model with first order absorption rate constant. Almost cent percent bioavailability values demonstrated the superiority of subcutaneous route over other nonvascular routes. Levofloxacin may be administered to cattle calves @ 10 mg/kg body weight and repeated at 24 h interval by SC route for treating majority of the susceptible microbial infections of veterinary importance. Key words: Cattle calves, pharmacokinetics, subcutaneous.
INTRODUCTION Levofloxacin, a fluorinated 4-quinolone containing a six-member (pyridobenzoxazine) ring, is active against most aerobic Gram-positive and Gram-negative organisms with moderate activity against anaerobes (Davis and Bryson, 1994) and potent bactericidal activity against organisms such as Pseudomonas, Enterobacteriaceae and Klebsiella (Klesel et al., 1995). It distributes well to target tissues and fluids in the respiratory tract, skin, urinary tract, prostate, other soft tissues and its uptake by cells make it suitable for use against intracellular pathogens. Based on the pharmacological profile, levofloxacin can be a promising tool for the treatment of bacterial infections in cattle calves which form an important component of the Indian livestock. In view of easy administration, slow absorption and maintenance of therapeutic concentrations for a longer duration, the present study was undertaken to determine the pharmacokinetic profile of levofloxacin following subcutaneous administration in cattle calves. MATERIALS AND METHODS Experimental animals Six healthy female cattle calves, 3-6 months (4785 kg) old, were quarantined and maintained following standard managemental practices and provided concentrate, green fodder and wheat- straw with ad libitum fresh water at the Dairy Farm of University. The animals were dewormed using albendazole (5 mg/kg) 21 days before the start of actual experiment. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC).
Drugs
For preparation of the standard curves of levofloxacin, pure levofloxacin was purchased from SigmaAldrich. Injectable formulation of levofloxacin (Meriflox; Wockhardt Limited., Mumbai) was injected in female cattle calves in subcutaneous (SC) space on the lateral aspect of neck at the dose rate of 10 mg/kg body weight. Collection of blood samples Blood samples were collected into heparinised tubes by jugular venipuncture before drug administration and at predetermined time intervals i.e. 2.5, 5, 10, 15, 30, 45 minutes, 1, 1.5, 2, 3, 4, 6, 8, 12, 18 and 24 hours post drug administration. The blood samples were centrifuged at 3000 rpm for 20 min to separate plasma which were stored in storage vials at -20C until further assay. Extraction of levofloxacin from plasma Extraction of levofloxacin from plasma was carried out as per the modified method of Neilson and Gyrd-Hansen (1997). Plasma (0.5 ml) was deproteinized by adding of 100 l perchloric acid (0.6M), vortexed at high speed for 1 minute and then centrifuged at 10,000g for 5 min. The clear supernatant (200 l) was collected in a microcentrifuge tube and 100 l of HPLC grade water was added. This mixture was then filtered through Millipore 0.22 m cellulose acetate membrane filter, and an aliquot of 20 l of the sample was injected into HPLC system for analysis. Chromatographic conditions For determination of levofloxacin levels in plasma, modified HPLC method of Gao et al. (2007) was used. The HPLC system (Waters, USA) comprised of two Waters 515 HPLC pump, rheodyne manual loop injector with a 20
Kumar et al.
l loop, Waters 2996 photodiode array detector and Empower software. C18 reverse phase column (particle size 5 m; 4.6 x 100 mm, Waters XBridgeTM) was used as a stationary phase. The mobile phase consisted of acetonitrile, water, phosphoric acid and triethylamine (16:83:0.6:0.45, v/v/v/v) with pH adjusted to 2.34. The flow rate was 0.6 ml/min and the run time was 5 min. The samples were quantified at a wavelength of 294 nm. Calibration curves Levofloxacin stock solution (100 g/ml) was prepared in 0.1 N NaOH in HPLC grade water. The standard curve of levofloxacin was prepared from the stock solution of levofloxacin by diluting with pooled plasma from the untreated cattle calves. The standard curve of levofloxacin was linear in the range of 0.05 to 6.4 g/ml. These methods were found to be linear and reproducible and the correlation coefficient (R^2) was 0.999, the intra-day and inter-day coefficient of variance was less than 5 per cent, mean recovery was more than 90 per cent. These standard curves were used for determination of levofloxacin concentrations in the unknown plasma samples. Cattle calf blank plasma produced no endogenous interferences at the retention time of levofloxacin. Pharmacokinetic analysis of data The plasma levofloxacin concentration time profile of each animal following drug administration were used to determine the pharmacokinetic variables describing the absorption, distribution and elimination characteristics in cattle calves with the help of a non-linear iterative curve fitting computer programme (Pharmkit) and other parameters were determined using the equations described by Gibaldi and Perrier (1982) and Baggot (1977). The best compartment model was selected on the basis of sum of squares, positive and negative residuals correlation coefficient values and Akaike Information criterion. The dosage regimens of levofloxacin were suggested using pharmacokinetic and pharmacodynamic indices of antimicrobial (Walker, 2000; Dudley, 1991). RESULTS AND DISCUSSION Mean plasma concentrations of levofloxacin at different time intervals following the drug administration by subcutaneous route are presented in Fig. 1. An appreciable and clinically effective concentration of levofloxacin (1g.ml-1) in plasma could be detected within at 0.04 h and the peak plasma concentration of levofloxacin was observed at 1 h following SC administration. Levofloxacin could be detected in plasma up to 12 h after drug administration. Evaluation of the results of observed plasma levels of levofloxacin indicated that the data could be best fitted to a one compartment open model with the first order absorption and adequately described by the equation:
38
Cp = Be-t - A2 e-kat Where Cp is the plasma concentration of levofloxacin at time t, A2 and B is zero time plasma drug concentration intercepts of the disposition curves, Ka and are the first order rate constant for absorption and elimination phases, respectively and e is the base of natural logarithm. The resulting kinetic parameters have been presented in Table 1. Following SC injection of levofloxacin, absorption of the drug was slow perhaps due to low vascularity of the subcutaneous tissue but elimination was apparently fast. This might be due to the limited entry of the drug to the peripheral compartment due to low initial plasma levels. This contention is also supported by significant reduction in the apparent volume of distribution of levofloxacin following SC administration. The absorption half-life (t 1/2Ka) following SC administration was longer (0.750.18h) than 0.340.07 h in buffalo calves (Ram et al., 2010), thus suggesting species variation in absorption kinetics. Elimination halflife (t1/2ke) of levofloxacin (2.570.29 h) was almost comparable to that of 3.050.17 h in cross bred calves (Kumar et al., 2009), 3.270.31 h in buffalo calves (Ram et al., 2008), 3.470.86 h in camel (Goudah, 2008) and 2.94 0.78 h in stallions (Goudah et al., 2008). The AUC values are indicative of the systemic bioavailability of the drug. In cattle calves, the AUC values (g.mL-1h) were higher to that of 21.31 1.24 in lactating goats (Goudah and Abo-El-Sooud, 2008), 7.66 0.72 in cross bred calves (Dumka and Srivastava, 2006), 8.81
TABLE 1: Pharmacokinetic parameters of levofloxacin following a single subcutaneous administration at a dose of 10 mg/kg in cattle calves. Parameters(units) B (g.mL ) Ka ( h-1) Ke ( h-1) T1/2ka (h) T1/2ke (h) AUC (g.mL-1 h ) AUMC (g.mL-1 h2) MAT ( h ) MRT ( h ) Vd/F (mL.kg-1 ) Cmax (g.mL-1) Tmax (h) CL/F (mL.kg-1. h-1) F (%)
-1
MeanSE 8.39 1.63 1.24 0.28 0.29 0.03 0.75 0.18 2.57 0.29 28.61 6.40 93.7116.19 0.59 0.62 3.460.39 1.470.20 3.440.32 1.590.29 0.410.06 97.5019.66
B=Zero time intercept of elimination slope in the one compartment model, Ka =absorption rate constant, T1/2Ka = absorption half-life, T1/2e = elimination half-life, Cl/F = Clearance of drug, Vd/F=Apparent volume of distribution, AUC = Total area under the concentration time-curve, AUMC = Total area under the first moment concentration time-curve, MRT = Mean residence time, MAT = Mean absorption time, Tmax = time post drug administration at which peak concentration is achieved, Cmax = observed peak plasma concentration, F = bioavailability
kinetics of levofloxacin in calves
0.37 in buffalo calves (Ram et al., 2008) and 13.633.11 in camels (Goudah, 2008). Bioavailability (F) is one of the important determinants of drug therapeutic efficacy. Now a days, subcutaneous route of administration is becoming more popular in clinical practice for several reasons including ease of administration and the pharmacokinetic variables after intramuscular and subcutaneous administration have been found to be almost comparable, and even sometimes, slightly better in favour of SC route. Therefore, subcutaneous route should be preferred over the intramuscular route for better availability, ease of administration and being less painful to the animal. In the present study, The overall bioavailability (calculated on the basis of IV data being published elsewhere) was much higher and was almost 100 % following SC administration . Lower bioavailability values have been reported in calves (44.3-56.6%) in earlier studies (Ram et al., 2010; Dumka and Srivastava, 2006). Cent percent bioavailability along with lower Vdarea values implies that minimal amount of the drug reaches the sink/ reservoir and more of the levofloxacin remains available to the central compartment for exerting the antibacterial action in the body following the SC route of administration. Due to concentrationdependent bactericidal and post-antibiotic effect of fluoroquinolones (Castelli et al., 1989), blood concentrations of these drugs are not required to be maintained above the minimum inhibitory concentration values throughout the duration of therapy. It has been established that for concentration dependent floroquinolones, the AUC/MIC ratio is the most important predictor of efficacy with a clinical cure rate greater than 80% when this ratio is higher than 100-125 (Lode et al., 1998). A second predictor of efficacy for concentrationdependent antibiotics is the Cmax/MIC ratio, considering that values>10 lead to better clinical results (Toutain et
al., 2002). High Cmax/MIC values are necessary in order to avoid the emergence of bacterial resistance (Walker, 2000). The critical breakpoints that determine the efficacy of floroquinolones are suggested as Cmax/MIC >10 and AUC/MIC> 125 (Toutain et al., 2002; Walker, 2000). The MIC of levofloxacin has not yet been determined for bacteria isolated from cattle calves. To cover most of the susceptible organism, in the cattle population, the MIC90 of 0.06 to 0.12 g/ml of levofloxacin has been taken into consideration (Marshall and Jones, 1993). Based on the pharmacokinetic data generated in this study and taking the extremes of MIC 90 as 0.06-0.12g.ml -1 , a dosage of 10 mg/kg levofloxacin SC in cattle calves would result in following AUC/MIC and Cmax/MIC ratios in the range of 238.4-476.8 and 28.7-57.3 for SC administration. Based on the calculated Cmax/MIC and AUC/MIC, a levofloxacin dose of 10 mg/kg once daily can effectively be used by subcutaneous route of administration. Considering the first pharmacodynamic index and bioavailability, the subcutaneous route is almost at par with the intravenous administration. So, taking into the account the advantages of subcutaneous administration, it may be proposed that levofloxacin be preferentially used by subcutaneous route of administration at a dose of 10mg.kg-1 repeated at 24h intervals.
REFERENCES Baggot, J. D. (1977). Principles of drug disposition in domestic animals. The basis of veterinary clinical pharmacology. Ist Edn., W.B. Saunders Co., Philadelphia, U. S.A. pp.144-189. Castelli, M., Bertolini, A., Baggis, G., Aresca, P., Bossa, R. and Galatulas, I. (1989). Bactericidal and cytotoxic effects of combination of norfloxacin and 5-fluorouracil. Anticancer Res. 9: 49-52. Davis, R. and Bryson, H. M. (1994). Levofloxacin. A review of its antibacterial activity, pharmacokinetics and therapeutic efficacy. Drugs. 47(4): 677-700. Dudley, M. N. (1991). Pharmacodynamics and pharmacokinetics of antibiotics with special reference the fluoroquinolones. American J. Med. 91 (Suppl 6A): 45-50. Dumka, V. K. and Srivastava, A. K. (2006). Pharmacokinetics, urinary excretion and dosing regimen of levofloxacin following a single intramuscular administration in cross bred calves. J. Vet. Sci. 7(4): 333-337. Gao, X., Yao, G., Guo, N., An, F. and Guo, X. (2007). A simple and rapid high performance liquid chromatography method to determine levofloxacin in human plasma and its use in a bioequivalence study. Drug Discovery and Therap. 1(2): 136-140. Gibaldi, M. and Perrier, D. 1982. Pharmacokinetics, 2nd Edn. Marcel Dekker Inc. New York.
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Goudah, A. (2008). Pharmacokinetics of levofloxacin in male camels (Camelus dromedarius). J Vet. Pharmacol. Therap. 32: 296-299. Goudah, A. and Abo-El-Sooud, K. (2008). Pharmacokinetics, urinary excretion and milk penetration of levofloxacin in lactating goats. J. Vet. Pharmacol. Therap. 32: 101-104. Goudah, A., Abo El-Sooud, K., Shim, J. H., Shin, H. C. and Abd El-Aty, A.M. (2008). Characterization of the pharmacokinetic disposition of levofloxacin in stallions after intravenous and intramuscular administration. J. Vet. Pharmacol. Ther. 31 (5): 399-405. Klesel, N., Geweniger, K. H., Koletzki, P., Isert, D., Limbert, M., Markus, A., Riess, G., Schramm, H. and Iyer, P. (1995). Chemotherapeutic activity of levofloxacin (HR 355, DR 3355) against systemic and localized infections in laboratory animals. J. Antimicrob. Chemother. 35: 805-819. Kumar, S., Kumar, S., Kumar, V., Singh, K.K. and Roy, B. K. (2009). Pharmacokinetic studies of levofloxacin after oral administration in healthy and febrile cow calves. Vet. Res. Commun. DOI 10.1007/s 11259-009-9237-0. Lode, H., Borner, K. and Koeppe, P. (1998). Pharmacodynamics of fluoroquinolones. Clinical Infect. Dis. 27: 33-39.
Marshall, S.A. and Jones, R.N. (1993). In vitro activity of DU-6859a, a new florocyclopropyl quinolone. Antimicrobial Agents and Chemotherapy 37: 27472753. Nielsen, P. and Gyrd-Hansen, N. (1997). Bioavailability of enrofloxacin after oral administration to fed and fasted pigs. Pharmacology and Toxicology 80: 246250. Ram, D., Dumka, V.K., Sandhu, H.S. and Raipuria, M. (2010). Pharmacokinetics and dosage regimen of levofloxacin in buffalo calves after single subcutaneous administration. Vet. Arhiv 80 (2): 195-203. Ram, D., Dumka, V.K., Sharma, S.K. and Sandhu, H.S. (2008). Pharmacokinetics, dosage regimen and in vitro plasma protein binding of intramuscular levofloxacin in buffalo calves. Iranian Journal of Veterinary Resarch, Shiraz University 9(2): 23. Toutain, P. L., Del Castillom J. R. E. and Bousquet-Melou A. (2002). The pharmacokinetic pharmacodynamic approach to a rational dosage regimen for antibiotics. Res Vet Sci 73: 105-114. Walker, R. D. (2000). The use of Fluoroquinolones for companion animal antimicrobial therapy. Australian Veterinary Journal. 78: 8490.
40
Research Article
PHARMACOKINETICS OF PEFLOXACIN IN E.COLI (K88 STRAIN) ENDOTOXIN INDUCED FEBRILE GOATS

A. K. NATH, R.K.ROY, D.C.ROY* AND R. GOGOI
Department of Veterinary Pharmacology and Toxicology College of Veterinary Science, Assam Agricultural University, Khanapara ,Guwahati -781 022, India * Corresponding author: Email: roydula@gmail.com
ABSTRACT The present investigation was carried out to determine the disposition kinetics of pefloxacin in endotoxin induced febrile goats. Six clinically healthy male local goat of Assam were taken for the study. Pefloxacin (5mg/kg) was administered as single i.v. dose. Fever was induced experimentally by i.v administration of E.coli (K88 strain) endotoxin solution at a dose rate of 0.25 g/Kg body weight. Fever condition decreased the therapeutic persistence of pefloxacin in plasma from 600 min (10h) to 480 min (8h). Induced fever increased the values of Cp0, ClB, , , Kel and T/C ratio but lowered the values of t, t, AUC and Vd(area) of pefloxacin. The T/C ratio was higher in febrile (3.81) than afebrile (3.23) goats which indicated an increase in distribution of pefloxacin into extravascular tissues. Key words: Assam, endotoxin, goat, kinetics, pefloxacin.
INTRODUCTION Pefloxacin is a fluoroquinolone antimicrobial agent used against wide range of both gram positive and gram negative microorganisms. Although a few of studies on pharmacokinetics of pefloxacin in cattle (Patil etal., 1996), sheep (Moutafchiva and Djouvinov, 1997), goat (Roy et al., 1997; Malik et al., 2002) are available yet the reports of its disposition kinetics during febrile condition are scarcely available. Therefore, the present study was undertaken to investigate disposition kinetics of pefloxacin in both afebrile and endotoxin induced febrile state of local goat of Assam. MATERIALS AND METHODS Experimental animals Six clinically healthy male local goats of Assam, weighing between 9.2 to 11.7 kg of 8 to 9 months old were used for the study. The animals were procured locally, housed in the departmental animal shed, dewormed and acclimatized for 15 days prior to the start of the experiment. The animals were maintained on grazing, concentrate and gram feeding and water ad libitum. The same animals were used in cross over design to study both febrile and afebrile condition but not earlier than a gap period of two weeks of the last administered dose. Experimental design Pefloxacin (5mg/kg) was administered as single i.v. dose into the right jugular vein of healthy goats. Prior to the i.v. injection of pefloxacin, a blood sample (2.5ml) was collected in a dried heparinised (0.2mg/ml blood) test tube which was considered as blank. Similar samples (2.5 ml) from each animal were collected in heparinised test tubes by left jugular vein puncture after pefloxacin administration at 2, 5, 10, 20, 30, 45, 60, 120, 240, 360,
480, 600 and 720 min. For febrile condition, fever was induced experimentally by i.v administration of E.coli (K88 strain) endotoxin solution at a dose rate of 0.25 g/kg body weight. Pefloxacin was given intravenously after 1h of endotoxin administration through right jugular vein. Blood samples were collected prior to and after administration of pefloxacin at different predetermined time intervals as mentioned above. The plasma fractions were separated by centrifugation (3000 rpm for 30 min) and stored at 50C until analysed within 24h. For quantitative determination of pefloxacin concentration in plasma, the spectrophotometric method as described by Jha et al. (1996) was followed. Plasma pefloxacin concentration versus time profile of each goat was used to calculate the various pharmacokinetic determinants of pefloxacin, using the methods of Gibaldi and Perrier (1982). RESULTS AND DISCUSSION The plasma concentrations of pefloxacin persisted in afebrile and febrile goats till 10h (0.800.04g/ml) and 8h (0.820.04g/ml) of post administration, respectively. Mean plasma concentrations were plotted on a semilogarithmic scale as a function of time, exhibited distinct distribution and elimination phases. Initially the pefloxacin concentration declined rapidly followed by a slow disappearance, indicating that data could be best described by a two compartment open model in consonance with the first order kinetics. Pefloxacin has been reported to follow two compartment open model in cows (Patil et al., 1996), sheep (Moutafchiva and Djouvinov, 1997), goats (Roy et al., 1997). The various
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TABLE 1: Pharmacokinetic determinants of pefloxacin in afebrile and febrile goats following a single intravenous dose of 5mg/kg body weight (MeanSE).
Determinants C0P (g/ml) A (g/ml) (min-1) t (min) B (g/ml) (min-1) t (min) AUC (g.min/ml) Vd(area) (L/kg) Vc (L/kg) Kel (min-1) tkel (min) K12/K21 (ratio) ClB (ml-1.kg-1.min) T/C (ratio) Afebrile febrile 25.14 1.52 30.03 1.46 19.63 1.37 24.41 1.23 0.2652 0.0212 0.3195 0.0204 2.70 0.23 2.21 0.15 5.51 0.18 5.61 0.25 0.0049 0.0002 0.0080 0.0004 140.27 5.98 87.98 5.06 1194.73 76.09 781.67 17.29 0.8528 0.0248 0.8095 0.0359 0.216 0.022 0.168 0.008 0.0210 0.0005 0.0385 0.0023 33.04 0.7987 18.69 1.23 2.97 0.17 3.35 0.07 4.27 0.26 6.41 0.13 3.23 0.17 3.81 0.09
and bile, leading to lesser persistence of pefloxacin in plasma in febrile goats. It is concluded from this investigation that febile condition decreased the therapeutic persistence of pefloxacin in plasma from 600 min (10h) to 480 min (8h) in afebrile goats than healthy one. Induced fever increased the values of Cp0, ClB, , , Kel and T/C ratio but lowered the values of t, t, AUC and Vd(area) of pefloxacin. The high T/C ratio indicated its higher distribution in extravascular tissues in febrile (3.81) than afebrile (3.23) goats. ACKNOWLEDGEMENT Authors are thankful to the Dean, Faculty of Veterinary Science and Head, Department. of Pharmacology and Toxicology, AAU, Khanapara, Guwahati781 022 for providing necessary facilities. REFERENCES Gibaldi, M. and Perrier, D. (1982). Pharmacokinetics.2nd edn.Marcel Dekker Inc. New York. Jha, K., Roy, B.K. and Singh, R.C.P. (1996). The effect of induced fever on the bio-kinetics of Norfloxacin and its interaction with Probenecid in Goats. Vet. Res. Commun. 20: 473. Malik, J.K., Rao, G.S., Ramesh, S., Muruganandan, S., Tripathi, H.C. and Schukla, D.C. (2002). Pharmacokinetics of Pefloxacin in Goats after intravenous or oral administration. Vet Res Commun.26:141. Manna, S., Mandal, T.K. and Chakravarty, A.K. (1993). Modification of disposition kinetics of oxytetracycline by paracetamol and endotoxin induced fever in goats. Indian J.Pharmacol. 25: 199. Miert, Van A.S.J.P.A.M., Gogh, Van H. and Wit, J.G. (1976).The influence of pyrogen induced fever on absorption of sulpha drugs. Vet.Rec.11:480. Moutafchiva, R. and Djouvinov, D. (1997). Pharmacokinetics of pefloxacin in sheep. J.Vet. Pharmacol. Therap. 20: 405. Patil, R.V., Gatne, M.M., Somkuwar, A.P. and Ranade, V.V. (1996). Pharma-cokinetics of milk conc. of pefloxacin injection (pelwin) in lactating cows. Indian vet. J. 73: 1130. Roy, B.K., Pandey, S.N., Sinha, K.P., Thakur, D.K. and Singh, K.K. (1997). Pharmacokinetics of Pefloxacin in Goats. Indian J. Pharmacol. 29 : 435.
A= zero time intercept of distribution phase; B = zero time intercept of elimination phase; = Distribution/absorption rate constant; = Elimination rate constant; t = Distribution/absorption half life; t=Elimination half life; Vd area = Apparent volume of distribution; ClB= Total body clearance, AUC = Total area under the time concentration curve, C0P = Zero time plasma concentration, MRT = Mean residence time.
pharmacokinetic determinants (Mean SE) of afebrile and febrile goats were calculated with standard procedures and are presented in Table 1. Fever altered pharmacokinetic determinants of pefloxacin. The mean zero time plasma concentration of pefloxacin in febrile goat was 20 percent higher than in afebrile one. A similar observation was made by Jha and co-workers (1996) using norfloxacin in goats. On the contrary, lower oxytetracycline plasma concentrations were reported in febrile goats (48.30 1.60 g/ml) than in afebrile one (83.600.80 g/ml. Manna et al., 1993). Induced fever increased the values of total body clearance, elimination rate constant, distribution rate constant and the ratio of T/C and K12/K21. This results in corresponding lower values of t , t, AUC and tkel. This indicates that pefloxacin persisted in plasma for a shorter period of time in febrile goats than in afebrile one. A significant increase in AUC value but decrease in ClB value of norfloxacin was observed by Jha et al. (1996). Fever caused shorter persistence of pefloxacin in plasma. Fever alters the physiological status of the body by increasing the basal metabolic rate, heart rate and blood flow to both kidney and liver and consequently glomerular filtration rate (GFR) (Miert et al.,1976). So there is a possibility of rapid excretion of pefloxacin through urine
42
Research Article
ACUTE TOXICITY AND NEUROBEHAVIOURAL EFFECTS OF ACETAMIPRID IN MICE

V. KARTHIKEYAN, S. K. JAIN* AND J. S. PUNIA
Department of Pharmacology and Toxicology, LLR University of Veterinary and Animal Sciences Hisar - 125 004, Haryana, India *Corresponding author e-mail: drskjainhau@gmail.co ABSTRACT The maximum tolerated dose of acetamiprid, a pyridyl methylamine nicotinoid insecticide was found to be 46 mg/ kg, i.p. in Swiss albino male mice by conducting pilot dose range finding studies. Various gross behavioural profiles viz. spontaneous motor activity, forced locomotor activity, cocaine induced locomotor activity and amphetamine induced locomotor activity were deteimined. Acetamiprid decreased spontaneous motor activity and forced locomotor activity in dose dependent manner and antagonized cocaine and amphetamine induced locomotor behaviour. The findings indicated that acetamiprid alters the normal behaviour of mice having moderate toxic potential in mice. Key words: Acetamiprid, amphetamine, mice, neurobehaviour.
INTRODUCTION Acetamiprid, pyridyl methylamine is a new nicotinoid insecticide that has been recently introduced for use. It acts in a different manner by mimicing acetylcholine and induces abnormal excitement in the insect by interrupting the normal synaptic transmission, consequently to excitation and paralysis followed by death of the insects (Bai et al., 1991). Acetamiprid is highly selective and provides outstanding control over sucking pests such as aphids and white flies in vegetables. Especially, it has shown excellent activity against peach fruit moth, and apple leaf miners, and citrus leaf miners and highly effective for flea control in cats and dogs (Tomizawa and Casida, 2005). Therefore, this compound is widely used in agricultural practices and thus there are more chances that the persons handling it or animals may accidentally come in contact with this compound from recently applied fields or through its residues leading to toxicity. The purpose of studying acute toxicity is to obtain information on the biologic activity of a chemical and gain insight into its mechanism of action. Behaviour is the final integrated expression of nervous function. Locomotor activity has been suggested as behavioural endpoint (Lawrence, 1978). Therefore, the present study was undertaken to investigate the acute toxicity and neurobehavioural changes induced by acetamiprid in mice. MATERIALS AND METHODS Experimental design Swiss albino male mice weighing 20-25g were procured from Disease Free Small Animal House, LLR University of Veterinary And Animal Sciences, Hisar and housed in the polyacrylic cages in groups of six animals per cage and maintained at room temperature with a natural
light-dark cycle. The animals were provided feed and water ad libitum. All the experiments were conducted in noise free laboratory conditions. The prior approval of Institutional Animal Ethical Committee for the protocol of this study was obtained. Technical grade Acetamiprid was procured from Topical Agrosystem (India) Pvt. Limited, Chennai. Maximum tolerated dose Maximal tolerated dose (MTD) of acetamiprid by intraperitoneal route was determined conducting pilot dose range findings studies. Small groups of animals (n=6/dose) were administered a single dose of acetamiprid and observations were recorded at various time intervals. A range of doses was used initially, including a few lethal doses and then several iterations were selected to determine dose that would produce clear signs of toxicity but not result in lethality. Gross observable behaviour The effect of acetamiprid on gross observable behavioural profiles was studied using Irwin schedule as described by Turner (1965). The mice were distributed randomly in 3 groups (n = 6). The control group was administered 10 ml/kg of gum acacia solution (2%) and treatment groups received acetamiprid at the dose rate of 1/5 th or 2/5 th of MTD i.p. The procedure involved manipulative phase in which animals were subjected to least provoking stimuli. Animals were observed for behavioural activity profiles at various time intervals (2, 10, 20, 40, 60, 120, 240, 360 and 1440 min) and scores were assigned as per Irwin schedule as described by Turner (1965). Spontaneous motor activity (SMA) It is a measure of exploratory behaviour of animal. The SMA was monitored using a Any-Maze Advanced Video Tracking Software of Stoelting Co., USA for behavioural experiments. This tracks the animal
Karthikeyan et al.
movements according to the manual setting. Animals were divided randomly in three groups of six animals each. Control group received 10 ml/kg of gum acacia solution (2%), while treatment groups received 1/5th or 2/5th of MTD of acetamiprid i.p. Immediately after the treatment, animals were placed in a cage with suitable background to allow clearly visibility of the animals. Each animal was recorded for about 4 to 5 minutes and Any-Maze was set to track the animal for 250 seconds each and record various components of locomotor behaviour viz. total distance travelled (m), total distance travelled by animal head (m), average speed (m/s), total time mobile (s), total time immobile (s), total mobile episodes, total immobile episodes, maximum speed (m/s), rotations of animal body, path efficiency. Forced locomotor activity It is a measure of strength and co-ordinated movements of animal. The effect of acetamiprid on forced locomotor activity (FLA) was studied using rota rod (Acceler, Rota rod 7750, UGO Basile, Italy) as described by Dunham and Miya (1957). The mice were randomly divided in three groups of six animals each and trained to remain on the horizontal rod of rota rod apparatus rotating at twenty five revolutions per minute. As soon as mice fell off the rota rod, they were immediately placed back on the rod. The training session was terminated when mice remained on the rod continuously for two minutes. Next day, the effect of acetamiprid was studied thirty min after the treatment. Control group received 10 ml/kg of gum acacia solution (2%), while treatment groups received 1/ 5th or 2/5th of MTD of acetamiprid i.p. Thirty min after the treatment, the mice were given three consecutive trials of two min each on the rota rod. The cumulated time spent on the rota rod was recorded. Cocaine induced increased locomotor activity The effect of acetamiprid on cocaine induced increased locomotor activity was studied in mice. Mice were randomly divided in three groups each having six animals. Control group received 10ml/kg of gum acacia solution (2%), while treatment groups received 1/5th or 2/ 5th of MTD of acetamiprid i.p. thirty min prior to cocaine (15.0 mg/kg, s.c.) administration. Then the animals were observed for various components of locomotor behaviour using Any- maze Advanced Video Tracking software for behavioural experiments. Amphetamine induced increased locomotor activity Mice were randomly divided in three groups each having five animals. Control group received 10 ml/kg of gum acacia solution (2%), while treatment groups received 1/5th or 2/5th of MTD of acetamiprid i.p. thirty min prior to amphetamine (0.7 mg/kg, s.c,) administration. Then animals were observed for various components of locomotor behaviour using Any - maze Advanced Video Tracking software for behavioural experiments.
RESULTS Various iterations of acetamiprid (50, 48, 46, 44, 42, 40 mg/kg, i.p.) were used while determining the maximum tolerated dose and was determined to be 46 mg/kg by intraperitoneal route in adult Swiss albino male mice. It produced dose dependent onset and severity of toxic symptoms in mice. The major gross observable symptoms induced by acetamiprid were decrease in alertness and grooming, continued restlessness, ataxia, tremors, complete change in body and limb posture. At higher dose (50 mg/kg), animals also showed writhing and exophthalmia. In some animals open mouth breathing and vocalization was observed. In terminal stage, some animals showed clonic convulsions followed by tonic extension of hind limbs before death. However, stereotypy and straub tail were not observed in mice at any of the dose levels used in the study. Mice started showing symptoms of toxicity as early as 5 to 10 min following i.p. administration of acetamiprid. Mortality, if any, was recorded in 30 min up to 90 min. Animals which showed severe sign and symptoms for more than 2 h, survived and slowly became normal. Effects of 9.2 mg/kg and 18.4 mg/kg (1/5th and 2/ th 5 of MTD), i.p. of acetamiprid were observed and gross observable behavioural profiles were noted. The treated animals did not show significant change in behavioural profiles at any dose level either by manipulating or without manipulating the animal (Tables 1 and 2). The time of onset, peak effect and duration of acetamiprid effect in treated mice was found to be 5 -10 min, 20-30 min and 2 h, respectively. Acetamiprid decreased the spontaneous motor activity in mice. Various components of locomotor behaviour were affected by both lower (9.2 mg/kg) and higher (18.4 mg/kg), i.p. dose levels of acetamiprid ( Table 3, Fig. 1 and 2). Forced locomotor activity was also decreased at both the doses (Table 4). Acetamiprid antagonised the cocaine and amphetamine induced effect on various components of locomotor behaviour in a dose dependent manner (Tables 5 and 6, Fig. 3 and 4). However, the antagonistic effect was significant for cocaine induced behaviour. DISCUSSION The rapid absorption of acetamiprid from the peritoneum of mice probably resulted in the rapid onset of severe toxic symptoms in mice and death occurred due to respiratory failure. It has been reported that in imidacloprid toxicity, respiratory failure in mice might be due to both central paralysis and peripheral blockade of muscles of respiration (Hardman et al., 1996). Various gross observable sign and symptoms produced by acetamiprid in mice viz. decrease in alertness,
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Behavioural effect f acetamiprid
Fig. 1: Track plots showing the position of the centre of the animal SMA
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Fig. 2: Group occupancy plots of the animals' centre position and head position-SMA
Fig. 3: Group occupancy plot of the animals' centre position and head position - Cocaine induced
Fig. 4: Group occupancy plot of the animals' centre position and head position- Amphetamine induced
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TABLE 1: Effect of 1/5th of MTD (9.2 mg/kg, i.p.) of acetamiprid on gross observable behaviour of mice by manipulation. Scores at various time intervals (min) after drug administration 5 4 0 4 0 4 4 0 0 0 0 0 4 4 0 4 4 4 4 0 4 0 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 10 20 40 60 120 240 360 1440 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00
Activity profiles
Normal Score
Alertness Stereotypy Grooming Restlessness Touch response Pain response Startle response Straub tail Tremors Twitches Convulsions Body posture Limb position Ataxia Righting reflex Grip strength Pinnal reflex Corneal reflex Writhing Snout withdrawal Raubichaud test
Values are Mean SEM of six observations.
TABLE 2: Effect of 2/5th of MTD (18.4 mg/kg, i.p.) of acetamiprid on gross observable behaviour of mice by manipulation. Scores at various time intervals (min) after drug administration 5 4 0 4 0 4 4 0 0 0 0 0 4 4 0 4 4 4 4 0 4 0 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 10 20 40 60 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 120 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 240 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 360 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00 1440 4.00.00 0.00.00 4.00.00 0.00.00 4.00.00 4.00.00 0.00.00 0.00.00 0.00.00 0.00.00 0.00.00 4.00.00 4.00.00 0.00.00 4.00.00 4.00.00 4.00.00 4.00.00 0.00.00 4.00.00 0.00.00
Activity profiles
Normal Score
Alertness Stereotypy Grooming Restlessness Touch response Pain response Startle response Straub tail Tremors Twitches Convulsions Body posture Limb position Ataxia Righting reflex Grip strength Pinnal reflex Corneal reflex Writhing Snout withdrawal Raubichaud test
Values are Mean SEM of six observations.
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TABLE 3: Effect of acetamiprid on spontaneous motor activity (SMA) in mice. Components Control Total distance travelled (m) Total distance travelled by animal head (m) Average speed (m/s) Total time mobile (s) Total time immobile (s) Total mobile episodes Total immobile episodes Maximum speed (m/s) Rotations of animal body Path efficiency 3.390.61 6.671.06 0.010.00 235.5511.84A 2.372.37B 1.170.17B 0.170.17B 0.180.03 6.170.75A 0.040.01 Dose (mg/kg, i.p.) 1/5th of MTD (9.2) 2.120.58 4.320.43 0.000.00 188.2318.49A 61.7718.49A 3.830.91A 3.000.89A 0.250.12 5.001.03A 0.040.00 2/5th of MTD (18.4) 1.320.43 2.290.35 0.000.00 143.5318.35B 102.3217.34A 4.500.50A 4.000.68A 0.150.04 2.170.98B 0.050.02
Values are MeanSEM of six observations and means bearing different superscripts differ significantly (P<0.05). TABLE 4: Effect of acetamiprid on forced locomotor activity Dose (mg/kg, i.p.) Control 9.2 18.4 Time (sec) spent on rotarod 113.864.64A 78.5919.60B 41.998.08C
Values are MeanSEM of six observations and means bearing different superscripts differ significantly (P<0.05). TABLE 5: Effect of acetamiprid on cocaine (15 mg/kg, s.c.) induced locomotor activity Components Control(cocaine) Total distance travelled (m) Total distance travelled by animal head (m) Average speed (m/s) Total time mobile (s) Total time immobile (s) Total mobile episodes Total immobile episodes Maximum speed (m/s) Rotations of animal body Path efficiency 9.582.19 17.593.01A 0.040.00A 228.4614.65A 8.860.86B 1.600.60B 0.600.60B 0.310.06 14.204.55A 0.030.01B
A
Dose (mg/kg, i.p.) 1/5th of MTD+cocaine 7.721.70 15.794.05A 0.030.00A 202.4019.88A 47.2419.68B 3.200.86B 2.200.86B 0.530.23 6.201.85A 0.040.00B
A
2/5th of MTD+cocaine 2.040.90B 4.781.15B 0.000.00B 142.6621.52B 107.3621.51A 4.800.80A 3.800.80A 0.220.06 2.200.58B 0.090.02A
Values are MeanSEM of six observations and means bearing different superscripts differ significantly (P<0.05). TABLE 6: Effect of acetamiprid on amphetamine (0.7 mg/kg, s.c) induced locomotor activity. Components Total distance travelled (m) Total distance travelled by animal head (m) Average speed (m/s) Total time mobile (s) Total time immobile (s) Total mobile episodes Total immobile episodes Maximum speed (m/s) Rotations of animal body Path efficiency Values are MeanSEM of six observations 2.170.53 5.191.03 0.000.00 175.1216.01 73.9416.37 4.800.37 3.800.37 0.200.03 4.002.00 0.070.02 Dose (mg/kg, i.p.) Control(amphetamine) 1/5 th of MTD+ amphetamine 2/5 th of MTD+ amphetamine 1.700.37 4.160.63 0.000.00 150.225.68 99.2012.20 4.000.71 3.600.51 0.160.02 3.401.17 0.070.01 2.090.28 4.690.49 0.000.00 162.7010.62 87.0210.84 4.800.73 3.800.73 0.150.01 4.000.45 0.030.01
restlessness, ataxia, tremor and convulsions could be due to the agonist action of acetamiprid on nAChR or due to direct action on autonomic ganglia or neuromuscular junction. Tomizawa and Casida (2005) reported that the mammalian toxicity of neonicotinoids is centrally mediated
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since the symptoms of poisoning are similar to that of nicotine and agonist action in the vertebrate 42 nAChR, the primary target in brain. Except urination, no muscarinic effect was observed in mice indicating that acetamiprid is not active on the atropine blocking sites and urination might
be probably due to irritation in the urinary tract caused by acetamiprid. Exophthalmia in mice could be due to sympathetic stimulation by direct acting action on autonomic ganglia and writhing due to irritation to tissues or stimulation of the sensory receptors. Activation of mesencephalic dopamine pathway results in increase in locomotor activity ( Robbins and Everitt, 1982). Disruption of normal activity of dopamine cells in the ventral tegmental area or substantia nigra, with lesions or pharmacological manipulation, can inhibit locomotor activity ( Koob et al,. 1981). Nicotine is known to pass through the blood brain barrier readily (Oldendorf, 1971). Acute administration of nicotine activates the cortical region and diminishes locomotor activity in animals (Schechter and Rosecrans, 1972). Similar motor incoordination and decrease in SMA have been reported in isoproturon toxicity in mice ( Sarkar, 1990) suggesting that reduction in spontaneous motor activity and effect on motor coordination may be through involvement of distinct inhibitory action on motor performances. Administration of cocaine leads to increased ambulatory activity and dopaminergic system plays an important role in mediating the ambulatory and rewarding activities of cocaine (Roy et al., 1978). One of the neural substrate mediating these ambulation-acceleration activities of cocaine like psychostimulants as well as amphetamine is increase in the dopamine level in nucleus accumbens ( Bradberry and Roth, 1989; Kuczenski and Segal, 1989). Therefore, phenomenon of enhancement of dopamine receptor sensitivity can be regarded as a kind of possible mechanism of behaviour sensitization to psychomotor stimulatory drugs. Acetamiprid acts on nicotinic receptors in insects. Nicotine is known to potentiate the effect of amphetamine induced behaviour ( Bhatwadekar et al., 1999) and has been attributed to dopamine releasing effect of nicotine. However, in the present study, acetamiprid produced antagonistic effect indicating that it did not behave exactly like nicotine in mammals. The results of this study suggested that acetamiprid alters the normal behaviour of mice and also does not behave exactly like nicotine in mice having toxic potential and is a moderate risk insecticide. REFERENCES Bai, D., Lummis, S.C.R., Leicht, H.W., Breer and Sattelle, D.B. (1991). Actions of imidacloprid and related nitromethylene on cholinergic receptors of an identified insect motor neuron. Journal of Pesticide science. 33: 197-204. Bhatwadekar, N.A., Logade, N.A., Shirsat, A.M., Kasture, V.S. and Kasture, S.B. (1999). Effect of nicotine on behaviour mediated via monoamine neurotransmitters. Indian Journal of Pharmacology.
31: 410-415. Bradberry, C.W. and Roth, R.H. (1989). Cocaine increases extracellular dopamine in rat nucleus accumbens and ventral tegmental area as shown by in vivo microdialysis. Neuroscience Letters. 103: 97-102. Dunham, M.W. and Miya, T.S. (1957). A note on simple apparatus for detecting neurological deficit in rats and mice. Journal of the American Pharmacist Association. 46: 208-209 Hardman. G.L., Limbird, L.E., Molinoof, P.B., Ruddon, R.W. and Gilman, A.G. (1996). Goodman and Gilmans The Pharmacological basis of therapeutics. 9th ed. McGraw-Hill, New York. Koob, G.F., Stinus, L. And Lemoal, M. (1981). Hyperactivity and hypoactivity produced by lesions to the mesolimbic dopamine system. Behavioural Brain Research. 3:341-359. Kuczenski, R. and Segal, D. (1989). Concomitant characterisation of behavioural and striatal neurotransmitter response to amphetamine using in vivo microdialysis. Journal of Neuroscience. 372: 21-30. Lawrence, R. (1978). Use of activity measures in behavioural toxicology. Environmental Health Perspective. 26: 9-20. Oldendorf, W. (1971). Brain uptake of metabolites and drugs following carotid arterial injections. Transactions of the American neurological Association. 96:46-50. Robbins, T.W. and Everitt, B.J. (1982). Functional studies of the central catecholamines. Review of .Neurobiology. 23: 303-365. Roy, S.N., Bhattacharya, S. and Pradhan, S.N. (1978). Behavioural and neurochemical effects of repeated administration of cocaine in rats. Neuropharmacology. 17: 559-564. Sarkar, S.N. (1990). Toxicological investigation of isoproturon with special reference to fetal toxicity. Ph.D. Dissertation, Indian Veterinary Research Institute, Izatnagar, U.P. Schechter, M.D. and Rosecrans, J.A. 1972. Behavioural tolerance to an effect of nicotine in the rat. Archives of International Pharmacodynamics and Theapeutics. 195:52-56. Tomizawa, M. and Casida, J. E. (2005). Neonicotinoid insecticide toxicology: mechanism of selective action. Annual Review of Pharmacology and Toxicology. 45:247-268. Turner, R.A. (1965). Screening methods in pharmacology. Academic Press, New York, London. pp 22-41.
Received on: 27-06-2012 Revised on: 12-09-2012 Accepted on: 22-12-2012
Research Article
COMPARATIVE ANALYSIS OF PESTICIDE RESIDUES IN FODDER AND BUFFALO PLASMA COLLECTED FROM THREE DIFFERENT DISTRICTS OF PUNJAB
V. K. DUMKA, H. S. SANDHU, S. RAMPAL, RAJDEEP KAUR* AND KAMALPREET KAUR
Department of Veterinary Pharmacology and Toxicology Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana - 141 004 *Corresponding author: E-mail: rajksidhu@yahoo.com
ABSTRACT
The use of pesticides is comparatively more in certain crops while in some it is negligible. The farmers use pesticides more frequently and in increased doses than the recommended doses or procedures which leads to the presence of high amount of residues in food commodities including animal fodder. As a result there is persistence of pesticide residues in food chain which further poses serious threat to animal and human health. In this study, comparative analysis of pesticide residues was done in three different districts of Punjab and results revealed maximum use of pesticides in Bathinda district among three. Key words: Pesticides, Punjab, residues, survey.
INTRODUCTION In the present scenario of global food crises, the extensive use of agrochemicals including pesticides and fertilizers is unavoidable for increasing and sustaining crop production. Even the pesticides with longer persistence characteristics are also being used, which endanger our human and animal health through the pesticide contamination of food stuff and environment (Muhammad et al., 2009). The large-scale use of pesticides in modern agriculture has caused serious concern due to the presence of their residues in the environment. Besides combating insect pests, insecticides also get accumulated in many parts of the ecosystem and exert toxic effects on organisms including animals and human. Animals are exposed to these hazardous chemicals through consumption of contaminated fodder and water. Increasing incidence of cancer, chronic kidney diseases, suppression of the immune system, sterility in male and females, endocrine disruption, neurological disorders, have been attributed to chronic pesticide poisoning (Hosie et al. 2000). Currently, there are about 165 pesticides registered for use in India, of which 40% are organochlorines (FAO, 2005). Pesticides have become integral part of agronomic practices in Punjab. Bathinda and Moga districts of Punjab constitute an important cotton belt of the country, grows largely cotton and rice crop the two crops known for excessive use of pesticides. Pesticide residues persist in field soils and can enter other crops including maize, sorghum etc which are used as animal fodder. Consequently pesticide residues enter food chain and affect animal and human health. Therefore, the present study was conducted to analyze the pesticide residue concentration in animal blood samples and fodder being fed to such animals in various districts of Punjab.
50
MATERIALS AND METHODS The survey was undertaken in three districts of Punjab viz Bhatinda, Moga and Ludhiana. Amongst these, Bathinda and Moga districts fall under the high-pesticideuse areas whereas Ludhiana district was kept as control (low-pesticide-use area). In each district, five villages were randomly selected and from each village five samples of fodder and buffalo blood were collected in sterilized containers. Therefore, total of twenty-five samples from five villages of each district was collected and analysis of pesticide residues was done in laboratory. Fodder samples were first grinded properly and 50g of each sample was taken for extraction which was done in organic solvents including acetone, dichloromethane and hexane followed by clean-up procedure using column chromatography that included use of florisil and anhydrous sodium sulphate. Final volume of 10ml was prepared in acetone and hexane and stored in closed vials at -200C till further analysis. Blood samples were centrifuged at 3000 rpm for 15 min to obtain plasma. Plasma samples were processed by the protocol developed by Gill et al. (1996) with some modifications. The gas chromatograph, Perkin Elmer (Clarus 500) equipped with nitrogen phosphorus detector and electron capture detector was used for the detection of various pesticide residues. Concentrations obtained were compared with standard curve prepared by using specific concentration of different pesticide standards and results were obtained by using following formulaConcentration (ppm) = Pesticide standard injected(ng) Area of the standard Final vol of sample extract(ml) Initial vol (ml) x Area of sample Sample extract injected(l) x
RESULTS AND DISCUSSION Pesticides were identified from their respective
Pesticide residues in fodder and blood
retention time and confirmed by comparison with authentic standards. The results of reproducibility of recovery, suggested that extraction and cleanup procedure could be considered reliable enough for the analysis of plasma and fodder. Analysis of pesticide residues in fodder samples collected from three different districts revealed that maximum concentration of pesticide residues was found in fodder collected from villages of Bathinda followed by Moga and Ludhiana (Table 1). This indicates maximum use of pesticides in fields of Bathinda district to raise crop yield. In recent years, many people residing in villages of Bathinda district are facing problem of cancer and as a result the area has earned the dubious name of being the
Fig 1: Distribution of pesticide residues (ppm) in the fodder samples from Bathinda district of Punjab
Cancer Belt and studies are going on to ascertain that use of indiscriminate pesticides can be the initiating factor for the dreadly disease. Residues of pesticides including Heptachlor, Fipronil, Fenitrothion, dieldrin, aldrin, endrin, - HCH, o,p-DDD, endosulphan, L-cyalothrin, fenvalerate and deltamethrin were found in concentration above the Accetable Daily Intake (ADI) limits (Fig 1). Serum collected from buffaloes also contain similar residues, but in lesser concentrations. In the state of Punjab, buffalo which contributes to its 70% milk production is an important dairy animal. Quality of its milk and contamination of milk with various toxicants would have a lot of bearing on human health. In fodder samples of Moga district, pesticide residues of - HCH, - HCH, - HCH,fipronil, heptachlor, fenitrothion, aldrin, dieldrin, p,p-DDD, o,p-DDD, p,p-DDE, endosulphan, butachlor, endrin, L-cyalothrin, fenvalerate and deltamethrin were found to be above the Acceptable Daily Intake limits (Fig 2). Fipronil is a highly effective, broad-spectrum insecticide used for the control of a wide range of agricultural, public health and veterinary pests (Rhone-Poulenc, 1996). Organochlorine pesticides (including accompanying residues and metabolites) are ubiquitous in the environment because of their widespread use. On the other hand, the samples collected from Ludhiana, a less-pesticide-use area, revealed a lesser number of residues, of which, only heptachlor and butachlor were found to above ADI limits in serum of animals whereas in fodder samples levels of - HCH, heptachlor, fenitrothion, fipronil, dieldrin, aldrin and endrin were above the ADI limits (Fig 3) but in a significantly lower concentration in comparison to Bathinda and Moga districts. -
TABLE 1: Pesticide residues (ppm) in the fodder samples collected from Bathinda, Moga and Ludhiana districts of Punjab. S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Pesticides - HCH - HCH - HCH - HCH Heptachlor Fenitrothion Fipronil Dieldrin Aldrin p,p-DDD o,p-DDD p,p-DDT p,p-DDE Endosulphan-SO4 Butachlor Endrin L-cyalothrin Fenvalerate Deltamethrin ADI (ppm) 0.10 0.10 0.10 0.10 0.0001 0.005 0.0002 0.0001 0.0001 0.01 0.01 0.01 0.01 0.006 0.05 0.0002 0.002 0.02 0.01 Bathinda 0.0222 0.0099 0.0107 0.2264 0.5592 1.1333 0.6155 0.1542 0.0093 0.0036 0.0245 0.0024 0.0026 0.0500 0.0436 0.0779 0.0383 0.0231 0.0156 0.004 0.005 0.003 0.107 0.144 0.521 0.549 0.043 0.003 0.001 0.002 0.001 0.002 0.003 0.003 0.030 0.020 0.002 0.001 Moga 0.02014 0.019 0.07001 0.03 0.00486 0.002 0.12865 0.04 0.74471 0.05 0.05625 0.002 0.69125 0.28 0.07042 0.03 0.14720 0.11 0.21575 0.03 0.03167 0.02 0.00847 0.004 0.01750 0.007 0.16117 0.104 0.29520 0.11 0.00392 0.002 0.11625 0.02 0.13667 0.021 0.20375 0.04 Ludhiana 0.02559 0.00563 0.01022 0.25733 0.37333 0.63050 0.02417 0.13334 0.00757 0.00275 0.00145 0.00361 0.00667 0.01361 0.016 0.004 0.004 0.062 0.213 0.466 0.005 0.042 0.004 0.001 0.001
0.002 0.003 0.003
Dumka et al.
Fig 2: Distribution of pesticide residues (ppm) in the fodder samples from Moga district of Punjab
Fig 3: Distribution of pesticide residues (ppm) in the fodder samples from Ludhiana district of Punjab
REFERENCES FAO. (2005). Proceedings of the Asia regional Workshop.Regional Office for Asia and the Pacific, Bangkok. Gill, U. S., Schwartz, H. M. and Wheatley B. (1996). Development of a method for the analysis of PCB congeners and organochlorine pesticides in blood/ serum. Chemosphere 32: 1055-1061. Hosie, S., Loff, S., Witt, K., Niessen, K., and Waag, K.L (2000). Is there a correlation between organochlorine compounds and undescended
testes? Euro. J. Pediatric Surgery 10: 304309. Muhammad, S. Z., Muhammad, J. K., Muhammad, Q. and Abdul, R. (2009). Pesticide residue in the food chain and human body inside Pakistan J. Chem Soc. Pak., 31(2): 284-291 Rhone-Poulenc. (1996). Fipronil Worldwide Technical Bulletin. Rhone-Poulenc.
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Research Article
PARALYTIC EFFECT OF PEGANUM HARMALA LINN. ALCOHOLIC EXTRACT ON FASCIOLA GIGANTICA

S. W. HAJARE1, M. K. LONARE2 AND DINESH KUMAR3
1,2
Assistant Professor, Department of Veterinary Pharmacology and Toxicology, Post Graduate Institute of Veterinary and Animal Science, Akola -444104, M.S., India; 3 Principle Scientist, Division of Veterinary Pharmacology & Toxicology, IVRI, Izatnagar-243122, U.P., India: 1 Corresponding Author: E-mail: sunilwhajare@rediffmail.com ABSTRACT
Peganum harmala Linn (Harmal) is an herb native to arid and semiarid regions of Central Asian deserts. The effect of P. harmala alcoholic extract (PHAE) seed was investigated in vitro on the spontaneous muscular activity and AChE activity of Fasciola gigantica. The extract induced flaccid paralysis at 300g/ml concentration in isometrically mounted liver flukes in tissue organ bath. However, extract did not markedly modify the AChE activity of the liver flukes following 4h incubation. It is AchE independent paralytic effect on liver fluke in vitro.
Key words: AChE activity, Fasciola gigantica, Peganum harmala, spontaneous muscular activity.
INTRODUCTION Fasciolosis mainly caused by Fasciola gigantica in Indian sub-continent (Bhatia et al., 1989) is responsible for heavy economic loss to livestock owners. Synthetics anthelmintic are currently used as the most effective means for the control of fasciolosis. But these anthelmintics are not available in some of the rural areas of developing countries or have some serious disadvantages such as risk of misuse, development of drug resistant (Singh et al., 2002), adverse drug reactions, high cost, environmental pollution and residual effect. Plant based anthelmintics offer an alternative that can overcome some of these problems and are sustainable and environmentally acceptable (Hammond et al., 1997). A large number of medicinal plants in India have been reported to possess anthelmintic activity and they have been used traditionally (Nadkarni, 1954). Peganum harmala Linn. (Rutaceae) is a common plant used traditionally to treat several diseases and has many medicinal properties such as antioxidant, hepatoprotective, antidepressant and anthelmintic activities. (Khaliq et al., 2008 & 2009; Hamden et al., 2009; Herraiz et al., 2010). There is no study reported on the effect of P. harmala seeds on liver flukes F. gigantica and on fluke acetyl cholinesterase (AChE) activity. Therefore, the present study was undertaken to evaluate the effect of P. harmala alcoholic extract on in vitro spontaneous muscular activity and AChE activity of F. gigantica. MATERIALS AND METHODS Plant material and preparation of extract P. harmala seeds were collected from the local market and were identified botanically for their authenticity before use. The seeds were dried under shade and ground to fine powder. Further, it was extracted with 70% ethanol under reflux. Yield of P. harmala alcoholic extract (PHAE)
of seeds was found 7.06%. Stock solutions of the extract having strength of 10 mg/ml in tween-80 and distilled water were prepared. Parasites Mature and healthy flukes were collected from the bile ducts of the freshly slaughtered buffaloes from local abattoir in insulated container containing sterile modified and warm Hedon-Fleig (H.F.) solution (NaCL119.82mM; KCL-4.01mM; MgSO4-0.29mM; CaCl2-0.40mM; NaHCO 3 -17.8mM; Glucose-22.3mM; Streptomycin sulphate-6900 units @ 10mg/liter and benzyl penicillin9900 units/liter). Flukes were maintained at 381C in BOD incubator until use. in vitro muscular activity of Fasciola gigantica Spontaneous muscular activity (SMA) of mature flukes was recorded by mounting it isometrically in the tissue bath, using force displacement transducer connected to a pen writing recorder (Polyrite, Medicare, India) as per the method of (Fairweather et al., 1983) for F. hepatica and modified by (Kumar et al., 1995) for F. gigantica. The SMA was recorded, 30 min after equilibration under the resting force of 0.5g. After 15 min of equilibration period, the fluke was exposed to different cumulative concentration (30, 100 and 300g/ml) of PHAE and response was recorded. Each concentration was allowed to act for a period of 15 min. Six flukes were mounted isometrically to examine effect of cumulative concentration of PHAE. Isometrically mounted flukes were also exposed to Tween-80 (Final concentration 0.1%) at an interval of 15 min and for a period of 1h to eliminate the possibility of its effect on SMA of the flukes. Estimation of acetyl cholinesterase activity of F. gigantica The F. gigantica were incubated in different concentrations (30 and 100g/ml) of PHAE in H-F solution for 4h. The control group was also taken with only normal
Hazare et al.
TABLE 1: Effect of cumulative doses of PHAE on SMA of F.gigantica. Groups Control P. harmala Dose Tween-80 (1%) 30 g/mL 100 g/mL 300 g/mL Frequency / minute 4.360.11 4.460.20 4.800.17 0.00 Amplitude/ tension (g) 0.370.02 0.490.02** 0.640.06** 0.00
TABLE 2: Effect of different doses PHAE on AChE activity (mM of ACh hydrolyzed/g wet weight/h) of F. gigantica. Groups Control Hexachlorophene P. harmala Dose Tween-80 (1%) 10-5 M 30 g/mL 100 g/mL AChE activity 71.345.51 72.805.84 71.736.54 65.213.91
n=6; values are expressed as MeanSEM; tension 0.5 g. **= p>0.05 as compared with control.
n=6; values are expressed as MeanSEM
H-F solution. Following 4h incubation, flukes were used for estimation of AChE activity (Fishman and Green, 1961). Minimum six observations were made for each of the four groups. Statistical analysis All the values are expressed as Mean SE. The statistical analysis of the data was carried out by one way ANOVA with Studentized Range Post-Hoc test. A probability level of P<0.05 was considered significant. RESULTS AND DISCUSSION The alcoholic extract cause statistically significant concentration dependent increase in frequency of SMA at 30 (4.460.20 conc./min) and 100 (4.800.17 contractions/ min) g/ml concentrations as compared to the control frequency (4.360.11 contractions/min). At 300g/ml concentration, the frequency became nil. The amplitude of contractions was increased significantly and in concentration dependent manner (0.490.02g at 30g/ml and 0.640.06g at 100g/ml conc.) as compared to control (0.370.02g). Irreversible flaccid paralysis was produced at 300g/ml concentration of PHAE (Table 1). At this concentration, flukes did not recover from the paralysis for period of 30 min of recording with two successive washes with normal H-F solution at 15 min interval. In earlier studies acetylcholine (ACh) has been shown to produce inhibitory effect on the rhythmicity of F. gigantica at higher concentrations (10-4 and 10-3M) (Tripathi and Kumar, 1997) and caused flaccid paralysis of many flatworm parasites (Ribeiro et al., 2005). Similarly, in the present study, the extract flaccidly paralyzed the fluke. In the absent of substantial information on the effect on neurotransmitters on the spontaneous muscular activity of trematodes, it can be speculated that the paralytic effect of the P. harmala extract might be due to the presence of some cholinergic agent and/or an agent possessing the activity to inhibit AChE activity of the fluke. Because, Harmine, a betacarboline amine alkaloid isolated from P. harmala has shown its antileishmanial properties both in vitro and in vivo (Lala et al., 2004). Total alkaloid of P. harmala showed a marked effect as a treatment for haemosporidian namely, T. sergenti, T. annulata and B. bigemina infection in cattle (Hu et al., 1997). SMA of isometrically mounted F. hepatica (Holmes
54
and Fairweather, 1984) has been used as an index of neuromuscular activity to evaluate the role of neurotransmitter in neuromuscular physiology of helminthes. In the present study we have used this index to examine concentration dependent inhibitory effect of the PHAE on SMA of F. gigantica to ascertain its anthelmintic action in vitro. The SMA of F. gigantica is grossly similar to that of whole S. mansoni (Mellin et al., 1983) and F. hepatica (Fairweather et al., 1983). The changes produce on SMA of isometrically mounted flukes by drugs/chemical/extracts demonstrate the involvement of neuromuscular system on account of rapidity of action. The SMA can be quantified in terms of frequency and amplitude of rhythmic contractions. It can be compared before and following drug treatment. SMA of F. gigantica shows full (excitatory) and dull phases similar to that of F. hepatica (Fairweather et al., 1983) with apparent change in baseline tension and amplitude of SMA. However, Seeds of P. harmala have been shown to be efficacious in natural cystodal infection in goat (Akhter and Riffat, 1986) and in vitro against E. granulosus (Kang, 1994). Its constituent tetrahydroharmine has been found effective in nematode infection in goats (Akhter and Ahmad, 1991). P. harmala seed powder caused significant decrease in schistosomal cercarial production recorded in snail treated with its sub lethal concentrations (El-Ansary et al., 2001). Extract did not show any marked effect on AChE activity of treated flukes as compared to that of control flukes (71.345.51mM ACh hydrolyzed/g wet tissue /hr) following four hour incubation (Table 2). AChE activity has earlier been reported in F. hepatica and F. gigantica (Probert and Durrani, 1997) as well and inhibitors of the enzyme have been reported to markedly reduce rhythmic activity of flatworm parasites (Ribeiro et al., 2005). Many conventional anthelmintic including those used against F. hepatica and against F. gigantica. viz., oxyclozanide and rafoxanide act through inhibiting AChE enzyme of the parasite (Durrani, 1977). As higher concentration of ACh cause flaccid paralysis, inhibition of AChE might also cause increase in concentration of endogenous ACh which might finally cause flaccid paralysis. Surprisingly, the extract did not inhibit the activity of AChE enzyme of the fluke. Thus, it is interpreted that PHAE might possess some cholinomimetic constituent which could not be hydrolyzed
Effect of P. harmala on F. gigantica
by fluke AChE. Thus, it is concluded that the alcoholic extract of P. harmala seeds has paralytic effect on liver fluke in vitro and the effect appear to be independent of its effect on AChE activity of the fluke. ACKNOWLEDGEMENT The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar for providing necessary facilities to carry out research work. REFERENCES Akhtar, M.S. and Ahmad, I. (1991). Evaluation of antinematodal efficacy of tetrahydroharmine in goats. Veterinarski Archive 61: 307-311. Akhtar, M.S. and Riffat, S. (1986). A field trial of Paganum harmala Linn. seeds (Harmal) against natural cestodal infection in Betel goats. J. Pharmacol. Univ. Karachi Pakistan. 4: 7984. Bhatia, B.B., Upadhaya, D.S. and Juyal, P.D. (1989). Epidemiology of Fasciola gigantica in sheep in tarai region of Uttar Pradesh. J. Vet. Parasitol. 3: 25-29. Derakhshanfar, A. and Mirzaei, M. (2008). Effect of Peganum harmala (wild rue) extract on experimental ovine malignant theileriosis: pathological and parasitological findings. Onderstepoort J. Vet. Res. 75: 67-72. Durrani. M.S. (1977). Fasciola hepatica and Fasciola gigantica: Total cholinesterase, characteristic and effect of specific inhibitors. Expt. Parasitol. 42: 203-210. El-Ansary, A., Sammour, E.M., Soliman, M.S. and Gawish, A. (2001). In vivo, attenuation of schistosome cercarial development and disturbance of egg laying capacity in Biomphalaria alexandrina using sublethal concentrations of plant molluscicides. J. Egyptian Soc. Parasitol. 31: 657-669. Fairweather, I., Holmes, S.D. and Threadgold, L.T. (1983). Fasciola hepatica; a technique for monitoring in vitro motility. Expt. Parasitol. 56: 369-380. Fishman, W.H. and Green, S. (1961). Calorimetric estimation of Acetylcholine esterase activity. In: Methods Med. Research 9: 73-75. Hamden, K., Masmoudi, H., Ellouz, F., ElFeki, A. and Carreau, S. (2008). Protective effects of Peganum harmala extracts on thiourea induced diseases in adult male rat. J. Environ. Biol. 29:73-75. Hamden, K., Carreau, S., Ayadi, F., Masmoudi, H. and El-Feki, A. (2009). Inhibitory effect of estrogens, phytoestrogens and caloric restriction on oxidative stress and hepato toxicity in aged rats. Biomed. Environ. Sci. 22:381-387. Hammond, J.A., Fielding, D. and Bishop, S.C. (1997). Prospects for plant Anthelmintics in tropical
veterinary medicine. Vet. Res. Com.. 21: 213228. Herraiz, T., Gonzlez, D., Ancn-Azpilicueta, C., Arn, V.J. and Guilln, H. (2010). Beta- Carboline alkaloids in Peganum harmala and inhibition of human monoamine oxidase (MAO). Food and Chem. Toxicol. 48: 839-45. Holmes, S.D. and Fairweather, I. ( 1984). Fasciola hepatica: the effects of neuropharmacological agents on in vitro motility. Expt. Parasitol. 58: 194208. Hu, T., Fan, B., Liang, J., Zhao, S., Dang, P., Gao, F. and Dong, M. (1997). Observations on the treatment of natural haemosporidian infections by total alkaloid of Peganum harmala L. in cattle. Trop. Anim. Hlth. Prod. 29(4): 72S76S. Kang, J.F. (1994). In vitro cidal effect of ten Chines medicinal herbs against Echinococcus granulosus protscolices. Endemic Dis. Bull. 9: 22-24 Khaliq, T., Misra, P., Gupta, S., Reddy, K.P., Kant, R., Maulik, P.R., Dube, A. and Narender, T. (2009). Peganine hydrochloride dihydrate an orally active antileishmanial agent. Bioorganic and Med. Chem. Let. 19: 2585-2586. Kumar, D., Chandra, S. and Tripathi, H.C. (1995). In vitro motility recording of Fasciola gigantica. J. Vet. Parasitol. 9: 31-36. Lala, S., Pramanik, S., Mukhopadhyay, S., Bandyopadhyay, S. and Basu, M.K. (2004). Harmine: Evaluation of its antileishmanial properties in various vesicular delivery systems. J. Drug Target. 12: 165-175. Mellin, T.N., Busch, R.D., Wang, C.C. and Kath, G. (1983). Neuropharmacology of the Parasitic Trematode, Schistosoma Mansoni. Am. J. Trop. Med. Hyg. 32: 83-93. Nadkarni, K.M. (1954). Indian Meteria Medica. 3rd ed. Popular Prakashan, Bombay p. 242. Probert, A.J. and Durrani, M.S. (1977). Fasciola gigantica and Fasciola hepatica: Total cholinesterase, characteristic and effects of specific inhibitors. Expt. Parasitol. 42: 203-210. Ribeiro, P., El-Shehabi, F. and Patocka, N. (2005). Classical transmitters and their receptors in flatworms. Parasitol. 131:S19-S40. Singh, D., Swarnkar, C.P. and Khan, F.A. (2002). Anthelmintic resistance in gastrointestinal nematodes of livestock in India. J. Vet. Parasitol. 16(2):115-130. Tripathi, S.C. and Kumar, D. (1997). Effect of cholinergic drugs on rhythmic motility of Fasciola gigantica. J.Vet. Parasitol. 11:31-36. Received on: 01-10-2012 Accepted on: 12-11-2012
Research Article
HAEMATO-BIOCHEMICAL PROFILE AFTER REPEATED ADMINISTRATION OF PEFLOXACIN IN GOATS

NIRBHAY KUMAR*, BIPIN KUMAR1, S.D. SINGH AND C. JAYACHANDRAN
Department of Veterinary Pharmacology and Toxicology, 1Department of Veterinary Medicine, Bihar Veterinary College, Patna 800 014, India *Corresponding author: E-mail: drnkvet76@rediffmail.com ABSTRACT In the present study, pefloxacin was assessed for its adverse effects in goats when given at the dose rate of 10 mg/ kg, i.v. daily for seven consecutive days. Among hematological parameters, hemoglobin and differential leukocyte count differed non-significantly while total leucocyte count increased significantly (p<0.01) from day 0 onwards till day 10. However, the increase was within the physiological limit. Biochemical parameters like blood glucose, serum cholesterol, blood urea nitrogen, total protein, albumin, globulin and A/G ratio showed non-significant changes while alanine aminotransferase (ALT) and aspartate aminotransferase (AST) differed significantly (p<0.01) between pre-treatment and post-treatment. The changes in ALT and AST values were also within the normal physiological limits. The study revealed that the drug is quiet safe to be used for clinical trials of a week. Key words: Goat, hematological parameters, pefloxacin.
INTRODUCTION Fluoroquinolones are synthetic antimicrobial agents with an established position as highly effective group of drugs in modern veterinary medicine. They have broad spectrum of activity with excellent pharmacokinetic profile. Pefloxacin, a member of third generation fluoroquinolone is a broad spectrum, antibacterial agent having potent bactericidal activity against a wide range of gram-negative and gram-positive organisms. It is effective against various diseases such as gastro-intestinal, respiratory, urinary, genital, skin, soft tissues, bone and joint infections etc. The drug is metabolized in the body into its active metabolite, norfloxacin. Pefloxacin like other quinolones has toxic potentials in the muscle, tendon and synovial membrane (Kashida and Kato, 1997). One of the most common problems is the gastrointestinal disturbance (like nausea, vomiting, diarrhoea etc). Pefloxacin also induces arthropathy in juvenile animals (Machida et al., 1990). Thrombocytopenia at very high doses on prolonged administration in human has also been reported (Chichmanian et al., 1992). Photosensitivity and photoallergenicity of norfloxacin in some cases of guinea pigs has also been described (Horio et al., 1994). Although many reports of toxicity of pefloxacin are available, studies regarding safety of the drug in clinical trials in animals especially goats are scanty. Hence, the present study was undertaken to evaluate its toxicity, if any, on the hematological and biochemical parameters in goats after repeated intravenous administration at double of the therapeutic dose for a week. MATERIALS AND METHODS The present study was conducted on five clinically healthy female goats of non-descript breed of 1.5 to 2
56
years of age and weighing about 2025 kg body weight. All the goats were dewormed with albendazole immediately after procurement. They were maintained under standard managemental conditions and acclimatized in the College Animal House for a period of 15 days prior to the experiments. Pefloxacin (Pelox infusion as pefloxacin methane sulfonate dihydrate equivalent to 4 mg/ml of pefloxacin base in 5% dextrose solution) manufactured by M/s. Wockhardt Limited, Mumbai was used for the study. The drug was administered intravenously at the dose rate of 10 mg/kg body weight (double of the therapeutic dose) for 7 consecutive days. Blood samples were collected routinely on 0, 2, 4, 8 and 10th day during the study. For determining different haematological values, blood was collected from the experimental animals from the jugular vein by venepuncture in small sterile vials having the anticoagulant EDTA @ 1.5 mg/ml of blood. Blood was collected from the ear vein of goat for total leucocyte count (TLC) and differential leucocyte count (DLC) estimation. Fluoride oxalate mixture was used as an anticoagulant for blood sugar estimation. Other biochemical parameters like blood glucose (Frankel et al., 1970), BUN (Wootton, 1964), total protein and albumin (Reinhold, 1953), ALT and AST (Reitman and Frankel, 1957) were estimated using commercially available kits from Nice and Span Diagnostics Co. The data were expressed as Mean S.E. and subjected to Students ttest (Snedecor and Cochran, 1976). RESULTS AND DISCUSSION Table 1 shows the effect of pefloxacin on haematological parameters when given at the dose rate of
Biochemical profile of pefloxacin in goats
TABLE 1: Effect of pefloxacin (10 mg/kg i.v. daily for 7 days) on haematological parameters in goat (Mean+ S.E.M., n=5) Parameter (Unit) Hb (g/dl) TLC (x103/mm3) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophill (%) Basophill (%) Day 0 8.50 +0.20 9.80a+0.218 39.57 52.47 4.22 3.00 0.79 (n=2)
+
Day 2 8.48+0.21 10.30ab+0.21 39.47 52.10 4.05 3.62 0.60 (n=1)
Day 4 8.52+0.22 10.785bc+0.24 40.00 53.02 3.99 2.62 0.40 (n=1)
Day 8 8.52+0.19 11.325c+0.18 39.66 53.10 3.64 3.14 0.50 (n=1)
Day 10 8.50+0.20 11.30c+0.18 39.74 52.90 4.28 2.47 0.50 (n=2)
Different superscripts denote significance at p<0.01. TABLE 2: Effect of pefloxacin (10 mg/kg i.v. daily for 7 days) on biochemical parameters in goat (Mean+S.E.M., n=5) Parameter (Unit) Blood sugar (mg/dl) BUN (mg/dl) Serum cholesterol (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) A/G ratio ALT (IU/L) AST (IU/L) Day 0 59.34+0.87 17.7+0.76 81.92+2.86 7.22+0.08 2.43+0.09 4.79+0.06 0.504 11.80a+0.56 66.80a+3.39 Day 2 59.13+2.17 17.40+0.94 77.26+2.51 7.19+0.06 2.38+0.08 4.81+0.06 0.494 15.40b+0.66 84.90b+3.80 Day 4 63.35+1.42 18.11+1.06 78.03+4.50 7.16+0.05 2.40+0.10 4.76+0.06 0.503 19.60c+0.76 112.10c+3.69 Day 8 62.20+0.83 18.47+0.99 80.01+4.44 7.16+0.05 2.36+0.06 4.81+0.06 0.491 26.80d+0.66 150.40d+3.99 Day 10 62.90+0.67 17.82+0.67 79.44+3.76 7.23+0.13 2.41+0.06 4.82+0.08 0.499 25.60d+0.43 146.40d+3.90
Different superscripts denote significance at p<0.01.
10 mg/kg i.v. for seven consecutive days in goats. The study revealed that there was no significant change in haemoglobin (gm/dl). Value of TEC significantly increased from day 2 onwards till day 10 without a significant change in DLC. The TLC and DLC values remained within the normal range during the study. Following repeated administration of pefloxacin at double of the therapeutic dose (i.e. 10 mg/kg b.wt.) for 7 consecutive days, the alterations recorded in the haematological parameters were non-significant between pretreatment and post treatment values. Similar findings in haematological parameters were also noted by Sachan et al. (2000) in broiler chicken when pefloxacin was administered to at doses of 5, 10 and 40 mg/kg orally. Another fluoroquinolone, ofloxacin also produced similar changes in haematological values when it was given @ 10 mg/kg twice daily for 10 days treatment in healthy male volunteers (Stein et al., 1991). Pallavacini et al. (1989) observed that pefloxacin and ofloxacin in concentrations of 0.5 to 50 g/ml did not induce inhibition of human myelopoiesis in vitro. Table 2 represents the biochemical values when pefloxacin was given at double of the therapeutic dose for seven days. Pefloxacin did not reveal any effect on values of blood sugar, BUN and serum cholesterol on different intervals in comparison to values on day 0. Pefloxacin was found to have non-significant effect on total protein, albumin, globulin and A/G ratio and the values of total protein, albumin, globulin and A/G ratio remained around normal on different days during the study. The value of ALT and AST increased significantly on day 2, 4 and 8
showing its mild hepatotoxic effects. The findings of the biochemical parameters like ALT, AST, BUN and total bilirubin in the present study were in agreement with the results found in day old chicks by Sachan et al. (2002). Increased levels of AST might have been due to some muscle damaging effects of pefloxacin like other fluoroquinolones. Since the increase in AST level is not very pronounced, it can be evaluated as a relatively safer drug. Sridevi et al. (2002) reported that pefloxacin at the high dose rate of 15 and 20 mg/kg b.wt. produced toxic symptoms in pups following prolonged (4 weeks) administration while 10 mg/kg b.wt. was comparatively safer. Chondrotoxicity, athropathy, muscle damage etc. have been reported only after prolonged administration of pefloxacin by several authors (Kashida and Kato, 1997; Machida et al., 1990). Thus , it is concluded that pefloxacin @ 10 mg/kg, i.v for 7 days is quiet safe for clinical uses in goats. ACKNOWLEDGEMENTS The authors are thankful to Associate Dean-cumPrincipal, Bihar Veterinary College, Patna for providing facility and necessary help for this study. REFERENCES Chichmanian, R.M., Spreux, A., Bernard, E., Garraffo, R. and Fuzibet, J.G. (1992). Thrombocytopenia due to pefloxacin (peflacine): dose dependent toxicity. Therapie. 47: 419-21. Frankel, S., Reitman, S. and Sonnerwirtha, A.C. (1970). Gradiwhols Clinical Laboratory Methods and
Kumar et al.
Diagnosis (The C V Inospby Co, St Louis): 82-83. Horio, T., Miyauchi, H., Asada, Y., Aoki, Y. and Harada, M. (1994). Phototoxicity and photoallergenicity of quinolones in guinea pigs. J. Dermatol. Sci. 7: 130-35. Kashida, Y. and Kato, M. (1997). Toxic effects of quinolone antibacterial agents on the musculskeletal system in juvenile rats. Toxicol. Pathol. 25: 635-43. Machida, M., Kusajima, H., Aijima, H., Maeda, A., Ishida, R. and Uchida, H. (1990). Toxicokinetic study of norfloxacin induced arthropathy in juvenile animals. Toxicol. Appl. Pharmacol. 105: 403-12. Pallavicini, F., Antinori, A., Federico, G., Fantomi, M. and Nervo, P. (1989). Influence of two quinolones, ofloxacin and pefloxacin, on human myelopoiesis in vitro. Antimicrobial Agents Chemother., 33: 12223. Reinhold, J.G. (1953). Total proteins, albumins and globulins. In Standard methods of clinical chemistry, edited by Reine M (Academic Press, New York) : 88. Reitman, S. and Frankel, S.A. (1957). Colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Path. 28: 56.
Sachan, A., Jayakumar, K., Honnegowda, Gowda, R.N.S. and Narayana, K. (2000). The effect of pefloxacin on haematological parameters in clinical toxicity study in broiler chickens. Ind. Vet. J. 77: 307309. Sachan, A., Jayakumar, K., Honnegowda, Umesh, M.H., Venkatesha Udupa and Narayana, K. (2002). Safety evaluation of pefloxacin in day old broilder chicken. Ind. Vet. J. 79: 387-88. Snedecor, W.G. and Cochran, W.G. (1976). Statistical Methods (Oxford and IBH Publishing Company, Calcutta). Sridevi, V., Reddy, K.S., Kalakumar, B. and Reddy, G. (2002). Evaluation of experimentally induced pefloxacin toxicity in pups. Ind. Vet. J. 79: 38586. Stein, G.E., Flor, S.C. and Beals, B.S. (1991). Safety of multiple doses of ofloxacin in healthy volunteers. Drugs Exp. Clin. Res. 17: 525-29. Wootton, J.D.P. (1964). Microanalysis in Medical Biochemistry (J & A Churchill Ltd, London): 8384, 91-92, 101-105.
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Research Article
EVALUATION OF TOXICITY OF ENDOSULFAN FOLLOWING MULTIPLE ORAL ADMINISTRATION IN POULTRY

LATA GAYAL1, S.P.SINGH2 AND A.H.AHMAD3
1
PhD Scholar, 2Professor and Head, 3Professor; Department of Veterinary Pharmacology and Toxicology, C.V.A.Sc., Pantnagar-263145, Uttarakhand (India). 1 Corresponding author : E-mail-vetdoclata@gmail.com
ABSTRACT The present study was carried out to evaluate haemato-biochemical profile following multiple oral dose @16 mg/ kg bwt (1/5th of oral LD50) administration of endosulfan at an interval of 24h for five days in poultry birds. A significant (P<0.05) reduction in haematological values up to 24h post administration and thereafter gradual increase observed approaching normal values 96h post administration of endosulfan. The total serum proteins, albumin and globulin significantly (P<0.05) decreased upto 48h post administration gradually returned to normal value after 96h. The serum AST and ALT activities increased upto 24h gradually decreased to normal value after 96h post administration. A significant (P<0.05) increase in the values of serum creatinine and cholesterol upto 24h post administration and gradual decrease upto 96h post administration of endosulfan was observed. A significant reduction in serum glucose level upto 24h post administration was observed, however , the level increased gradually reaching to normal value upto 96 h post administration of the pesticide. It is concluded from this study that endosulfan produced mild to moderate haemotoxic, hepatotoxic and nephrotoxic effects in poultry birds. Keywords: Biochemical, endosulfan, haematological, hepatotoxic, nephrotoxic, poultry birds.
INTRODUCTION Endosulfan is a member of organochlorine group which mainly affects central nervous system, kidney, liver and blood chemistry. It is classified as a persistent organic pollutant (POP). It persists in the environment for extended periods of time. It volatilizes easily after application and is transported to areas distant from the application site. It has been detected in ice and snow samples of the arctic region and has a high potential for bioaccumulation and biomagnification. In animal husbandry, it is used against external parasites of domestic animals and poultry birds. This study was undertaken to evaluate the haematological and biochemical effects of endosulfan after 5 days multiple oral dose administration. MATERIALS AND METHODS Experimental design 20 male RIR birds weighing 10.5kg were divided into five groups namely Group I, II, III, IV and V comprising 4 birds in each group. Group I served as control free from pesticide. Birds of Group II, III, IV and V were administered 0.1ml of endosulfan (prepared by mixing 1ml of 35% EC with 1ml of olive oil) as multiple oral (the formulation was administered using thin plastic tube attached to a syringe) dose@16 mg/kg bwt (1/5th of LD50; 80mg/kg) for five days at an interval of 24h. The birds were reared under uniform management and husbandry conditions, maintained on poultry feed (as per NRC). The birds were fed commercial broiler ration free from any antibiotic. Fresh water was provided ad libitum during the entire period of experiment.
The birds were observed daily morning and evening for assessing their health status and other signs of clinical toxicity. Following multiple (5) oral dose administration of endosulfan@16 mg/kg bwt in poultry birds, blood samples were collected at 24, 48, 72 and 96 h after last dose from the left brachial vein or jugular vein. Biochemical analysis The blood samples were divided into two parts, one part was collected in heparinized tubes for hematological studies while second part in sterilized vials for collection of serum for analysis of biochemical parameters. Blood was collected from each bird in clean heparinized microcenrifuge tube (eppendorf ) and hematological parameters such as packed cell volume (Jain, 1986), haemoglobin (Jain, 1986), total erythrocyte count (Natt and Herric, 1952) and total leucocyte count (Natt and Herric, 1952) were estimated immediately after the collection of blood samples. 0.1N-HCl was used for estimating the blood haemoglobin concentration while, Natt-Herric diluting fluid was used for TEC and TLC estimation. Statistical analysis Statistical analysis of data was done by using one way ANOVA technique followed by Tukeys Multiple Comparison Test by using Graph pad prism statistical software. Statistically significant difference was considered at 5 and 1percent level. RESULTS A significant (p<0.05) decrease in Hb (g/dl) and
Gayal et al.
PCV (%) was observed in Group II as compared to control whereas there was a significant (p<0.05) increase in value in group V as compared to group II. A significant (p<0.05) decrease in the values of TEC (X106/l) and TLC (X103/l) in Group II and III were observed as compared to control whereas a significant (p<0.05) increase in the values of TEC and TLC in groups IV and V was observed as compared to groups II and III. There was a time dependent increase in the hematological parameters in endosulfan treated groups (Table 1). As given in Table1, a significant (p<0.05) decrease in value of serum proteins (g/dl) was observed in groups II, III and IV as compared to control whereas a significant (p<0.05) increase in serum proteins was observed in group V as compared to group II, III and IV. There was no significant difference observed in the values of serum albumin, globulin levels and A:G ratio of control as compared to test groups. A significant (p<0.05) increase in the value of ALT (U/L) and AST (U/L) in group II was observed as compared to control. ALT activity in group III was significantly higher than control. A significant (p<0.05) decrease in the value of ALT was observed in Group III, IV and V as compared to Group II. A significant (p<0.05) decrease in value of AST in Group V was observed as compared to Group II. A significant (p<0.05) decrease in the value of ALT in Group V was observed as compared to Group III. A significant (p<0.05) decrease in the values of glucose (mg/dl), urea (mg/dl) and uric acid (mg/dl) was observed in groups II and IV as compared to control. Values of glucose and urea in groups III and V were significantly (p<0.05) lower than control. A significant (p<0.05) decrease in the values of glucose (mg/dl), urea (mg/dl) and uric acid (mg/dl) was observed in group II and IV as compared to control. Values of glucose and urea in groups III and V
were significantly (p<0.05) lower than control. A significant (p<0.05) increase in the value of cholesterol (mg/dl) was observed in Group II, IV and V as compared to control. Value of cholesterol in group IV was significantly (p<0.05) higher than group III. DISCUSSION Hematological parameters Hb, PCV, TEC and TLC significantly decreased in endosulfan treated groups after 24 h post administration and gradually increased after 48, 72 and 96 h post administration of endosulfan as the residue concentration of pesticide gradually declined in the body in the present study. The reduction in Hb value indicated that the birds suffered from a mild degree of anemia. The anemic condition might have occurred due to interference in erythropoiesis, hemosynthesis and osmoregulatory dysfunction or due to an increase in the rate of erythrocyte destruction in haematopoetic organs (Varshaneya, 1983; Siddiqui et al., 1987). Khurana et al. (1996) observed significant reduction in total leucocyte count and absolute lymphocyte count in broiler chicken fed 30 ppm endosulfan for 8 weeks. The toxic effects of endosulfan in albino rat were also observed by Das et al. (2010) who reported a decline in RBC and and Hb count. In the present study, leucopenia might have been due to direct cytotoxic effects of pesticides on lymphocytes. Girdhar and Singhal (1989) reported decreased total leukocyte count in goats given lindane for 30 days. Earlier study with lindane, quinalphos, carbaryl, Fenvalerate, butachlor and isoproturon revealed leucopenia and lymphopenia in chickens (Garg, 2000; Gupta, 2001; Chauhan, 2003). The serum AST and ALT activities were increased significantly following multiple oral dose administration of
TABLE 1: Effect on hemato-biochemical parameters at different intervals following multiple (5) oral dose of endosulfan @ 16mg/ kg in poultry.
Groups Hb (g/dl) PCV (%) TEC(106/l) TLC(10 3/l) Cholesterol (mg/dl) Glucose(mg/dl) Uricacid(mg/dl) Urea (mg/dl) Creatinine (mg/dl) T.P. (g/dl) Albumin(g/dl) Globulin(g/dl) ALT (U/L) AST (U/L) I (Control) 12.200.26 35.800.94 2.750.10 21.250.77 99.12.97 38012.42 5.390.20 7.210.26 0.480.03 3.690.18 1.890.29 1.810.25 11.661.18 64.012.26 II (24 h) 7.850.65a 23.702.44a 1.610.15a 14.010.58a 150.3013.35a 188.36.47a 3.430.19a 3.260.29a 0.760.04a 2.500.19a 1.230.21 1.270.14 38.23 4.11a 144.7 20.33a
a d
III (48 h) 9.850.83 28.952.56 2.240.09ab 11.450.33a 114.610.22 211.813.44a 3.760.48 3.140.70a 0.280.02ab 2.290.34a 1.090.19 1.210.22 27.223.09ab 104.8 5.55
IV (72 h) 10.031.44 30.01.68 2.520.06b 17.820.68bc 163.48.08ac 187.819.67a 2.910.27a 3.130.42a 0.420.08b 2.610.19a 1.320.13 1.280.18 16.57 1.42b 105.2 6.19
b
V (96 h) CD 11.500.51b 35.01.29b 2.620.16b 19.271.40bc 152.310.34a 195.118.66a 6.200.76bcd 4.120.60a 0.410.02b 3.490.17bc 1.870.1 1.620.17 11.23 0.65bc 89.52 2.45b
One Way ANOVA d.f 15 15 15 15 15 15 15 15 15 15 15 15 15 15 F 7.24 6.78 14.88 22.81 8.19 30.97 10.11 12.94 15.6 7.91 3.69 1.79 22.23 8.72 1.89 5.74 0.35 2.51 29.02 44.94 1.32 1.46 0.14 0.68 0.59 0.59 7.41 29.94
Values in table are Mean SE (n=4); c significant (P<0.05) Vs group III;
significant (P<0.05) Vs group I; significant (P<0.05) Vs group IV
significant (P<0.05) Vs group II;
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Toxicity of endosulfan in poultry
endosulfan. These enzymes are used as an indicator of damage of vital organs in the body (Cornelius, 1989) and thus suggested that administration of these insecticides caused necrotic changes in the liver leading to leakage of the enzymes into the blood. Choudhary and Joshi (2002) also reported dose dependent increase in the activities of AST, ALT and acid and alkaline phosphatases after exposure of rats orally @ 5, 10 and 15 mg/kg of endosulfan for 15 to 30 days. A significant (p<0.05) decrease in value of serum proteins (g/dl) was observed after 24, 48 and 72 h post administration of endosulfan whereas no significant difference could be observed in the values of serum albumin and globulin levels. Khurana et al. (1996) have also observed significant suppression in total serum proteins in broiler chickens fed 30 ppm endosulfan for 8 weeks. Similarly, decline in the values of total serum proteins was also reported in endosulfan exposed carp Cyprinus carpio by Jenkins et al. (2003). A significantly higher level of creatinine at 24 h post administration of endosulfan was observed in poultry birds which indicated its nephrotoxic effect as creatinine is considered as one of the important biomarkers for nephrotoxicity. Similar results were also reported in endosulfan intoxicated poultry birds (Rawat, 2002; Kumar et al., 2010). The cholesterol level was significantly increased at 24 h post administration of endosulfan. Hypercholesterolaemia reported in this study might be attributed to the fact that endosulfan being lipophilic has tendency to interact with lipids and have inhibitory action on their metabolism resulting in an increase in the levels of cholesterol in the serum.A reduction in serum glucose levels was observed in this study following endosulfan exposure in birds. Reduction in glucose levels was also reported for endosulfan in the carp Cyprinus carpio Jenkins et al. (2003). It is concluded from this investigation that endosulfan following multiple (5) oral dose @ 16 mg/kg administration produce mild to moderate degree of hepatotoxic and nephrotoxic effects in poultry birds. REFERENCES Chauhan, P.P.S. (2003). Effect of chlorpyrifos and endosulfan on the production performance and immunity in chickens. M.V.Sc, thesis submitted to G.B.P.U.A.& T, Pantnagar, India. Choudhary, N. and Joshi, S.C. (2002). Effect of short term endosulfan exposure on haematology and serum analysis of male rats. Ind. J. Toxicol. 9 : 83-87. Cornelius, C.E. (1989). Liver function. In Kaneko, J.J. ed
Clinical Biochemistry of Domestic Animals. Academic Press Sandiego, New York, pp-386. Das, B., Pervin, K., Roy, A.K., Ferdousi, Z. and Saha, A.K. (2010). Toxic effects of prolonged endosulfan exposure on some blood parameters in Albino rat. J. Life Earth Sci. 5 : 29-32. Garg, S. (2000). Immunopathological effects of gammaBHC and Quinolphos in Chicken. M.V.Sc Thesis submitted to G.B.P.U.A&T.; Pantnagar, India. Girdhar, N. and Singhal, K.K. (1989). Subacute toxicity of lindane (gamma-benzene hexachloride) in ruminants. Indian J. Nutr. 62 : 133-139. Gupta, N. (2001). Impact of butachlor and isoprptoron on the immunity of chicken. M.V.Sc Thesis submitted to G.B.P.U.A & T.; Pantnagar, India. Jain, N.C. (1986). Schalms Veterinary Haematology. 4th Edn. Lea and Febringer, Philadeiphia. Jenkins, F., Smith, J., Rajanna, B.; Shameem, U., Umadevi, K., Sandhya, V. and Madhavi, R. 2003. Effect of Sub-Lethal Concentrations of Endosulfan on Hematological and Serum Biochemical Parameters in the Carp Cyprinus carpio. Bull. Environ. Contam. Toxicol. 70: 993997. Khurana, R., Chauhan, R.S. and Mahipal, S.K. (1996). Insecticide induced biochemical alteration in sheep. Indian J. Vet. Res. 8 : 31-38. Kumar, A., Singh, S.P. and Sharma, L.D. (2010). Immunotoxicological evaluation of chlorpyrifos following medication with Withania Somnifera in cockerels. J. Vety. Pharmacol. Toxicol. 9: 34-37. Natt, M.P. and Herrick, C.A. (1952). A new blood diluent for counting the erythrocytes and leucocytes of the chicken. Poult. Sci. 31: 735-778. Rawat, D. (2002). Survey on residues of different pesticides in Garhwal region of Uttaranchal and their toxicological evaluation in poultry. M.V.Sc. thesis submitted to G.B.P.U.A.& T. Pantnagar, India. Siddiqui, M.K.J., Anjum, F., Mahboob, M. and Mustafa, M. (1987). Effect of dimethoate on hepatic cytochrome P-450 and Glutathion-s-transferase activity in pigeon and rat. Indian. J. Exp. Biol. 29: 1071-1073. Varshaneya, C. (1983). Toxicological evaluation of malathion, lindane and endosulfan in Gallus domesticus with special reference to hepatic microsomal drug metabolizing systems. Ph.D Thesis submitted to G.B.P.U.A & T.; Pantnagar, India. Received on: 23-04-12 Accepted on: 26-06-12
Research Article
SUB ACUTE DERMAL TOXICITY OF BIFENTHRIN WITH SPECIAL REFERENCE TO HAEMATO-BIOCHEMICAL CHANGES IN RAT
MUNEER AHMAD DAR*, RAJINDER RAINA1, NRIP KISHORE PANKAJ2 , MUDASIR SULTANA 3, PAWAN KUMAR VERMA4 AND AADIL MEHRAJ5
1
Professor,
Asstt Prof., 3Professor and Head, Division of Pharmacology and Toxicology, FVSc & AH, R.S Pura, SKUAST -Jammu-181102. 5 PhD Scholar, Division of Pharmacology and Toxicology, GADVASU, Punjab. * Corrosponding author: E mail: darmuneer29@yahoo.in
2,4
ABSTRACT The study was carried out to investigate the toxic effects of bifenthrin on hematology and biochemistry in rats after repeated dermal application for a period of 30 days. Twenty four rats were randomly divided into four groups of six rats each. Group I and Group III served as control for Group II and Group IV to which bifenthrin was applied (1/100th LD50), respectively, for 20 and 30 days. Repeated dermal application of bifenthrin caused significant (P<0.01) decrease of Hb, PCV and TEC after 30 days of treatment. The value of TLC increased significantly (P<0.05) after 20 and 30 days. There was a significant (P<0.05) increase in MCV with marginal changes in values of MCH and MCHC. The AST activity increased significantly (P<0.01) only after 30 days of exposure. ALT and alkaline phosphate activities were increased both at 20 and 30 days with no changes in values of acid phosphate, BUN, creatinine and plasma proteins. The results of present study indicated potential of bifenthrin dermal application to alter haemato-biochemical indices in rats. Key words: Bifenthrin, haematobiochemical, rat, subacute toxicity.
INTRODUCTION Pyrethroids account for 30 per cent of insecticides used globally (Prasanthi et al., 2000). Though severe toxicity of pyrethroids has been uncommon in developed countries, it appears common in developing countries because of their extensive and intensive use for agricultural and domestic purposes (Kakko et al., 2003). Available literature suggests pyrethroids exposure is of great magnitude throughout the (Narashi, 2000). The present study was therefore undertaken to evaluate sub acute dermal toxicity of bifenthrin-type I pyrethroid on haematobiochemical indices in Wistar rats. MATERIALS AND METHODS Experimental design Bifenthrin ( Biflex R 2.5% EC), available commercially was obtained from local market for this study. Adult wistar rats (150-250 gm b.wt) of either sex were procured from Indian Institute of Integrative Medicine (CSIR), Jammu and acclimatized in the laboratory conditions for a period of 2 weeks. The animals were provided with standard pelleted food and water ad libitum. The protocol for conducting the experiments was duly approved by IAEC. Rats were randomly divided into four groups of six each. Group I and III served as control and were applied with water. Group II and IV were topically applied on interscapular region with bifenthrin (Biflex R 2.5% EC; FMC India Pvt. Limited, Tamil Nadu) @ 45mg/ kg for 20 and 30 days, respectively. Collection and processing of samples The rats were anaesthetized with diethyl ether
62
and two sets of blood samples-1 ml(EDTA added @ 2 mg/ ml) for haematological and 3 ml (heparin @ 5-10 IU/ml) for separation of plasma for biochemical analysis. Hb, PCV, TLC, TEC, MCV, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined by using standard reference methods (Benjamin, 2001). Biochemical parameters like AST, ALT, alkaline phosphate and acid phosphate (Srivastava and Bal,1994), creatinine and total plasma proteins (Hawk,1988) were determined after completion of trial. Statistical analysis The significance of difference between two means was determined by unpaired Students t-test at levels of 1(P < 0.01) and 5 (P < 0.05) percent (Snedecor and Cochran, 1967). RESULTS AND DISCUSSION Application of bifenthrin did not induce any prominent toxic signs in exposed rats. However, rats showed loss of body weight, signs of nervousness and abnormal gait after 15-18 days of dermal application. Dermal application of bifenthrin on skin caused scratching, licking and biting in rats and these signs lasted for 15-20 min. The skin in and around application site showed burn type wound after 3 weeks of daily application of bifenthrin. Repeated dermal application of bifenthrin did not produce significant changes in values of Hb and PCV after 20 days. However, both these indices were significantly decreased in bifenthrin exposed animals after 30 days of treatment. After 20 day of dermal exposure of bifenthrin, no significant alteration was observed in TEC which got
Dar et al.
TABLE 1: Effect of repeated dermal application of bifenthrin on haematobiochemical parameters in rats. Days of dermal application Parameters Haemoglobin (Hb) (g/dl) Packed cell volume (PCV) (%) Total erythrocytic count (TEC) (106/mm 3) Mean corpuscular volume (MCV) (fl) Mean corpuscular haemoglobin MCH) (pg) Mean corpuscular haemoglobin concentration (MCHC) ( %) Plasma Aspartate aminotransferase (nmol pyruvate formed/min/ml) Plasma alanine aminotransferase (nmol pyruvate formed/min/ml) Plasma Alkaline phosphatase (nmol pyruvate formed/min/ml) Plasma Acid phosphatase (nmol pyruvate formed/min/ml) BUN (mg/dl) Creatinine (mg/dl) Protein (g/dl) 20 days Group I 11.330.71 39.551.97 8.380.49 47.391.26 13.530.43 28.560.41 35.242.71 21.292.11 121.642.80 4.010.42 12.360.62 1.020.03 6.380.56 Group II 10.070.26 36.921.43 7.590.41 48.101.86 13.400.65 27.840.71 39.712.19 30.642.04b 134.924.32a 5.040.52 14.160.73 1.110.07 6.640.46 30 days Group III 11.400.26 40.961.10 8.720.66 47.942.76 13.431.01 27.920.46 33.463.11 28.991.74 125.853.88 3.120.36 11.820.72 1.080.05 6.470.55 Group IV 9.730.0.40b 37.050.63b 6.500.29b 57.381.65a 15.060.93 26.271.48 43.931.23b 42.923.06b 144.844.74b 4.040.40 13.810.86 1.190.06 7.450.61
Values given are Mean SE of the results obtained from 6 animals unless otherwise stated a,b significantly different as compared to control values at 5% (P<0.05) and 1% (P<0.01) level of significance, respectively.
decreased significantly after 30 th day of treatment. Compared to control group, there was a significant increase of TLC both after 20 and 30 days of exposure. Bifenthrin treated animals @ 45 mg/Kg/day as compared to control showed significant increase of MCV after 30 days of treatment with no change in MCH and MCHC after both of these exposure periods (Table 1). The daily dermal application of bifenthrin caused significant increase in the value of aspartate aminotransferase and a significant increase from control after 30 days of dermal exposure (Table 1). The activities of alanine aminotransferase and alkaline phosphatase were significantly increased in comparison to control after 20 days and onwards till 30th day of dermal exposure. No significant alteration was observed in concentration of acid phosphate in bifenthrin treated animals as compared to control after both of these exposure periods. Marginal and non-significant increase of BUN and creatinine in treated animals as compared to control group were observed. Animals exposed to dermal treatment of bifenthrin revealed non-significant change in protein concentration of plasma (Table1). The fall in haemoglobin could be due to interference with heme synthesis as has been reported for certain toxins and drugs (Turk and Casted, 1997). Decline in values of Hb and PCV have also been reported in dermal toxicity of fenvalerate and decamethrin in rats (Mohamed, 1988) and deltamethrin, fenvalerate and cypermethrin treated mice (Tosluty et al., 2001 and Haratym-Maj, 2002). Significant decreased value of TEC are in concurrence with results of Mohamed (1988) who reported significant decreased value of TEC after dermal application of fenvalerate and decamethrin in rats. Low TLC value could be due corticosteroid mediated leucocytosis Liver plays important role in metabolism to
maintain energy level and structural stability of body (Guyton and Hall, 2002). Aspartate transaminase a mitochondrial enzyme plays a role in the metabolism of amino acid aspartate and is predominantly found in the liver, skeletal muscles and kidneys. Alanine aminotransferase is a cytosolic enzyme which is more specific for the liver than aspartate transaminase (Paliwal et al., 2009). The increase in transaminase activity in present study could be due to damage caused by ROS after treatment with bifenthrin. Significant increase of alkaline phosphatase activity was observed both after 20 and 30 days of bifenthrin application. However, no such alteration in acid phosphatase concentration was seen at either time periods in treated animals. Similar to our findings Yousef et al. (2006) has also reported significant increase in both alkaline and acid phosphatases in deltamethrin treated rats. The increase in phosphatases due to bifenthrin toxicity may be an adaptive sign against pyrethroids poisoning. Non-significant and inconsequential increase of both BUN and creatinine in plasma was observed after 20 and 30 days of dermal application of bifenthrin. Similar results of non-significant increase in BUN have been reported by Shah and Gupta (2001) in rats following oral administration of permethrin for 30 days. Garg et al. (2004) also reported similar trend in fenvalerate treated broiler chicks. The results revealed non-significant increase in proteins of blood, both after 20 and 30 days of dermal exposure. These results are in concurrence to the findings of Shah and Gupta (2001) reporting non-significant alteration in total protein level in permethrin treated rats. Raina et al. (2009) observed significant increase in total protein in cypermethrin treated rats. It is concluded from this study that of bifenthrin dermal application @ 45 mg/ kg revealed potential to alter haemato-biochemical indices in rats.
Toxicity of bifenthrin in rat
REFERENCE Benjamin, M. M. (2001). Outline of Veterinary Clinical Pathology. Kalyani Publishers, New DelhiLudhiana. Garg, U. K., Pal, A. K., Jha, G. J. and Jadhao, S. B. (2004). Haemato-biochemical and immunopathological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks. Internat. Immunopharmacol. 4:1709-22. Guyton, A. C. and Hall J. E. (2002). Text book of Medical Physiology, 9th ed. Prism Book (Pvt) Ltd., Bangalore, India, pp Xliii+ 1148. Haratym-Maj, A. (2002). Hematological alterations after pyrethoid poisoning in mice. Ann. Agric. Environ. Med. 9: 199-206 Hawk, O. S. (1988). Practical Physiological Chemistry. 13th edn, pp: 555. Kakko, I., Toimela, T and Tahti, H. (2003). The synaptosomal membrane bound ATPase on a target for neurotoxic effects of pyrethroids , permethrin and cypermethrin. Chemosphere. 51: 475-480. Mohamed, Z. A. (1988). Changes in rat blood profile and blood chemistry after repeated dermal application of fenvalerate and decamethrin. Egyptian J. Food Sci. 16: 79-86. Narashi, T.(2000). Neuroreceptors and ion channel as the basis for drug action: Past, present and future. J. Pharmacol. Exp. Therap. 294 (1):1-26. Paliwal, A., Gurjar, R. K. and Sharma, H. N. (2009). Analysis of liver enzymes in albino rat under stress of -cyhalothrin and nuvan toxicity. Toxeminar 12: 70-73.
Prasanthi, K., Muralidharan and Rajini, P. S. (2000). Fenvalerate induced oxidative damage in rat tissue and its attenuation by dietary sesame oil. Food Chem. Toxicol. 43: 299-306. Raina, R., Verma, P. K., Pankaj, N. K. and Vinay Kant. (2009). Ameliorative effect of -tocopherol on cypermethrin induced oxidative stress and lipid peroxidation in wistar rats. Internat.J.Med.Med. Sc. 1(9): 396-399. Shah, M. A. A. and Gupta, P. K. (2001). Subacute toxicity studies on permethrin-a synthetic pyrethoid insecticide with particular reference to biochemical changes in rats. Indian J. Toxicol. 8 (1): 61-67. Snedecor, G. W. and Cochran, W. G. (1967). Statistical Methods. 6th ed. Ames: Iowa State University Press. Srivastava, A.K. and Bal, M.S. (1994) Microanalysis in Pharmacology. 1st edn. Ludhiana. Punjab Agricultural University.75-80. Tos-luty, S., Haratym-May, A., Latuszynska, J., Obuchowska, P. D., Tokaska-Rodak, M. (2001). Oral toxicity of deltamethrin and fenvalerate in swiss mice. Ann. Agric. Environ. Med. 8: 245254 Turk, J. R. and Casted, S. W. (1997). Clinical biochemistry in Toxicology. In: Kaneko, J. J., Harvey, J. W. and Brus, M. L. (ed). Clinical Biochemistry of Domestic Animals. 5 th edn. pp 829-44. Academic Press, San Diego. Yousef, M. I., Awad, T. I. and Mohamed, E. H. (2006) Deltamethrin induced oxidative damage and biochemical alterations in rat and its attenuation by vitamin E. Toxicology. 227 (3): 240-247.
Received on : 15-07-2012 Accepted on : 12-09-2012
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Research Article
PREGNANCY-DEPENDENT ALTERATIONS IN FREQUENCY AND AMPLITUDE OF MYOMETRIAL SPONTANEITY IN BUFFALOES

ABHISHEK SHARMA, SOUMEN CHOUDHURY, UDAYRAJ P NAKADE, RAJKUMAR SINGH YADAV AND SATISH KUMAR GARG*
Department of Pharmacology and Toxicology, College of Veterinary Sciences and Animal Husbandry U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Gau Anusandhan Sansthan, Mathura- 281 001, U.P., India. *Corresponding author: E-mail: profsatish@gmail.com
ABSTRACT
Myometrial autorhythmicity regulates physiology of pregnancy and labour. Present study revealed that buffalo uteri exhibit consistent pattern of myogenic spontaneity irrespective of the stages of pregnancy, however, the nature of uterine autorhythmicity differed between cycling non-pregnant (diestrous) and different stages of pregnant uterus. The amplitude of spontaneous contractions was found to be increased as the pregnancy advanced and the late pregnant (6-8 months) uteri exhibited maximum spike heights (9.67 0.67 g; n=30) compared to non-pregnant (1.95 0.25 g; n=20), early pregnant (2.25 0.45 g; n=18) and mid pregnant (3.83 0.47 g; n=22) uteri. But the frequency (BPM) of myogenic spontaneity significantly (P<0.05) decreased with advancement of pregnancy and it was calculated as 0.71 0.05 in non-pregnant (n=12), 0.43 0.04 in early pregnant (n= 13), 0.30 0.02 in mid pregnant (n= 33) and 0.11 0.006 in late pregnant (n= 33) uterus. Based on our observation, it may be inferred that pregnancy-dependent alterations in the pattern of buffalo myometrial spontaneity help the uterus to be in almost quiescent state during most of the gestation period so as to accommodate the growing fetus and maintain pregnancy and possible reversal of this characteristics is responsible for induction of labour and parturition. Key words: Autorhythmicity, buffalo, myometrium, pregnant.
INTRODUCTION Molecular and cellular signaling mechanisms controlling uterine activity during pregnancy and reproductive disorders is not well understood both in human beings and animals. Human uterine muscle remains in relatively quiescent state for majority of the pregnancy state and towards the end term series of events result in initiation of pre-term labour and development of powerful rhythmic contractions during delivery (Tribe, 2001). Combinations of hormonal, chemical and mechanical signals interact to down- or up regulate different contractile pathways during different stages of pregnancy. Myometrium is a phasic smooth muscle that exhibits spontaneous and agonist-induced contractions. The rhythmicity and generation of such contractions is intimately related to oscillations in [Ca2+]I (Wray, 2002) and its excitability, in part, is governed by the resting membrane potential which is determined by opposing inward (Na+, Ca2+) and outward (K+, Cl-) ionic fluxes and generation of slow waves and superimposed action potentials (Inoue et al., 1990). The resting membrane potential gradually becomes more depolarised as term approaches. Parkington et al. (1999) reported that the resting membrane potential increased from around 70 mV at 29 weeks to 55 mV at term and during labour and these changes are associated with an increased frequency of contractions
and excitability of the myometrium is enhanced. In view of paucity of any information on uterine activity of buffaloes during different stages of pregnancy, the present study was undertaken to evolve effective therapeutic strategy for treatment or prevention of preterm labour or other pathophysiological states of myometrium in buffaloes. MATERIALS AND METHODS Tissue collection and preparation Complete uteri along with the ovaries from cyclic (non-pregnant) and pregnant buffaloes were collected from the local abattoir of Mathura. The uteri were cut open in case of mid-or late stage pregnancy and foetus were taken out to determine the stage of pregnancy by measuring the curved-crown versus rump (CRV) length of foetus by applying the formula as suggested by Soliman et al. (1970). Uterine strips were dissected out from the midcornual region of buffalo uterus and the myometrial strips of about 3 mm x 1cm were prepared and transferred to a petri dish containing Ringer Locke solution (RLS). Both the ends of tissue strip were tied with thread and mounted in a thermostatically controlled (37.0 0.5OC) organ bath (Ugo Basile) of 10 ml capacity containing continuously aerated physiological salt solution (RLS).
Calibration of physiograph and recording of spontaneous myogenic activity
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Tissue tension changes were measured with the help of highly sensitive isometric force transducer and recorded in a PC using Lab Chart Pro V5.41 software programme (AD Instruments, Australia). After calibration, individual myometrial strips were subjected to a constant resting tension of 2 gm and allowed to equilibrate for at least 1.5 to 2 hrs before recording the isometric tension in the tissue. Absolute tension of spontaneous myogenic activity (amplitude in gm) and frequency (beats per min; BPM) in myometrial strips from non-pregnant and different stages (early, mid, late) of pregnant animals were calculated using Lab Chart Pro V5.41 software. Results were expressed as mean SEM. Multiple mean values among different groups were analysed by one-way ANOVA followed by Tukeys post hoc test using Graph pad prism 4.0 software and significance difference at 5% level were considered. RESULTS AND DISCUSSION The pattern of spontaneous motility of isolated myometrial strips of non-pregnant (diestrous stage) and different stages of pregnant buffaloes are illustrated in Fig. 1. Following equilibration for about 2 hr, myometrial strips exhibited a regular pattern of spontaneity which was characterized by consistent amplitude and frequency. The average amplitude of myogenic spontaneity in nonpregnant and early pregnancy (1-3 months) stage was found to be 1.95 0.25 g (n=20) and 2.25 0.45 g (n=18),
respectively. However, as the pregnancy advanced, there was increase in the height of spikes of spontaneous contractions and it was calculated to be 3.83 0.47 g (n=22) in mid pregnancy (3-6 months) and 9.67 0.67 g (n=30) in late pregnancy (6-8 months) stages (Table.1 and Fig.2). Frequency (BPM) of myometrial spontaneity during early pregnancy stage (1-3 months) and nonpregnant (Fig. 2) was found to be 0.43 0.04 BPM (n= 13) and 0.71 0.05 BPM (n= 12), respectively. But as the pregnancy advanced towards mid stage (3-6 months), there was decrease in the frequency of myogenic spontaneity (Fig. 2) and it was found to be 0.30 0.02 BPM (n= 33) while towards the later stage of pregnancy (6-8 months), time interval between the consecutive spikes of myometrial contractions further increased and it was found to be 0.11 0.006 BPM (n= 33) as shown in Fig. 2 and Table 1. Thus, the present study on buffalo myometrium revealed that the average amplitude (gm) and frequency (BPM) of myometrial spontaneity are differed during different stages of pregnancy. Like other phasic smooth muscles uterus also exhibits autorhythmicity and the rhythmic electrical and mechanical activity regulate the physiology of pregnancy and labour. Interstitial cells of Cajal (ICC) like specialized pacemaker cells are considered to be responsible for generation of spontaneous rhythmicity in variety of smooth muscles. Recently presence of similar cells resembling
Fig 1: Representative physiograph recordings of the spontaneous myometrial contraction of non pregnant (A), early pregnant (B), mid pregnant (C) and late pregnant (D) buffaloes.
66 Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/65-68
Myometrial spontaneity in buffaloes
Fig 2: Representative physiograph recordings of the spontaneous myometrial contraction of non pregnant (NP), early pregnant (EP), mid pregnant (MP) and late pregnant (LP) buffaloes.
to ICC has been demonstrated in rat and human uterus (Duquette et al., 2005). However, these cells did not show the spontaneous electrical activity and thus the uterine pacemaking was suggested to be an intrinsic feature regulated by some other mechanisms (Shmygol et al., 2004). Hyperpolarization of myometrial cells during pregnancy makes the uterus less excitable and contributes to its quiescence, whereas when the tissue becomes more depolarised towards the term, the excitability of myometrium is enhanced. It is now well established that the resting membrane potential (RMP) of tissues is primarily determined by opposing inward (Na+, Ca2+) and outward (K+, Cl-) ionic fluxes (Tribe, 2001) and changes in expressions of ion channels regulate these inward and outward currents during different stages of pregnancy in myometrial cells (Yoshino et al., 1997). Increased Na+ current density towards the term in pregnant rat myometrium (Inoue and Sperelakis, 1991;
Yoshino et al., 1997) has been reported and the enhanced inward sodium current with faster kinetics is responsible for frequent repetitive spike generation to facilitate simultaneous excitation and contractions in parturient uterus while reduction in both sodium and calcium inward current was observed in postpartum rat myometrial cells (Yoshino et al., 1997). Therefore, the possibility of similar changes in the ionic currents during different stages of pregnancy and cycling buffaloes and their involvement in regulating myometrial spontaneity cannot be ruled out. Calcium oscillatory mechanisms are well documented in number of smooth muscles (McHal et al., 2006). Intracellular release of Ca2+ was reported to be responsible for this oscillation in urethra and ryanodinesensitive, rather than IP3-sensitive store, is suggested as prime oscillator. Probable involvement of sodium/calcium exchange mechanism to trigger the intracellular Ca2+ release and thereby activating T- and L-type voltage operated calcium channels to promote membrane depolarization was documented as underlying mechanisms for Ca2+-oscillation in smooth muscle (McHale et al., 2006). Withdrawl of Ca2+ from the physiological salt solution or addition of L-type Ca2+ channel blockers consistently attenuated or abolished the spontaneous contraction in myometrium (Parkington et al., 1999; Tribe, 2001); thus the involvement of these Ca2+ channels/exchangers mechanisms in generating myometrial spontaneity and their regulation by pregnancy needs further investigations. Regulation of myometrial spontaneity by BKCa channels were reported earlier in rat and human myometrium (Anwer et al., 1993) and the decrease in Ca2+ sensitivity of BKCa channels was considered to be the underlying mechanism for enhancing membrane excitability in laboring human myometrium (Khan et al., 1993; 2001). However, Kv channels, rather than BKCa channels, were suggested to play crucial role in regulating basal rhythmicity in rat myometrium especially during mid and late pregnancy (Aaronson et al., 2006). Involvement of KATP, Kv and BKCa channels in regulating myometrial spontaneity in non-pregnant cyclic diestrous buffaloes were
TABLE 1: Amplitude and frequency of myometrial spontaneity in non-pregnant and pregnant buffaloes. Group Non-pregnant Early pregnant (0-3 months) Mid pregnant (3-6 months) Late pregnant (6-8 months) Amplitude (g) 1.95 0.25 (20) 2.26 0.45 (18) 3.83 0.47 (22) 9.67 0.67*# (30) % change in amplitude 15.9 96.41 395.9 Frequency (BPM) % change in frequency 0.71 0.05 (12) 0.43 0.04* (13) 0.30 0.02*# (33) 0.11 0.006*# (33) 39.44 57.75 84.51
Values expressed are Mean SEM. Figures in parentheses indicate the number of observations. Data were analysed by one-way ANOVA followed by Tukeys post-hoc tests. *P< 0.05 vs non-pregnant, #P<0.05 vs early pregnant and P< 0.05 vs mid pregnant.
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previously documented by Choudhury et al., (2010, 2011). We have shown that in the presence of glybenclamide, 4AP and iberiotoxin, myometrial membrane is excited and either there was change or increase in spontaneity in buffalo uterus. Thus changes in the expression of these channels may be the underlying mechanism for alterations in myogenic spontaneity of buffalo myometrium. Based on the results of the present study it may be inferred that the pregnancy-dependent alterations in amplitude and frequency of myometrial spontaneity may be regulated by altered characteristics and expressions of ion channels governing the membrane potentials. As uterine function are greatly influenced by reproductive hormones, thus possible endocrine regulation of these ion channels in maintaining uterine rhythmicity cannot be ruled out. With the advancement of pregnancy stages, increase in the spike height of myometrial spontaneity may facilitate forceful uterine contractions required for expulsion of growing fetus during parturition while feeble contractions (low height contractions) are responsible for keeping the uterus in quiescent state during early and mid pregnancy states. However, the precise mechanism(s) responsible for these alterations in myogenic spontaneity during nonpregnant and different stages of pregnant buffalo uterus needs further investigations. REFERENCES Aaronson, P. I., Sarwar, U., Gin, S., Rockenbauch, U., Connolly, M., Tillet, A., Watson, S., Liu B. and Tribe R.M. (2006). A role for voltage-gated, but not Ca2+-activated, K+ channels in regulating spontaneous contractile activity in myometrium from virgin and pregnant rats. Br. J. Pharmacol. 147: 815824. Anwer, K., Oberti, C., Perez, G.J., Perez-Reyes, N., Mcdougall, J.K., Monga, M., Sanborn, B.M., Stefani, E. and Toro, L. (1993). Calcium-activated K+channels as modulators of human myometrial contractile activity. Am. J. Physiol. 265: C976 C985. Choudhury, S., Garg, S.K., Singh, T.U., and Mishra, S.K. (2010). Cellular coupling of potassium channels with 2- adrenoceptors in mediating myometrial relaxation in buffaloes (Bubalus bubalis). J. Vet. Pharmacol. Therap. 33: 22-27. Choudhury, S., Garg, S.K., Singh, T.U., and Mishra, S.K. (2011). Functional and molecular characterization of maxi-K + channels (BKCa) in buffalo myometrium. Anim. Reprod. Sci. 126(3-4): 173178. Duquette, R.A., Shmygol, A., Vaillant, C., Mobasheri, A., Pope, M., Burdyga, T. and Wray, S. (2005).
Vimentin-positive, c-kit-negative interstitial cells in human and rat uterus: a role in pacemaking. Biol. Reprod. 72: 276283. Inoue, Y., Nakao, K., Okabe, K., Izumi, H., Kanda, S., Kitamura, K. and Kuriyama, H. (1990). Some electrical properties of human pregnant myometrium. Am. J. Obstet. Gynecol . 162 : 10901098. Inoue,Y. and Sperelakis, N. (1991). Gestational change in Na+ and Ca+2 channel current densities in rat myometrial smooth muscle cells. Am. J. Physiol. 260: C658-662. Khan, R.N., Matharoo-Ball, B., Arulkumaran, S. and Ashford, M.L. (2001). Potassium channels in the human myometrium. Exp. Physiol. 86: 255264. Khan, R.N., Smith, S.K., Morrison, J.J. and Ashford, M.L. (1993). Properties of large conductance K+channels in human myometrium during pregnancy and labour. Proc. R. Soc. Lond. Ser. B. 251: 915. McHal, N., Hollywood, M., Sergeant, G. and Thornbury, K. (2006). Origin of spontaneous rhythmicity in smooth muscle. J. Physiol. 570(1): 2328. Parkington, H.C., Tonta, M.A., Brennecke, S.P. and Coleman, H.A. (1999). Contractile activity, membrane potential and cytoplasmic calcium in human uterine smooth muscle in the third trimester of pregnancy and during labor. Am. J. Obstet. Gynecol. 81: 1145-1151. Shmygol, A., Burdyga, T., Duquette, R., Mobasheri, A., Vaillant, C. and Wray, S.(2004). Spontaneous electrical activity in subpopulation of freshly isolated rat uterine myometrial cells. J. Physiol. 555: C167. Soliman, M.K. (1970). Studies on the physiological chemistry of the allantoic, amniotic fluids of buffaloes at the various periods of pregnancy. Ind. Vet. J. 52: 106-112. Tribe, R.M. (2001). Regulation of human myometrial contractility during pregnancy and labour: are calcium homeostatic pathways important. Exp. Physiol. 86(2): 247254. Wray, S. (2002). Role of the sarcoplasmic reticulum in uterine smooth muscle. In: What is the Role of the SR in Smooth Muscle. Chichester, UK: Wiley Press for the Novartis Foundation. pp: 6-25. Yoshino, M., Wang, S.Y. and Kao, C.Y. (1997). Sodium and calcium inward currents in freshly dissociated smooth myocytes of rat uterus. J. Gen. Physiol. 110: 565577.
68
Research Article
PROTECTIVE ROLE OF VITAMIN E AND PHENYTOIN IN CHLORPYRIFOS INDUCED DELAYED NEUROPATHY IN HEN
K. KAVITHA1, B. KALA KUMAR1,. S.S.Y.H QUADRI2, Y.N REDDY3 AND M. ALPHA RAJ4
1
Dept.of Pharmacology & Toxicology, 3Dept. of Microbiology, College of Veterinary Science College of Veterinary Science, Rajendranagar, Hyderabad; 2Assistant Director, Dept. of Pathology, NIN, Hyderabad (A.P.); 4 Dept. of Pharmacology & Toxicology, College of Veterinary Science, Proddatur, A.P. * Corresponding author: E-mail-alpharajm@gmail.com
ABSTRACT The present study was carried out to evaluate the protective effect of vitamin E, an antioxidant and phenytoin, a calcium channel blocker against chlorpyrifos (CPF) induced oxidative damage . A total 72 adult white leghorn birds of 56 weeks age were divided into four groups randomly. Group 1 served as control, Group 2, 3 and 4 were administered single dose of CPF @ 350mg/kg s/c divided over a period of 24 hrs. Vitamin E @ 50 mg/kg p.o as prior treatment for 10 days and phenytoin @50 mg/kg p.o as prior treatment for 4 days were given in groups 3 and 4, respectively. Blood was collected on 3rd,7th,10th and 14th days of the treatment. Albumin, total protein in serum, GSHPx and GSHR in RBC lysate and SOD, CAT, GSH in spinal cord were assayed. Oxidative stress with CPF was manifested in terms of a significant (P<0.05) decrease in the levels of serum albumin, total protein, GSH and inhibition of GSH-Px and GSH-R activities and an increase in SOD and CAT activities, however, reversal of these parameters occured by vitamin E without any effect of phenytoin. It is concluded that oxidative stress could be one of the mechanisms of OP toxicity and antioxidants might provide a viable therapeutic regimen. Key words: Chlorpyrifos, OPIDN, oxidative stress, phenytoin, vitamin E, white leghorn,
INTRODUCTION Organophosphate insecticides are the most widely used in agriculture, public health and animal husbandry veterinary practices. Treatment of acute toxicity rescues the patient most often but this is followed by delayed neurotoxicity in susceptible species and human beings. Previously, it was assumed that neuro target esterase (NTE) is target for producing delayed neurotoxicity (Richardson, 1995) but now, it is hypothesized that increased intracellular calcium and reactive oxygen species (ROS) are involved in developing organophosphosphate induced delayed neurotoxicity (OPIDN) (Uchendu et al., 2012). Toxicity due to pesticides also produces cognitive alterations (Lopez-Granero et al., 2012) besides oxidative stress. Due to the involvement of oxidative stress, in the development of delayed neuropathy, the present study was aimed at assessing the role of free radicals in developing OPIDN in hen and its alleviation using an antioxidant vitamin E and phenytoin, a calcium and sodium channel blocker. MATERIALS AND METHODS White leghorns (layers) of 56 weeks age were acclimatized to the laboratory conditions for a period of 1 week. Permission was obtained from IAEC before performing the experiment. The birds of approximately equal weights were divided into, 4 groups of 18 birds each. Group 1 served as control; Group 2 served as chlorpyrifos (CPF) control @350mg/kg s/c; group 3 as vitamin E+CPF (Vitamin E prior treatment for about 10 days @ 50 mg/kg per orally) and group 4 as phenytoin + CPF (phenytoin prior treatment @50 mg/kg for 4 days per orally). Blood was collected from the wing vein on 3, 7, 10 and 14 days for estimating
serum albumin (BCG Dye binding method), total protein (Lowry et al.,1951) employing kits supplied by Qualigens, Mumbai, India. Glutathione peroxidase (GSH-Px) (Paglia and Valentine, 1967) and glutathione reductase (GSH-R) (Raghuramulu, 1983) were estimated in the RBC lysate. Spinal cord was collected in 10% Tris -HCl, pH 7.4, homogenized and centrifuged at 10,000 g for 15 min and the supernatant was used for the estimation of GSH (Moron et al., 1979), superoxide dismutase(SOD) (Marklund, 1974) and catalase (CAT) activity (Caliborne, 1985). The data was analysed using SPSS 15.0 V software by one way ANOVA and tested for significant (P<0.05) differences by Duncans multiple comparison test. RESULTS AND DISCUSSION Serum albumin is a biomarker, whose estimation is in vogue recently, for exposure to OP compounds. OP compounds covalently bind to albumin. In this experiment, there was a significant (P<0.05) fall in serum albumin in CPF treated groups 2,3 and 4 from 3rd day of the experiment (Table 1). The decrease in serum albumin was attributed to the binding of CPF to albumin. Added to decreased availability of albumin for binding, hepatotoxicity at supra critical LD50 dose might have also affected protein synthesis contributing to decreased albumin levels in this study. Group 2 recorded a sgnificant fall in serum albumin throughout the study. The results comply with the findings of Peeples et al. (2005). Total proteins were estimated to assess the hepatotoxicity of CPF which undergoes oxidative desulfuration to yield chloryrifos oxon (CPO) by hepatic microsomal enzymes (Richardson, 1995). In the present
Kavitha et al.
study, the serum total proteins concentration was reduced in the CPF treated groups 2, 3 and 4, however, level in group 3 was significantly higher than groups 2 and 4 (Table 1). Similar damage to the liver was observed by Osman (1999) who suggested increased production of ROS with CPF initiating oxidative damage and cytotoxic effects on all organs including liver. Further, Bebe (2003) postulated that oxidative stress in liver occurred due to decreased endogenous antioxidants leading to hepatic injury and interference with the full expression of hepatocyte protein synthesis. CPF is a xenobiotic that hastens up generation of super oxide anions. Superoxide dismutase and catalase enzymes were estimated as an indirect indicator of superoxide anion generation. Group 2 and 4 both registered an increase in the activities of the SOD and CAT indicating increased generation of super oxide anion under the influence of CPF. In contrast, prior administration of vitamin E followed by CPF injection resulted in a significant (P< 0.05) decrease in SOD and CAT activities compared to
CPF control group (Tables 1). The findings of the present study coincide with earlier investigations on vitamin E and dimethoate (John, 2001), ginger and malathion (Ahmed, 2004), melatonin, vitamin C and E against CPF in lungs (Karaoz, 2002), kidney (Oncu, 2002) and rat erythrocytes (Gultekin, 2001). Similar results of attenaution of CPF induced toxicity on liver and hematology was also reported (Acker et al., 2012). Glutathione, a tri-peptide thiol is the most important free radical scavenger playing a major role in antioxidant defense mechanism of the body. Glutathione with -SH group acts as a nucleophile donating electrons to electrophilic free radicals. Loss of electron would oxidize reduced GSH to oxidized (GSSG) form. This oxidoreduction inter play is under the aegis of GSHPx and GSHR enzyme system. In the present study, the GSH content was significantly (P<0.05) decreased in groups 2 and 4 on day 3 and 7 which recovered by day 10 (Table 1). In group 3, there was a significant (P<0.05) decrease in glutathione concentration though the percent decrease was
TABLE 1: Serum protein levels (mg/dl) and antioxidative parameters following oral administration of Vit E and phenytoin for 14-days in chlorpyrifos(350mg/kg, s/c) intoxicated layers. Group Serum albumin concentration (g/dl) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin + CPF) Serum total protein level (mg/dl) I (Control) II (CPF alone) III (Vit. E+CPF) IV (Phenytoin+CPF) Superoxide dismutase (SOD)activity (U/mg protein) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin + CPF) Catalase (CAT) activity ( moles/min/mg protein) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin + CPF) Glutathione (GSH) concentration ( g/g of protein) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin + CPF) Glutathione peroxidase (GSHPX)activity (U/ml) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin +CPF) Glutathione reductase (GSHR) activity (units/ml) I (Control) II (CPF alone) III (Vit. E + CPF) IV (Phenytoin + CPF) 3rd day 2.15 + 0.05 b 1.40 + 0.07 a 1.88 + 0.30 a 1.63 + 0.10 a 447.24 + 0.13d 323.36 + 0.35a 395.04 + 0.14c 328.04 + 0.11b 07.59 + 0.36 a 15.47 + 0.76 c 11.40 + 0.77 b 14.22 + 0.34 c 2.12 + 0.84 --a 4.50 + 0.30 c 3.84 + 0.32 b 4.39 + 0.39 c 5.41 + 0.26 c 2.49 + 0.50 a 3.60 + 0.15 b 2.61 + 0.11a 80.39 + 0.44 c 49.77 + 0.87 a 65.45 + 0.11b 52.50 + 0.90 a 40.97 + 0.63 c 21.97 + 0.12 a 28.13 + 0.67 b 22.88 + 0.10a 7th day 2.07 + 0.03b 1.58 + 0.03 a 1.93 + 0.03 ab 1.68 + 0.07 a 443.88 317.35 406.40 343.59 + + + + 0.89d 0.59a 0.52c 0.56b 10th day 2.13 + 0.05 b 1.63 + 0.01 a 1.95 + 0.05 ab 1.90 + 0.07 ab 447.93 348.61 407.85 385.21 07.41 10.69 08.15 10.90 + + + + 0.29d 0.28a 0.67c 0.68b
a b a b
14th day 2.20 + 0.08 b 1.82 + 0.07 a 2.05 + 0.04 b 1.95 + 0.08 ab 450.59 378.06 405.21 386.31 + + + + 0.51b 0.23a 0.32a 0.37a
08.42 + 0.33 a 12.86 + 0.62 b 09.12 + 0.38 a 12.07 + 0.84b 2.43 4.02 3.14 3.95 + + + + 0.84 a 0.98 c 0.98 b 0.13 c
+ + + +
0.45 0.60 0.30 0.36
07.49 + 0.34 a 10.20 + 0.63 b 08.46 + 0.33 ac 09.80 + 0.29 bc 2.24 + 0.18 a 3.04 + 0.25 c 2.42 + 0.12 ab 2.89 + 0.13 bc 5.08 3.33 4.41 3.84 82.35 52.15 75.71 59.94 + + + + + + + + 0.16 c 0.10 a 0.27 b 0.13 a 0.87 d 0.14 a 0.89 c 0.18 b
2.33 + 0.16 a 3.57 + 0.12 b 2.95 + 0.14 a 3.49 + 0.21b 5.22 + 0.99 c 3.33 + 0.15 a 4.53 + 0.19 b 3.48 + 0.35 a 79.67 50.39 73.32 56.20 + + + + 0.60 0.13 0.66 0.12
d a c b
5.14 + 0.87 c 2.59 + 0.13 a 4.10 + 0.15 b 2.61 + 0.89 a 80.82+ 0.18 d 45.42 + 0.15 a 70.09 + 0.58 c 55.05 + 0.12 b 41.98 + 0.11 c 20.32 + 0.82 a 30.43 + 0.84 b 22.42 + 0.88 a
41.56 + 0.10 c 25.31 + 0.75 a 32.32 + 0.83 b 26.72 + 0.10 a
42.12 + 0.11 d 27.27 + 0.90 a 34.07 + 0.13 b 28.25 + 0.42 a
Values are Mean + S.E. (n=4); Means with different superscripts are statistically significant (p<0.05)
70
Vitamin E in CPF induced neuropathy
less compared to group 2 indicating protective role of vitamin E in quenching the free radical and thus decreasing glutathione consumption. The present findings are in accordance with Salama et al. (2005) who reported similar results with glutathione. GSHR and GSHPx enzyme activity serve as biomarkers to assay regeneration of GSH and also for conversion of H2O2 formed from super oxide anion to water. CPF inhibited the activities of both the enzymes in group 2,3 and 4 on the third day. However, the percent inhibition in group 3 was less compared to groups 2 and 4 (Table 1). GSHPx activity was never restored to normal till the end of the experiment in all the three groups. GSHR activity was also inhibited in similar fashion, except for group 3 which ranged between groups 1and 2, manifesting the protection provided by vitamin E against CPF induced free radicals. Oncu (2002) with CPF and Bebe (2003) also observed fall in GSHP and GSHR activities in liver with CPF exposed rats. From the present study, it is concluded that CPF impairs antioxidant defenses, thus favoring the progression of stress due to ROS. Vitamin E could offer significant protection in CPF induced oxidative stress in hens, whereas phenytoin was not effective. REFERENCES Acker,C.I., Souza, A.C.G., dos Sanos, M.P., Mazzanti, C.M. and Nogueira, C.W. (2012). Diphenyl diselenide attenuates hepatic and hematologic toxicity induced by chlorpyrifos acute exposure in rats. Environ. Sc. Poll. Res., doi.:10.1007/ s11356-012-0882-4. Ahmed, M. and Hollingworth, R.M. (2004). Synergism of insecticides provides evidence of metabolism of mechanisms of resistance in the obliquebanded leaf roller Choristoneura arosaceana (Lepidoptera; Tortricidae). Pest-Manag.Sc. 60 : 465-473. Bebe, F.N. and Panemangalore, M. (2003). Exposure to low doses of endosulfan and chlorpyrifos modifies endogenous antioxidants in tissues of rats. Journal of Environ. Sc. Hlth. 38: 349-363. Caliborne, A.L. (1985). Assay of Catalase. In: Hand Book of Oxygen Radical Research; Greenwald RA (Ed), CRC press, Baco- Raton. John, S., Kale, M., Rathore,N., Bhatnagar, D., Kale, M.O., Rathoxe, N. and Bhatnagar, D.2001. Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes. School of Bio Chem. 12: 500-504. Karaoz, E., Gultekin, F., Dogan, M., Oncu, M. and Gokcimen A. (2002). Protective role of melatonin and a combination of vitamin C and Vit. E on lung toxicity induced by chlorpyrifos ethyl in rats. Exptl and Toxicol. Pathol. 54: 97-108.
Lopez-Granero, C., Canadas, F., Diana, C., Yu, Y., Gimenez, E. Luzano, R., Silva, A.D., Asclves, M. and Sanchez-santed, F. (2012). Chlorpyrifos Diisopropylphosphorofluoridate and Parathion induced behavioural and oxidative stress effects: Are they mediated by analogus mechanisms of ction? Toxicological Sciences, Sep.17. Epub ahead of publication. Lowry, O.H., Rosenbrough, M.J., Farr, A.L. and Randell, R.A. (1951). Protein mesasurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Marklund, S.L. and Marklund, G.(1974). Involvement of super oxide anion radical in the auto oxidation of pyrogallol and a convenient assay for super oxide dismutase. European J. Bioche. 47: 469 474. Moron, M.S., Depierre, J.W. and Mannervik, B. (1979). Levels of glutathione, glutathione reductase and glutathione - s- transferase in rat lung and liver. Biochem Biophy. Acta. 582: 67-68. Oncu, M., Gultekin, F., Karaoz, E., Altuntas, I. and Delibas, N. (2002). Nephrotoxicity in rats induced by chlorpyrifos ethyl and ameliorating effects of antioxidants. Human Exptl.l Toxicol. 21: 223-230. Paglia, D.E. and Valentine, W.N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70: 158 159. Peeples, E.S., Lawrence, M.S., Ellen, G.D., Reggie, S., Troy, V., Charles, M.T. and Oksana, L. (2005). Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry. Toxicological Sciences, 83: 303312 Raghuramulu, M. (1983). Manual of Laboratory Techniques. National Institute of Nutrition, ICMR JamaiOsmania, Hyderabad pp. 204-206. Richardson, R.J. (1995). Assesment of neurotoxic potential of chlopyrifos relative to other organophosphorus compounds: A critical review of the literature. Toxicology and Environmental Health, 44:135-65. Salama, A.K., Osman, K.A., Saber, N.A. and Soliman, S.A. (2005). Oxidative stress induced by different pesticides in the land snails, Helix aspersa. Pakistan Journal of Biological Sciences, 8: 9296. Uchendu, C., Suleiman, F.A. and Joseph, O.A. (2012). The organophosphorus pesticides, chlorpyrifos, oxidative stress and the role of some antioxidants: A review. African Journal of Agricultural Research, 7: 2720-28.
Research Article
STUDY ON IMMUNOTOXIC POTENTIAL OF ALPHACYPERMETHRIN IN WLH CHICKS

NAVNEET KUMAR PANDEY1 AND A.M.THAKER2
1
Assistant Director, Division of Toxicology, Krish Biotech Research Pvt Ltd, Kalyani, Nadia, West Bengal-741235. 2 Prof. & Head, VPT and Dean, C.V.Sc. & A.H., A.A.U. Anand, Gujarat-388001. 1 Corresponding author: Email- neetvet@gmail.com
ABSTRACT
The effect of repeated oral administration of alphacypermethrin on immune system of day old White Leghorn (WLH) chicks was assessed. Day old, 125 White Leghorn (WLH) chicks were randomly divided equally in five groups C1, C2, T1, T2 and T3. Birds of all the groups were administered New castle Disease (NCD) vaccine on day 7. Group C1 and C2 served as untreated control as well as vehicle control (corn oil), respectively. Birds of group T1, T2 and T3 were administered with alphacypermethrin at 14.0625 mg/kg, 18.725 mg/kg and 28.125 mg/kg body weight, respectively, for 28 days through oral gavage in corn oil. Blood samples were collected at two week intervals for evaluation of humoral and cell mediated response. Lymphoid organs like thymus, spleen, bursa and liver were weighed at the time of necropsy for estimation of absolute and relative organ weight. Repeated oral administration of aphacypermethrin decreased New Castle Disease (ND) vaccine antibody titer, total protein, serum globulin and albumin. Organ weight and body weight were significantly reduced but relative organ weight was unaffected. It is concluded that alphacypermethrin altered the immunological response in white leghorn cockerels after 28 days repeated oral exposure. Key words: Alphacypermethrin, cytotoxic, immunotoxicity, WLH chicks.
INTRODUCTION The lowered immunocompetence in animals and birds due to environmental pollutants my lead to increased susceptibility to infections, epidemics of disease and vaccine failures (Chauhan et al.1995). Immunotoxic effect of alphacypermethrin has been reported in animals, however, the information available in the literature regarding immunopathological effects of alphacypermethrin in birds is scanty. Therefore, the present investigation was planned to study the effects of alphacypermethrin on immune response of the birds. MATERIALS AND METHODS Experimental animals A total of 125, day old WLH chicks were procured and housed at Central Poultry Research Station, Anand Agricultural University, Anand, Gujarat-388001. Experimental Design Day old chicks were randomly divided into five groups (C1, C2, T1, T2 and T3) of 25 each. All the chicks were vaccinated with Mareks disease vaccine (Georgia strain) 0.2ml on zero day through subcutaneous route. On day 7 all the birds were vaccinated with New Castle Disease vaccine (Lasota strain). Chicks were given standard chick ration and water ad libitum. All standard managemental procedures were adopted to keep the birds free from stress. LD50 of alphacypermethrin (Technical grade; Meghmani Corporation Pvt Ltd, Ahmedabad) , taken in to consideration for this study was 562.5 mg/kg body weight
72
(Singh and Sharma, 2003). Group C1 and group C2 were untreated control and vehicle (corn oil) control respectively. Dose volume was administered at the rate of 1ml/kg body weight. Three different dose levels of alphacypermethrin in ascending order, i.e., 14.0625 (LD50/40), 18.725 (LD50/ 30) and 28.125 (LD50/20) mg/kg were formulated daily in corn oil and administered to group T1, T2 and T3 for a period of 28 days. Birds were observed for any toxic signs during entire period of experimentation. Immunological Parameters Six birds from each group were sacrificed after 14 days and 28 days of completion of experimentation. Blood sample was collected to separate serum, which was used for estimation of serum total protein, albumin and globulin levels and New Castle Disease (NCD) vaccine antibody titer by enzyme linked immunosorbent assay (ELISA) (Synder et al., 1984), Absolute and relative organ weights were also estimated. Cell mediated immune response was evaluated with DNCB dye dermal sensitization test (Khurana et al., 2000). Statistical analysis All the values were expressed as mean S.E.M. Statistical analysis was done by using SPSS 7.5 software. Statistical significance (P<0.05) of differences between two mean was assessed by one way ANOVA test. RESULTS AND DISCUSSION In the present study, after exposure of alphacypermethrin total serum protein, albumin and globulin were decreased significantly in treatment groups
Imunotoxicity of alphacypermethrin in chicks
as compared to control group expressed in Table 1. The fall in total serum protein could be due to the stressogenic effect of alphacypermethrin, or general toxic action that also leads to decrease in weight gain in the insecticides treated birds. Significant decrease in total protein and albumin content in serum indicating that alphacypermethrin caused significant damage to vital organs and also interfered with protein metabolism. Reduction in total serum protein has also been reported in cockerels following prolong feeding of fenvalerate-medicated ration at the rate of 4000 ppm (Singh et al., 2001). Serum globulin level was decreased in all treated groups following feeding of broiler chicks with 20 ppm fenvalerate (Garg et al., 2004). Globulin content in serum was also significantly decreased on day 28 of study indicating that alphacypermethrin affects the antibody production in dose dependent manner. Albumin to globulin ratio was significantly increased in T3 group on day 28 of experiment depicting the effect of alphacypermethrin on immune response. Chauhan and Mahipal, (1994) reported immunotoxicity of cypermethrin in poultry and was noted that when birds were fed with cypermethrin there was a significant reduction in serum globulin and gamma globulins, which is indicative of non-specific and general reduction in immunity. No effect on serum albumin was noticed due to cypermethrin in chickens (Khurana et al., 1996a). Premlata et al. (2006) and Garg et al. (2004) reported decreased serum globulin levels in all fenvalerate treated broiler chicks as compared to control, but there was no change in serum albumin level. Fenvalerate (20
mg/kg body weight) oral administration in male rats resulted in significant (P<0.05) alterations in plasma proteins (Demerdash et al., 2004). There was significant decrease in absolute thymus, liver, spleen and bursa weight as compared to control birds on 28 day of study as expressed in Table 1. Reduction in thymus weight also supported by hypotrophy of individual thymic lobes but the histopathological examination did not show any decrease in lymphocyte populations. Prater (2003) observed decrease in splenic and thymic organ weight and cellularity in dose-related manner in mice, when 220-1100 mg/kg body weight permethrin was dermally applied. The highest dose (55.4 mg/kg) of cypermethrin resulted in a significant increase of the relative liver weight (Institoris et al., 1999). Decrease in liver and bursa weight was significant and supported by gross and histopathological observation of these organs. Liver showed fatty changes, necrosis and vaccuolations, may be the cause of decreased liver weight. Rupture of bursal follicles and also decrease in follicular density was suggestive of decreased bursa weight due to alphacypermethrin toxicity in lymphoid organs. In the present study antibody titre against NDV was measured by ELISA to monitor humoral immune response and DNCB dye test was done to monitor cellmediated immunity. The results indicate significant decrease (P<0.05) in antibody titres against ND vaccine on both day 14 and 28 of study in alphacypermethrin treated groups as compared to control group of birds expressed in Table 1. There was dose dependent decrease
TABLE 1: Effect of alphacypermethrin on antibody titer and absolute organ weight in WLH chicks (Mean SE; n=6). Group/Dose (mg/kg b.wt) Total Protein (gm/dl) Albumin (gm/dl) Globulin (gm/dl) Alb : Glb NDV Antibody Titer Liver (gm) Spleen (gm) Thymus (gm) Bursa (gm) Total Protein (gm/dl) Albumin (gm/dl) Globulin (gm/dl) Alb : Glb NDV Antibody Titer Liver (gm) Spleen (gm) Thymus (gm) Bursa (gm) C1 Control) C2 (Vehicle control) T1 (14.0625) T2 (18.725 ) T3 (28.125) 14 days after treatment 2.470.04a 2.180.14 ab 2.140.09ab 2.090.11 b a b ab 1.310.09 1.150.07 1.190.03 1.210.04 ab 1.1640.117a 1.0430.087a 0.9780.191 a 0.8760.108a 1.230.21a 0.990.19a 1.490.11 a 2.000.84a b bc 7439.83787.94a 6658.00296.44 6141.64581.20 5641.64392.32c 2.650.24a 2.160.13a 2.550.26 a 2.360.18 a 0.120.02a 0.110.02a 0.130.01a 0.110.01a a a a 0.210.02 0.190.03 0.190.01 0.170.02a a a ab 0.610.06 0.460.01 0.510.04 0.440.05b 28 days after treatment 2.640.07a 2.380.18b 2.300.12ab 2.240.06 b a bc ab 1.590.13 1.160.11 1.210.15 1.280.08 c 1.5970.130 a 1.2080.155ab 1.1020.109bc 0.9780.085c 0.82 0. 09a 0.770.05a 1.040 .12a 1.340.13 b a a ab 3372.33422.78 3169.50131.43 3040.17275.57 2958.67212.35b 6.540.59a 4.630.32b 4.130.36 b 3.870.48b 0.470.02a 0.260.02b 0.280.02b 0.230.02b a b b 0.480.03 0.300.03 0.260.03 0.23.03b a ab ab 1.150.16 0.870.04 0.950.08 0.680.07b
2.480.04a 1.300.03a 1.1830.061 a 1.120.08a 7595.17411.37a 2.640.08a 0.120.01a 0.190.01a 0.630.04a 2.860.19ab 1.380.08ab 1.3810.085ab 0.940 08a 3430.50352.84a 6.97 0.51a 0.460.03a 0.490.02a 1.180.17a
Data in the column having different superscript differ significantly (p< 0.05).
Pandey and Thaker
TABLE 2: Effect of alphacypermethrin on DTH response (skin thickness, mm) in WLH chicks (Mean SE; n=6). Group/Dose (mg/kg b.wt) Skin thickness (mm) At 24 hrs At 48 hrs Lt side Rt side Lt side Rt side Lt side Rt side Lt side Rt side C1 (Control) 0.22 0.04a 0.150.02a 0.070.01 a 0.040.01 a 0.540.12a 0.510.12 a 0.440.17a 0.410.06 a C2 T1 (Vehicle control) (14.0625) After 14 days of treatment 0.180.03 ab 0.150.01 b 0.130.02 ab 0.090.02b 0.060.01 a 0.060.01 a 0.040.01 a 0.050.01 a After 28 days of treatment 0.500.11 a 0.420.07 ab a 0.500.12 0.370.12a a 0.440.16 0.260.07a 0.330.07 a 0.230.08 a T2 (18.725 ) 0.120.01 b 0.090.01b 0.050.01 a 0.040.01a 0.420.07 ab 0.390.08a 0.360.09 a 0.290.06 a T3 (28.125) 0.090.02 c 0.090.20b 0.050.01a 0.030.01a 0.360.05 b 0.360.09 a 0.270.08 a 0.270.09 a
Skin thickness (mm) At 24 hrs At 48 hrs
Data in the column having different superscript differ significantly (p< 0.05).
in antibody titer of ND vaccine in all the treatment groups. Reduction in antibody titers against vaccine antigen was an indication of suppression of humoral immune response and diminished antibody synthesis. The results indicated that alphacypermethrin was found to be immumnotoxic at the dose levels tested. In other studies, humoral immune response of chicks was judged by estimating HI titre against Lasota strain of NDV at the end of 11 week with highest titre (1600+/- 319) in control and lowest (906+/- 207) in chicks of high dose treatment group. Siroki et al. (1994) also reported the immunotoxicological effect of pyrethroid pesticides in mice. Suppression of humoral immune response was also observed by alphamethrin in chickens (Singh et al., 1997). There was significant difference in DTH response to DNCB dye between treatment and control groups on day 14 after 24 and 48 hrs following primary sensitization but on secondary sensitization on day 28, there was no significant increase in dermal thickness as expressed in Table 2. As upon secondary sensitization there was no significant change in skin thickness among treatment group as compared to control, which indicates that alphacypermethrin seems to be least deleterious as for the cell mediated immune response is concerned. The results indicated that alphacypermethrin was found to be least toxic or non toxic to cell mediated immune response at dose rate administered in this study. Suhash (2004) reported that DNCB skin sensitivity test did not show any significant difference in skin thickness on cypermethrin treatment at dose rate of 3 mg/kg, 9 mg/kg and 30 mg/kg for 28 days. Fenvalerate (Singhal et al., 2001), cypermethrin (Khurana et al., 2000) and alphamethrin (Singh et al., 1997) caused suppression of CMI in chickens. Khurana and Chauhan, (2000) reported suppression of CMI in lambs due to fenvalerate exposure. Patel et al. (1996) reported significant depression of cell mediated immunity and humoral immune responses in cypermethrin treated crossbred male calves at the dose rate of 60 mg/kg body
weight for 30 days. An immune system is one of the most sensitive target of pesticides owing to continuous proliferation and differentiation. The day old chicks are more susceptible to toxicity on immune system as the lymphoid organs remains in growing phase. It was evident that alphacypermethrin produced necrosis hemorrhages and fatty changes in liver. Spleen showed condensation and vacuolation. Thymus showed reticuloendothelial cell hyperplasia. Bursa showed rupture and atrophy of bursal follicles. It is concluded from this study that alphacypermethrin altered the immunological response following 28 days repeated oral exposure in white leghorn chicks. ACKNOWLEDGEMENT Meghmani Corporation Pvt Ltd, Ahmedabad, for providing technical grade of alphacypermethrin for conduct of the experiment. REFERENCES Chauhan, R.S. and Mahipal, S.K. (1994). Immunotoxicity of pesticides in Poultry: Advances in Veterinary Research and Their impact on Animal Health and Production. IVRI, Bareiley. Chauhan, R.S., Bhushan, B., Khurana, S.K. and Mahipal, S.K. (1995). Effect of pesticide on immunocompetence of animal and there public health significance in proceding of Indo-German conference on impact of Modern agriculture on Environment. pp. 169-178. Demerdash, A., Yousef, M.I., Kedwany, F.S. and Baghdadi, H. (2004). Fenvalerate induced changes in oxidative stress: Hematobiochemical parameters of male rats. J. Env. Sc and Hlth. 39 : 443 459. Garg, U.K., Pal, A.K., Jha, G.J. and Jadhao, S.B. (2004). Haemato-biochemical and immunopathophysiological effects of chronic toxicity with
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Imunotoxicity of alphacypermethrin in chicks
synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks. International. Immunopharmacol. 4:17091722. Institoris, L., Siroki, O., Undeger, U., Desi, I. and Nagymajtenyi, L. (1999). Immunotoxicological effects of repeated combined exposure by cypermethrin and the heavy metals lead and cadmium in rats. Intern. J. Immunopharmacol. 21(11): 735-743. Khurana, R., Chauhan, R.S., Sharma, R. and Mahipal, S.K. (2000). Immunotoxic effects of cypermethrin on cell mediated immune response in chickens. Intern. J. Ani. Sci. 15: 33-38. Khurana, R. and Chauhan, R.S. (2000). Immunopathological effects of fenvalarate on cell mediated immune response in sheep. J. Immunol. Immunopathol. 2: 56-59 Patel, B.J., Singh, S.P., Sharma, L.D. and Joshi, D.V. (1996). In vivo immunosupression by cypermethrin toxicity in crossbred calves. Indian J. Toxicol. 3(2): 1-7. Prater, M.R. (2003). Immunotoxicity of dermal permethrin and cis-urocanic acid: Effects of chemical mixtures in environmental health. Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Veterinary Medical Sciences. Premlata., Jain, S.K. and Punia, J.S. (2006). Hematologiclal and biochemical changes in subacute fenvalerate toxicity. Indian J. Ani. Sci. 76(3): 233-235.
Singh, S.P., Sharma, L.D. and Chauhan, R.S. (1997). Immunotoxic effect of acute toxic effect of alphamethrin in chicks .In: Proc. Nat. Symp. Adv. Vet. Path. Post Independence Era and XIV Ann. Conf. IAV, Izatnagar. 157-159. Singh, B.P., Singhal, L.K. and Chauhan, R.S. (2001). Fenvalerate induced cell-mediated and humoral immune alterations in chickens. J. Immunol. Immunopathol. 3: 59-62. Singh, S.P. and Sharma, L.D. (2003). Evaluation of median lethal dose alphacypermethrin in 8 week old WLH cockerels. J .Vety. Pharmacolo and Toxicol. 3: 90-93. Singhal, L.K., Singh, B.P. and Chauhan, R.S. (2001). Fenvalerate induced stress and its effect on cellmediated immunity ion chickens. Int. Symp. End Tech. Sec. CIGR Anim. Welf. Consider. Siroki, O., Institoris, L., Tatar, E. and Desi, I. (1994). Immunotoxicological investigationn of SCMF, a new pyrethroid pesticides in mice. Human and Exp. Toxicol. 13: 337-343. Suhash, P. (2004). Studied sub acute toxicological effect of cypermethrin in wistar male and female rats. Ph.D Thesis submitted to University of Agricultural sciences, Bangalore. Synder, D .B., Marquadt, W.W., Mallinson, E.T., Savage, P.K. and Allen, D.C. (1984). Rapid serological profiling by enzyme linked immunosorbent assay. III. Simultaneous measurement of antibody titre to infectious bronchitis, infectious bursal disease and New castle disease in single serum dilution. Avian Diseases. 28: 12 -24.
Research Article
DEVELOPMENT OF THERAPEUTIC MODULE FOR JATROPHA CURCAS SEED AND SEED OIL TOXICITY IN GOATS
AMIT SHUKLA1 AND S.P. SINGH2
1
Ph.D.scholar IVRI, Izatnagar, 2Prof & Head, Department of Veterinary Pharmacology and Toxicology, C.V.A.Sc. Pantnagar, Distt-U.S. Nagar, Uttarakhand, India. 1 Corresponding author: princu.dr@gmail.com
ABSTRACT
The present study was designed to evaluate acute toxicity of Jatropha curcas seed and seed oil and to develop its therapeutic module in goats. Acute toxicity study of Jatropha curcas seed and seed oil was evaluated using 15 goats equally and randomly divided in five groups. Group I served as control. Group II and IV were given single oral dose of Jatropha curcas seed @ 4 seed /kg b.wt. and seed oil @ 4ml/kg b.wt., respectively, followed by the therapeutic module comprising of sodium thiosulphate ( 0.1565 gm/kg ) and glutathione ( 0.5 mg/kg ) in for 14 days. Groups III and V were given only single oral dose of seed and seed oil at the similar doses, respectively. Mild to moderate diarrhoea, dullness, depression and letharginess were observed in groups III and V, whereas mild clinical signs were observed in few cases in treatment groups II and IV. A significant (P<0.05) decline in Hb, PCV, TEC ,TLC, total serum protein, albumin and globulin values in groups without treatment and an increase in serum creatinine, serum urea, cholesterol, AST , ALT and ALP were observed in all groups , however, the 14 days treatment with glutathione and sodium thiosulphate ameliorated hemato-biochemical parameters indicating the protective efficacy of therapeutic module in seed and seed oil intoxicated goats. Key words: Acute toxicity, glutathione, goats, Jatropha curcas, sod, thiosulphate.
INTRODUCTION Jatropha curcas Linn (Greek:-iatros-doctor, trophefood), known as Ratanjyot, Barbados nut, Purgeer boontjie etc. and popularly called diesel plant, is a member of the Euphorbiaceae family. Jatropha seed kernel contains 4060% oil with a fatty acid composition similar to that of oils used for human nutrition. However, the oil contains irritant phorbol esters which are the complex mixture of esters of tetracyclic diterpene phorbol, responsible for purgative , skin irritant and tumorogenic actions. The seeds from J. curcas have been reported to produce toxicity due to the toxin curcin ,a ricin like toxalbumin characterized by burning and pain in mouth and throat, vomiting, delirium, decrease of visual capacity and increased pulse with a high mortality rate in rodents and domestic animals (Singh et al., 2010). A number of efforts have been made to explore the therapy of Jatropha toxicity in man and animals without satisfactory outcome. In view of this fact, this study was undertaken to develop a therapeutic module for the treatment of acute toxicity of seed and seed oil in goats. MATERIALS AND METHODS Experimental design The Jatropha seed and seed oil were collected from Medicinal Plant Research and Developmental Centre (MRDC), G.B.P.U.A & T, Pantnagar. All the chemicals required for this study were procured from Hi Media. 15 goats of 16 to 18 months old weighing 28-32kg, were divided randomly and equally into five groups and experiment was designed as mentioned in Table1. Hemato-biochemical parameters
4.0 ml of blood will be collected from each goat in clean heparinized microcentrifuge tube and hematological parameters such as packed cell volume, hemoglobin, total erythrocyte count and total leucocytes count were estimated immediately after the collection of blood samples. 0.1N-HCl was used for estimating the blood hemoglobin concentration while Hayems RBC diluting fluid and Thomass WBC diluting fluid were used for TEC and TLC estimation (Jain, 1986). The serum total proteins albumin, globulin, total cholesterol, creatinine and urea were estimated by using ERBA diagnostic kits. Serum enzymes viz. aspartate aminotransferase, alanine aminotransferase (Moss and Henderson, 1994) and alkaline phosphatase were estimated by ERBA diagnostics kits. RESULTS Mild to moderate diarrhoea was observed in goats within 7 days post exposure in the group IV and V whereas it occurred after 10 days post administration of seeds in treatment groups II and III. Goats appeared dull and depressed with reduction in the appetite in all groups 5 days post exposure to seed and seed oil without any mortality. The effect of single dose of Jatropha curcas seed and seed oil with or without treatment on Hb, PCV,TEC and TLC values are presented in the Table2. There was a significantly (P<0.05) higher value of Hb, PCV, TEC and TLC in both of the seed and seed oil plus treatment group II and IV in comparison to seed and seed oil without treatment groups III and V at 14th day of study. As depicted in Table 3, there was no significant
76
Therapeutic module for Jatropha toxicity in goats
Effect on haematological parameters following single oral administration of Jatropha seed and seed oil with and without treatment for 14days in goats (Mean valueS.E. ,n=3).
Therapeutic module for Jatropha curcas toxicity in goats (n=3). Groups I II III IV V Treatment Control Seed (T) Treatment* Seed (WT) Seed oil (T) Treatment* Seed oil (WT) Dose -_ 4 seed /kg 4 seed /kg 4ml/kg 4ml/kg Day of dosing _ 1 st 2-13 1 st 1 st 2-13 1st day
DISCUSSION Earlier appearance of the clinical signs in seed oil than seed intoxicated goats in acute study indicated that seed oil is more toxic than seed . This might be due to the higher concentration of phorbol and diterpenes in Jatropha curcas seed oil . Seed and seed oil contains phorbol esters known for its purgative effect (Gandhi et al., 1995) that occurs due to its stimulating effect on kinase-C enhancing intracellular signal transduction process (Makkar and Becker, 1997). Jatropha seed also contain various other toxic principles i.e. curcin, phorbol esters, tannins etc (Ahmad and Adam, 1979; Lin et al., 2010) which cause direct GIT
Hb (g/dl)
TABLES 2:
GROUPS/ DAYS
IV
III
II
8.499 0.016 8.49 0.026 8.46 0.106 8.48 0.116 8.46 0.036 I
8.46 0.021A 7.44 0.030a bAB 7.68 0.017 a b ABG 7.48 0.017 abABGD 7.65 0.049a b ABCD
8.44 0.015A 7.03 0.003 abAB 6.47 0.002 a b ABG 7.16 0.024abABGD 6.74 0.001ab ABCD
change on total serum proteins after 7 and 14 days exposure in all treated groups as compare to the control. The total protein values in seed and seed oil with treatment groups II and IV were non significantly higher than the seed and seed oil without treatment group III and V after 14 days. Albumin level in seed oil plus treatment group was non significantly higher than the seed oil group without treatment. A significant increase in serum urea and creatinine levels in groups without treatment as compared to control after 14 day (Table 3), however, the serum urea levels in seed and seed oil plus treatment groups were significantly (P<0.05) lower than without treatment groups at both 7 and 14 day interval. A significant increase was also observed blood cholesterol on 14th day in all treated groups as compare to the 0 day reading, however, a significant decline (P<0.05) in serum cholesterol value in seed and seed oil plus treatment groups (II and IV) was observed on 7th day. As shown in Table 3, a significant (P<0.05) decline in the activity of the AST and ALT in treatment groups (II and IV) in comparison to the without treatment groups (III and V) was observed on 7th day interval indicating the efficacy of the therapeutic module. A significant (P<0.05) increase was observed in all the groups as compare to control at both 7 and 14 days interval, however, activity was less (P<0.05) in treatment groups (II and IV) in comparison to without treatment groups (III and V) on both 7th and 14th day interval.
TEC x106/ l
14
25.33 0.24 25.41 0.046 25.46 0.071 25.22 0.069 25.54 0.174
25.23 25.40 0.05A 0.012 23.73 21.35 0.23a bAB 0.156 abAB 22.37 18.66 0.028 a b ABG 0.012 a b ABG 21.69 20.21 0.039 abABGD 0.19 abABGD 25.15 20.66 0.182 a b ABCD 0.172 ab ABCD
PCV (%)
14
10.77 0.652 10.69 0.456a 10.69 0.344 a 10.65 0.756 a 10.67 0.089 a
10.72 10.73 0.567A 0.554 A 11.07 10.06 0.562 ab AB 0.478 ab AB 11.07 10.06 0.349 ab AB 0.978 ab ABC 10.58 8.84 0.934 ab ABD 0.076 ab ABCD 10.44 8.73 0.075 ab ABD 0.128 ab ABCD
* Treatment was comprised of sodium thiosulphate @ 0.1565gm/kg bw IV route and glutathione @ 0.5 mg/kg bw via IM route. T=with treatment; WT= without treatment.
Treatment with Glutathione @0.5 mg/kg BW and Sodium thio sulphate @ 0.1565gm /kg BW Mean bearing common superscript with small letters differ significantly (P<0.05)when compared vertically with in the same column & mean value bearing capital alphabets differ significantly(P<0.05)when compared horizontally with in the same row.
TLC (x103/ l)
14
10.43 0.015 10.83 0.436 10.42 0.024 10.34 0.546 10.33 0.239
10.41 0.038A 9.92 0.231a bAB 9.47 0.049 a b ABG 8.48 0.534 abABGD 9.46 0.317 a b ABD
10.40 0.054A 9.44 0.156 abAB 8.65 0.078 a b ABG 10.40 0.576 abBGD 8.20 0.412 ab ABCD
TABLE1:
14
Shukla and Singh
TABLE 3: Effect on different biochemical parameters following daily oral administration of Jatropha seed and seed oil with and without treatment for 14days in goats (Mean S.E. , n=3). Parameter/groups I II III IV V
Total serum proteins (TSP, g/dl) 0 days 7 14 Albumin (g/dl) 0 7 14 Globulin (g/dl ) 0 7 14 Urea (mg/dl)
0 7 14 Creatinine (mg/dl) 0 7 14 Cholestrol (mg/dl) 0 7 14 Aspartate aminotransferase (AST, U/L) 0 7 14 Alanine aminotransferase (AST, U/L) 0 7 14 Alkaline phosphatase (ALP , U/L) 0 7 14
6.830.571 6.810.475 6.790.605 3.250.721 3.810.925 3.170.126 20.230.088 20.200.024A 20.240.05 A
6.980.834 6.490.767 8.650.417 2.600.324 2.700.627 3.200.417
7.310.343 7.240.178 7.350.414 2.640.343 2.700.178 3.200.414
7.530.458 a 5.940.057 a b 7.081.946 b 2.320.238 2.820.451 2.940.236
7.530.645 5.610.648 6.731.582 3.030.945 2.450.048 3.291.212 21.460.617 a 47.260.581 a A,BCD 48.290.716 a A BD
21.72.804a 24.661.898a 25.230.829 a 33.400.692 a A,B 42.942.416 a A,BC 31.150.421 a ACD 33.601.191A B 46.731.421A BC 33.021.231 ACD
20.230.088 20.200.024A 20.240.05 A 1.200.057 1.180.005 1.1700.005 66.320.761 66.760.612 66.730.634
21.72.804a 24.661.898a 25.230.829 a a A,B a A,BC 33.400.692 42.942.416 31.150.421 a ACD A B A BC 33.601.191 46.731.421 33.021.231 ACD 0.7660.145a 1.3000.057b 4.4301.457a,b 68.850.432 a 80.730.03 ab 96.871.20 ab 1.0660.133a 1.3660.088b 6.3761.214a,b 66.961.01 a 88.140.379 ab 100.171.981 ab 1.2330.088 1.2000.057 4.4231.894 66.990.896 a 84.871.604 ab 94.042.261 ab
21.460.617 a 47.260.581 a A,BCD 48.290.716 a A BD 1.0660.185 a 1.1660.145 b 5.1851.782 a, b 66.691.32 a 89.120.438 ab 100.391.296 ab
31.8260.298 31.790.338A 32.140.008A
33.5062.589a 30.050.723 a 41.243.805 a 77.603.594 a AB 93.182.710 a ABC 61.104.066 ABC 98.2205.831 a 102.5710.85 a 72.349.663 a
32.061.926 a 70.547.475 a AC 69.055.981 a
16.760.088 16.650.129 16.380.07
16.604.96 25.487.86 42.926.78
16.931.814 37.667.581 49.7212.232
16.931.059 42.506.944 54.464.364
16.180.43 34.066.155 51.6412.322
55.700.657 55.620.405A 55.440.605 A
56.360.184 a 55.340.163a 56.010.768 a 56.750.175 a 64.530.757 a b AB 69.920.078b ABC 56.450.057 abBCD 81.220.048 a b ABCD 68.181.457 a,bB 79.701.434a,bBC 66.181.878 abBCD 91.221.582 a, bBCD
Mean bearing common superscript with small letters differ significantly (P<0.05) when compared vertically within the same column & mean value bearing capital alphabets differ significantly(P<0.05)when compared horizontally within the same row of the same parameter.
irritation and cellular toxicity (Lin et al., 2003) which might be responsible for toxicity manifestation such as hepatotoxicity and nephrotoxicity in goats in this study. Low level of clinical manifestation in groups treated with glutathione reveals the therapeutic potential of the therapeutic module. Glutathione plays a vital role in neutralizing the oxidative radical and remove the electrophiles ,thus diminishes their toxic effect on the cells. Hematological parameters such as Hb, PCV, TEC
78
and TLC decreased in seed and seed oil intoxicated groups in acute toxicity. Fall in Hb, PCV, TEC and TLC might be due to hemolytic activity of curcin. Furthermore, damage to the GIT could also have resulted in poor digestion and absorption of nutrients required for erythropoiesis (Chivandi et al., 2006). Leucopenia might be correlated with the stress caused by anti nutritional factors as tannins, saponins, phytates etc present in the Jatropha meal (Feldman et al., 2000). Reduction in the total protein, albumin and globulin
Therapeutic module for Jatropha toxicity in goats
were observed in the seed and seed oil intoxicated groups in acute toxicity study of Jatropha seed and seed oil. Alteration in protein profile indicates hepatorenal dysfunction and gastroenteritis which might have resulted due to presence of curcin and phytates in Jatropha seed and seed oil. Similar findings were reported by following Jatropha intoxication in rats (Awasthy et al., 2010; Abdel safy et al., 2011). There was an increase in urea and serum creatinine level in seed and seed oil intoxicated groups indicating the nephrotoxic potential of Jatropha as urea and creatinine act as indicators of the kidney damage. In addition, increased hepatic urea production from amino acid metabolism could also be responsible for an increase in urea concentration in the serum .Treatment significantly reduced the level of urea and creatinine in both seed and seed oil with treatment groups II and IV. Our results are in agreement with the findings of Gadir et al. (2003) in goats following oral administration of Jatropha seed at the dose of 1 and 0.25g/kg/day and Awasthy et al. (2010) in rats. A significant increase in serum cholesterol level was observed in both seed and seed oil intoxicated groups in acute toxicity study . Liver is the major site of cholesterol synthesis and metabolism. Hepatic cholesterol homeostasis is maintained by equilibrium between the activities of the hydroxyl methyl glutaryl Coenzyme A reductase and acyl coenzyme A cholesterol acyl transferase . The rise in activity of serum AST , ALT and ALP enzymes could be attributed to damaged structural and cellular integrity of the hepatocytes and due to centrilobular necrosis which in turn increased the leakage of the liver specific enzymes . Being cytoplasmic in location, these enzymes are released into systemic blood circulation after cellular damage of hepatocytes (Ahmed and Khater, 2001). It is concluded from the above study that the seed oil was more toxic than seed and the therapeutic module comprising sodium thiosulphate and glutathione revealed protective efficacy against their toxicity in goats. ACKNOWLEDGEMENT The LSRB, DRDO, NEW Delhi duly acknowledged for financial assistance for conducting this study. REFERENCES Abdel-safy, S., Nasr, S. , Abdel, R. and Habeeb, S. (2011). Effect of various levels of dietary Jatropha curcas seed meal on rabbits infested by the adult ticks of Hyalomma marginatum marginatum I. Animal performance, anti-tick feeding and haemogram. Trop. Anim. Hlth. Prodn. 43(2): 347 Ahmed, M.B. and Khater, M.R. (2001).The evaluation of the protective potential of Ambrosia maritima extract on acetaminophen-induced liver damage. J. Ethnopharmacol. 75: 69-174.
Ahmed, O.M. and Adam, S.E. (1979). Effects of Jatropha curcas on calves. Vet. Pathol. 16: 476-82. Awasthy, V., Vadlamudi, V., Koley, K., Awasthy, B. and Singh, P.. 2011. Biochemical changes after shortterm oral exposure of Jatropha curcas seeds in wistar rats. Toxicology international. 17 (2): 6770. Chivandi, E., Erlwanger, K.H., Makuza, S.M., Read, J.S. and Mtimuni, J.P. (2006). Effects of dietary Jatropha curcas meal on percent packed cell volume, serum glucose, cholesterol and triglyceride concentration and alpha-amylase activity of weaned fattening pigs. Res. J. Anim. Vet. Sci. 1: 1824. Feldman, B., Joseph, G., Zinkl, J. and Jain, N.C. (2000). Schalms Veterinary Hematology, 5th ed., Lippincott Williams & Wilkins Co. Philadelphia. Gadir, A., Onsa, T.O., Ali, W.E.M., El Badwi, S.M. and Adam, S.E.I. (2003). Comparative toxicity of Croton macrostachys, Jatropha curcas, Piper abyssinica seeds in Nubian goats. Small Rum. Res. 48: 6167. Gandhi, V., Cherian, K. and Mulky, M. (1995). Toxicological studies on Ratanjyot oil. Food Chem. Toxicol. 33: 3942. Jain, N. (1986). Schalms Veterinary Haematology. 4th Ed. Philadeiphia, Lea and Febringer. Lin, J., Yan, F., Tang, L., and Chen, F. (2003). Antitumor effects of curcin from seeds of Jatropha curcas. Acta Pharmacol. Sinica. 24: 241246. Lin, J., Zhou, X., Wang, J., Jiang, P. and Tang, K. (2010). Purification and characterization of curcin, a toxic lectin from the seed of Jatropha curcas. Prep. Biochem. Biotechnol. 40(2): 107-18. Makkar, H.P.S. and Becker, K. (1997). Potential of Jatropha curcas seed meal as a protein supplement to livestock feed, constraints to its utilization and possible strategies to overcome constraints to its utilization. In Proceedings Jatropha 1997: International symposium on Biofuels and Industrial Products from Jatropha curcas and other tropical oil seed plants, February 2327, Managua, Mexico Moss, D. and Henderson, A. (1994). Clinical enzymology, In Tietz Textbook of clinical chemistry, 3rd ed., C.A. Burtis and E.R. Ashwood, Eds. W.B. Saunders, Philadelphia. 617-721. Singh, R., Singh, D. and Mahendrakar, A. (2010). Jatropha poisoning in children. Med. J. Armed Forces India. 66: 8081.

Research Article
PROTECTIVE EFFECT OF TRIANTHEMA PORTULACASTRUM ON CADMIUM INDUCED TOXICITY IN RATS

Y. PAVAN KUMAR1, K. ADILAXMAMMA2, U. VENKATESWARLU1, T.S. CHANDRASEKHARA RAO3 AND M. ALPHA RAJ2
Department of Veterinary Pharmacology & Toxicology, College of Veterinary Science, Tirupati - 517 502 Department of Veterinary Pharmacology & Toxicology, College of Veterinary Science, Proddatur - 516 360; 3 Dean of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati - 517 502. Corresponding Author:E-mail- adilaxmamma9301@yahoo.co.in
1
ABSTRACT The protective role of alcoholic extract of T. portulacastrum was studied in cadmium induced toxicity in rats. Fifty male albino Wister rats weighing (160-180) g were divided into five groups of ten each. Group I served as control; Groups II, III, IV and V received CdCl2 @ 8 mg/kg b. wt. per orally from Day 1 to Day 15. Groups III, IV and V also received alcoholic extract of T. portulacastrum @ 200 mg/kg b. wt, Nefroliv @ 100mg/kg b. wt. and Vitamin E @ 50 mg/kg b. wt. respectively by intra gastric intubation from Day 1 to Day 30. The serum biochemical parameters (ALT, AST, BUN, Creatinine, GSH), erythrocyte antioxidant enzymes (GPx, GR, CAT) were monitored on Day 15 and on Day 30. At the end of Day 30 animals were sacrificed and liver and kidney samples were collected for estimation of lipid peroxidation, superoxide dismutase and histopathological examination. Administration of CdCl2 resulted in increase of serum enzymes (ALT, AST, BUN, Creatinine) and lipid peroxidation in liver and kidney. The erythrocyte antioxidant enzymes (GPx, GR, CAT), SOD, GSH were decreased. Treatment with alcoholic extract of T. portulacastrum, Nefroliv and vitamin E significantly ameliorated (P<0.01) toxic effects of CdCl2 by restoring biochemical and histopathological changes to normal. It is concluded that alcoholic extract of T. portulacastrum exhibited protective property in CdCl2 induced toxicity, which was comparable to Nefroliv and Vitamin E. Key words: Cadmium, nephrotoxicity, rats, trianthema portulacastrum.
INTRODUCTION Cadmium, a heavy metal well known to be highly toxic to both human and animals, distributed widely in the environment and is responsible for multiple pathologies (Hall et al., 2012). The metal is widely distributed both by natural and anthropogenic sources, of which anthropogenic sources adds 3-10 time more to the contamination (Patra et al., 2011). Some of the toxic effects of cadmium exposure include testicular atrophy, renal dysfunction, hepatic damage, hypertension, central nervous system injury and anemia including high blood pressure, brain damage, diarrhea, infertility, immune suppression, and development of cancer including generation of reactive oxygen species (ROS) (Bagachi et al., 2000). Chronic exposure to cadmium (Cd) causes hepatotoxicity and nephrotoxicity (Sarkar et al., 1995) through accumulation mainly in the liver and kidney. Further, the role of cadmium in carcinogenesis, inhibition of DNA repair and apoptosis was also reported (Hanahan and Weinberg, 2000). Cadmium has strong affinity for biological structures containing -SH groups with indirect effects on multiple cellular and enzymatic systems resulting in poor health (Helbig et al., 2008). Indigenous medicinal plant Trianthema portulacastrum Linn. (Family: Aizoaceae) has been used in the treatment of edema of liver and spleen (Stohs et al., 2000) and is also reported to have antioxidant property
80
(Kumar et al., 2005), therefore, it was evaluated for its potential to mitigate the oxidative stress, hepatotoxicity and nephrotoxicity induced by cadmium in this study. MATERIALS AND METHODS Experimental animals Male albino rats of Wistar strain weighing 160 180 g were obtained from the department of Laboratory Animal Medicine, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram milk colony, Madhavaram, Chennai. and acclimatized for 15 days before the conduct of the experiment. Approval was obtained from IAEC for conducting this study. Drugs and chemicals Cadmium chloride was procured from SRL Pvt. Ltd, Mumbai, India. Nefroliv was procured from Indian Herbs Research and Supply Co. Ltd. Darra Shivpuri, Saharanpur, UP, India. All chemicals were of analytical grade (AR) and procured from Qualigens , S R L Pvt. Ltd and S D Fine Chemicals Ltd. Plant materials and preparation of extract Whole plant of T. portulacastrum was collected from the local market and surrounding areas of Tirupati, Andhra Pradesh, India and authenticated by the Department of Botany, S. V. University, Tirupati. Whole plant of T. portulacastrum was dried in
Cadimum toxicity in rats
Groups
I II III IV V
RESULTS AND DISCUSSION The serum enzymes AST and ALT were reported to serve as biomarker indices of the extent of tissue damage, especially liver, kidney and heart (Kaneko et al., 2008). The concentrations of AST and ALT in the plasma of rats fed with CdCl2 were significantly (P<0.01) elevated indicating cadmium related injury to the liver, kidney and heart (Hu et al., 1991). The levels of BUN and creatinine in the plasma of rats are indicators for kidney functions (Hu et al., 1991). In the present study, the serum creatinine and BUN levels were significantly increased in CdCl2 control group as compared to the remaining groups, suggesting impairment of renal function (Hwang et al., 2001). The decreased serum values of ALT and AST in amelioration group suggest the protective role of alcoholic extract of T. portulacastrum on liver (Mandal et al., 1997) and decreased
Values are Mean + S.E. (n=6) Two way ANOVA followed by Duncans multiple comparison test using SPSS 15.0 v Values with different superscripts are significantly different (p<0.01) Lowercase alphabets for vertical comparison; Uppercase alphabets for horizontal comparison.
TABLE 1: Effect of T.Portulacastrum on bio chemicals paratmeters in cadmium induced toxicity in rats.
AST (U/L)
Day 30
ALT (U/L)
Day 15
30.83 62.83 33.33 44.33 32.67
+ 0.82 + 1.51dA + 1.12bA + 1.87cA + 1.91abA
aA
30.67 57.67 32.33 35.67 30.17
+ + + + +
0.61 1.32cB 0.46aA 1.32bB 0.82aB
aA
114.50 + 1.72 166.17 + 1.91eA 121.67 + 2.44cA 137.50 + 1.76dA 105.17 + 2.51aA
shade. Later it was powdered and extracted (1.5 kg) with 6 L of 70 % alcohol in a soxhlet extractor for 18-20 hours. The extract was distilled and concentrated to dryness under reduced pressure and controlled temperature (40-50oc) and finally freeze-dried. The ethanolic extract yielded a weight of 150 g (10 % w/w). Experimental design Fifty rats were randomly divided into five groups, each containing 10 rats. Group I received 0.5 % carboxy methyl cellulose (CMC) p.o. for 30 days as the vehicle. Group II to V received aquous solution of CdCl2 @ 8 mg/ kg b.wt. /day p.o. for 15 days. Feed and water was withdrawn 6 hrs before the administration of drug. Group III was given alcoholic extract of T. portulacastrum @ 200 mg/kg b.wt./day, p.o., from day 1 to day 30. Similarly Nefroliv in 0.5 % CMC and Vitamin E (in olive oil) were administered to groups IV and V @100 mg/kg b. wt. and 50 mg/kg b.wt., respectively, from day 1 to day 30. Blood was collected from retro-orbital sinus under ether anesthesia on Day 15 and Day 30 in both EDTA added and non-EDTA added vials. Serum was used for the analysis of biochemical parameters like ALT, AST, BUN, Creatinine (Span Diagnostic Ltd) and total glutathione (Moron et al., 1979) where as erythrocytes were used for the analysis of oxidative parameters, viz. glutathione peroxidase (GPx) (Paglia and Valentine, 1967), glutathione reductase (GR) (Raghuramulu, 1983), catalase (CAT) (Aebi, 1984). At the end of experiment animals were euthanized and liver and kidney were collected for histopathology (Singh and Sulochana, 1997) and assay of superoxide dismutase (SOD) (Marklund and Marklund, 1974) and lipid peroxidation (LPO) (Yagi and Rastogi, 1979). The data was subjected to statistical analysis by applying two way ANOVA using SPSS 15.0 v software. Significant differences between groups and days were tested using Duncans multiple comparison test and significance was set at P<0.05.
35.44 + 1.01bcA 17.11 + 1.23aB 37.33 + 1.88cB 32.24 + 0.89bB 37.15 + 1.85cB
levels of BUN and creatinine suggest nephroprotection. The nephroprotection could be attributed to its diuretic activity of the constituents namely, potassium nitrate and punarnavine (Khan, 2003). The antioxidant defense profile was studied in order to assess the extent of cadmium induced free radical damage in the biological system. Cadmium was reported to inhibit the activities of most of the anti-oxidant enzymes including thiols (Helbig et al., 2008), leading to the intra cellular accumulation of reactive oxygen species with subsequent damage to liver and kidney. Cadmium depletes glutathione and protein bound sulfhydryl groups resulting in enhanced production of reactive oxygen species such as superoxide ions, hydroxyl radicals and hydrogen peroxides. These reactive oxygen species result in increased lipid peroxidation (Stohs et al., 2000). The peroxidative damage to the cell membrane may cause injury to cellular components due to the interaction of metal ions with the cell organelles (Sarkar et al., 1995). The results of GSH, GPx, GR and CAT activities were shown in Table 1 and 2, which showed a significantly reduced (P<0.01) in
GSH (mg/100ml) Creatinine (mg/dl) BUN (mg/dl)
Day 30 Day 15 Day 30 Day 15 Day 30 Day 15 Day 30 Day 15
bA
114.50 + 1.29 162.33 + 2.17eB 120.50 + 1.32cA 125.83 + 1.71dB 99.00 + 1.60aB
bA
20.31 45.54 22.41 25.44 27.51
+ 0.45 + 1.13eA + 0.43bA + 0.48cA + 0.34dA
aA
20.13 43.14 20.32 22.06 25.52
+ 0.35 + 0.93dB + 0.35aB + 0.67bB + 0.42cB
aA
0.638 0.808 0.625 0.703 0.652
+ 0.015 + 0.019cA + 0.013aA + 0.019bA + 0.011aA
aA
0.632 0.793 0.600 0.615 0.590
+ 0.007 35.98 + 0.021cA 14.83 + 0.013abB 33.73 + 0.020abB 29.17 + 0.009aB 33.58
bA
+ 1.20 + 1.09aA + 0.95cA + 1.08bA + 1.13cA
cA
Kumar et al.
TABLE 2: Effect of T. Portulacastrum on erythrocyte antioxidant enzymes in cadmium induced toxicity in rats Groups Day 15 I II III IV V 25.14 14.57 23.21 20.59 21.13 + + + + + 0.86 0.30aA 0.60cA 0.51bA 0.81bA
dA
GPx (U/ml) Day 30 25.00 15.80 24.78 23.92 24.81 + + + + + 0.96 0.26aB 0.66bB 0.29bB 0.57bB
bA
GR (U/ml) Day 15 21.28 + 0.33 7.14 + 0.11aA 17.21 + 0.18cA 13.39 + 0.19bA 21.70 + 0.18dA
dA
CAT (KU/g Hb) Day 30 Day 15

cA
Day 30
dA
20.91 + 0.91 9.10 + 0.41aB 19.71 + 0.30cB 17.97 + 0.69bB 23.72 + 0.56dB
23.25 14.89 21.14 19.37 21.99
+ + + + +
0.98 0.51aA 0.82cA 0.58bA 0.80cdA
22.67 14.83 22.89 21.62 23.12
+ + + + +
0.51bA 0.46aA 0.63bB 0.67bB 0.52bB
Values are Mean + S.E. (n=6) Two way ANOVA followed by Duncans multiple comparison test using SPSS 15.0 v Values with different superscripts are significantly different (p<0.01) Lowercase alphabets for vertical comparison; Uppercase alphabets for horizontal comparison. TABLE 3: Effect of T. Portulacastrum on LPO and SOD in Liver and Kidney in Cd induced toxicity in rats Groups I II III IV V LPO (n mol of MDA formed/g tissue) Day 30 (Liver) 34.60 87.14 32.55 32.71 32.53 + + + + + 1.18a 1.80b 0.49a 0.93a 0.79a Day 30 (Kidney) 32.62 + 0.51a 44.15 + 1.35b 32.01 + 0.73a 33.74 + 0.90a 32.52+ 0.52a SOD (units/mg protein) Day 30 (liver) 6.717 + 0.154bc 3.897 + 0.171a 6.607 + 0.131bc 6.342 + 0.180b 6.925 + 0.115c Day 30 (kidney) 5.372 + 0.130c 2.612 + 0.073a 5.040 + 0.075bc 4.997 + 0.057b 5.788 + 0.112d
Values are Mean + S.E. (n=6) Two way ANOVA followed by Duncans multiple comparison test using SPSS 15.0 v Values with different superscripts are significantly different (p<0.01) Lowercase alphabets for vertical com parison; Uppercase alphabets for horizontal comparison.
cadmium treated group II when compared to control group. GSH is a tri-peptide, non enzymatic antioxidant involved in the detoxification of heavy metals as well as reactive metabolites of xenobiotics by forming conjugates. Cadmium was reported to bind exclusively to thiol groups thereby decreasing the GSH levels and interfering with the antioxidant activity (Helbig et al., 2008). Cadmium induced oxidative damage has been demonstrated by the increase in lipid peroxidation and inhibition of enzymes required to prevent such oxidative damage ( Patra et al., 2011 and Jurczuk et al., 2004) viz., GSH, SOD and CAT. These changes seem to be due to the generation of ROS ( Wang et al., 2004 and Gobe and Crane, 2010). GPx, a selenium dependent antioxidant enzyme, removes both H2O2 and lipid peroxides by catalyzing the conversion of lipid hydroperoxide to hydroxy acids in the presence of GSH. Decreased GPx activity observed in this study could be due to attributed to the exhaustion or inactivation of the enzyme due to excessive free radicals (Prasad et al., 2003). The oxidative stress induced by cadmium, favours lipid peroxidation and further leads to the depletion of GSH in tissues (El-Maraghy et al., 2001) and decreased activities of SOD and CAT (Ognjanovic et al., 1995). With the supplementation of the alcoholic extract of T.portulacastrum, Nefroliv and Vitamin E, the antioxidant constituents were significantly increased (P<0.01) on both Day 15 and Day 30 when compared to II group suggesting antioxidant role of the plant. LPO and SOD concentrations
82
in liver and kidney were assayed and tabulated in Table 3. The concentration of MDA was assayed in liver and kidney on Day 30. The MDA concentration was significantly (P<0.01) increased in liver and kidney suggesting the oxidative damage to lipids in cell membranes. In liver and kidney, SOD concentration was decreased significantly (P<0.01) when compared to control group suggesting an oxidative damage to the organs. Treatment with alcoholic extract of T. portulacastrum, Nefroliv and Vitamin E restored the values of LPO and SOD to normal. The decreased LPO levels might be due to the active flavonoids and other ingredients of Nefroliv (Chatterjee and Agarwala, 2003). Decreased levels of LPO suggest the antioxidative property of alcoholic extract of T. portulacastrum. SOD constitutes an important link in the biological defense mechanism through disposition of endogenous cytotoxic superoxide radicals, which are deleterious to PUFA and structural proteins of plasma membrane. SOD catalytically scavenges the superoxide radicals and thus renders cytoprotection against free radical induced damage. It is reported that accumulated cadmium in the tissues, interacts with metal moieties of SOD (Cu, Zn, Mn) and inhibits the enzyme activity (Nagaraj et al., 2000). The increased concentration of GR, GPx, CAT and SOD in rats that received alcoholic extract of T. portulacastrum is by virtue of its antioxidative properties that acted on ROS liberated during the oxidative stress induced by the CdCl2 (Moron et al., 1979). Vitamin E protects critical
Cadimum toxicity in rats
cellular structures against damage caused by free oxygen radicals and reactive products of LPO. Vitamin E (tocopherol) inhibits peroxidation of membrane lipids by scavenging lipid peroxyl radicals, as a consequence of which it is converted into a tocopheroxyl radical (Arita et al., 1998). The gross lesions in kidney of cadmium treated group II rats were areas of congestion and paleness. Following treatment, the above changes were mild to normal suggesting the protective role of Trianthema portulacastrum, Vitamin E and Nefroliv in cadmium induced nephrotoxicity. Degenerative changes were observed in the histopathological examination of the liver and kidney of group II rats (Brzoska et al., 2003). The histopathological sections of liver and kidney in groups III, IV and V showed very mild areas of congestion with normal histological appearance in liver and kidney suggesting the role of Trianthema portulacastrum, Vitamin E and Nefroliv in preserving functional integrity of membranes by preventing the alteration of their phospholipid structure by free radical induced cadmium toxicity. Treatment with T. portulacastrum, Vitamin E and Nefroliv countered the toxic effect of cadmium suggesting nephroprotective role. However, the possible mechanism by which alcoholic extract of T. portulacastrum exerts nephroprotection could be attributed to its free radical scavenging property along with its diuretic property (Kumar et al., 2005). It is concluded from this study that CdCl2 produced significant nephrotoxicity as evinced by increased BUN and creatinine levels along with degenerative changes in the kidneys due to free radical generation i.e., oxidative stress which is evident by the increase in LPO and decrease in SOD, CAT, GSH, GPx and GR. Coadministration of alcoholic extract of T. portulacastrum along with CdCl2 prevented both functional and histological renal changes induced by CdCl2 in rats. ACKNOWLEDGEMENTS The authors would like to thank College of Veterinary Science, Tirupati and Sri Venkateswara Veterinary University for providing the infrastructure and necessary financial support for carrying out this research. REFERENCES Aebi, H. (1984). Catalase invitro: In: Packer, L. editor Methods in Enzymology. New York Academic Press. 105: 21-26. Arita, M., Sato, Y.,Arai, H. and Inoue, K. (1998). Binding of -tocopheryl quinone, an oxidized form of tocopheryl, to glutathione S- transferase in the liver cytosol. FEBS Letters. 436:424-426. Bagchi, D., Bagchi, M., Stohs, S. J., Ray, S. D., Kuszynski, C. A. and Pruess, H. G. (2000). Free radicals and grape seed proanthocyanidin
extract: importance in human health and disease prevention. Toxicol. 148: 187- 97. Brzoska, M. M., Jakoniuk, M., Marcinkiewicz, P. and Sawicki, B. (2003). Liver and Kidney function and histology in rats exposed to cadmium and ethanol. Alcohol and Alcoholism. 38: 2-10. Chatterjee, S. and Agrawala, S. K. (2003). Effect of ELKP1 on experimental diabetic Nephropathy. Phytomedicine, 4: 43-49. El-Maraghy, Gad, M. Z., Fahim, A. T. and Hamdy, M. A. (2001). Effect of cadmium and aluminium on the antioxidant status and lipid peroxidation in rat tissues. J. Biochem. Mol. Toxicol. 15: 207214. Gobe, G. and Crane, D. (2010). Mitochondria, ROS and Cadmium toxicity in kidney. Toxicol. Letters. 198:49-55. Hall., J. Haas, K. and Freedman, J.C. (2012). Role of MTL1, MTL-2 and CDR-1 in mediating cadmium sensitivity in C.elegans. Toxicologcal Science, 128: 418-26. Hanahan, D. and Weinberg, R.A. (2000). The hall marks of cancer. Cell. 100:57-70. Helbig, K., Grosse, C. and Nies, D.H. (2008). Cadmium toxicity in glutathione mutants of Escherichia coli. J. Bacteriol. 190:5439-54. Hu, C.C., Yem, C.J., Jang, M. L., Liu, C.B., Chen, W.K. and Chung, C. (1991). Cadmium induced serum biochemical changes in subchronically exposed rats. Chung Shah Medical Journal, 2: 97-101. Hwang, D. F. and Wang, L.C. (2001). Effect of taurine on toxicity of cadmium in rats. Toxicology, 167: 173180. Jurczuk, M., Brzoska, M.M., Jakoniuk, J.M., Sidorczuk, M.G. and Karpinska, E.K. (2004). Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol. Food and Chemical Toxicol. 42: 429-438. Kaneko, J.J., Harvey, J.W. and Bruss, M. (2008). Clinical biochemistry of domestic animals 6th edition; Academic Press, New York. Khan, S. A. (2003). Characterization and standardization of some traditional plant drugs. Ph.D thesis submitted to Jamia Hamdard University. Available at http://www.jamiahamdard.edu/thesis_traditional plantdrugs.asp Kumar, G., Banu, G. S. and Pandian, M. R. (2005). Evaluation of the antioxidant acitivity of Trianthema portulacastrum Linn. Indian J. Pharmacol. 37: 33133. Mandal. A., Bandyopadhyay, S. and Chatterjee, M. (1997). Trianthema portulacastrum Linn reverses hepatic lipid peroxidation, glutathione status and activities of related antioxidant enzymes in carbon
Kumar et al.
tetrachloride induced chronic liver damage in mice. Phytomed. 4: 239-244. Marklund, S. and Marklund, G. (1974). Involvement of superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase. Europ. J. Biochem. 47: 469-474. Moron, M. S., Depierre, J. W. and Mannervik, B. (1979). Levels of glutathione, glutathione reductase and glutathione-S-transferase in rat lung and liver. Biochem. Biophysics Acta. 582: 67-68. Nagaraj, M., Sunitha, S. and Varalakshmi, P. (2000). Effect of lupeol, a pentacyclic Triterpene on the lipid peroxidation and antioxidant status in rat kidney after chronic exposure. J.f Appl. Toxicol. 20: 413417. Ognjanovic, B., Zikic, R.V., Stajn, A., Saicic, Z.S., Kostic, M.M. and Petrovic, V.M. (1995). The effects of selenium on antioxidant defense system in liver of rats exposed to cadmium. Physiol. Rev. 44: 293-300. Paglia, D. E. and Valentine, W. N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clinic. Med. 70: 158-169. Patra, R.C., Rautray, A.K. and Swarup, D. (2011). Oxidative stress in lead and cadmium toxicity and its amelioration. Vet. Med. International. No.457327. Prasad, S.R., Nagaraj, M. and Varalakshmi, P. (2003). Combined efficacies of lipoic acid and meso 2,3-
dimercaptosuccinic acid on lead induced erythrocytes memberane lipid peroxidation and antioxidant status in rats. Human and Experiment. Toxicol. 22: 183-192. Raghuramulu, M. (1983). Manual of laboratory techniques. National Institution of Nutrition (ICMR) 1983; Hyderabad pp 204-206. Sarkar, S., Yadav, P., Trivedi, R., Bansal, A. K. and Bhatnagar, D. (1995). Cadmium induced lipid peroxidation and the status of the antioxidant system in rat tissues. J. Trace Elements Med. Biol. 9: 144. Singh, U.B. and Sulochana, S. A. (1997). Practical manual of histological and histochemical techniques. Kothari publications, Bombay. pp 1-41. Stohs, S.J, Bagchi, D., Hassoun, E. and Bagchi, M. (2000). Oxidative mechanism in the toxicity of chromium and cadmium ions. Journal of Environ. Pathol. Toxicol. Oncol. 19: 201. Wang, Y., Fang, J., Leonard, S.S. and Rao.K.M.K. (2004). Cadmium inhibits the electron transport chain and induces ROS. Free Radical Biol.Med. 36:143443. Yagi, K. and Rastogi, R. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95: 351-358.
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Research Article
DISPOSITION KINETICS OF CEFTRIAXONE AFTER INTRAMUSCULAR ADMINISTRATION IN GOATS

NAVEEN KUMAR1, B.K. ROY2, VIJAY KUMAR3, WASIF AHMAD4 AND N. K. PANKAJ5
MVSc Scholar, 2University Professor and Chairman, 3Senior Research Fellow, 1,2,3Department of Pharmacology and Toxicology, College of Veterinary Science and A.H., BAU, Ranchi- 834006, Jharkhand; 4,5Department Pharmacology and Toxicology College of Veterinary and Animal Sciences, U.S. Nagar, Pantnagar-263145, Uttarakhand, India. 1 Corresponding author: E-mail: knaveen7v@gmail.com ABSTRACT Plasma concentrations and pharmacokinetics of ceftriaxone were determined in Black Bengal goats after intramuscular administration at a single dose of 50 mg/kg body weight. Blood samples were drawn from the jugular vein at predetermined time intervals after drug injection. Plasma was separated and analyzed for ceftriaxone by reverse-phase high performance liquid chromatography (HPLC). The plasma concentration-time data for ceftriaxone were best described by two-compartment open pharmacokinetic model. The elimination half-life (t1/2), area under the plasma concentration-time curve (AUC), volume of distribution (Vdarea), mean residence time (MRT) and total systemic clearance (ClB) were 2.380.19 h, 163.405.43 g/h/ml, 0.150.07 L.kg-1, 3.270.10 h and 5.080.14 L.h-1.kg-1, respectively. The plasma concentration of ceftriaxone was detected 4.480.21 g/ml for upto 8 h. Ceftriaxone appears to be useful for the treatment of animal diseases associated with pathogens sensitive to this drug. Key words: Ceftriaxone (CTA), goats, HPLC, pharmacokinetics.
1
INTRODUCTION Ceftriaxone is a third generation cephalosporins, bactericidal agent, inhibits synthesis of bacterial cell wall through binding to the transpeptidase enzyme (Stoeckel et al., 1981) and shows a time- dependent bactericidal effect. It has total elimination through urine and bile with reciprocal compensation if one of the routes is faulty. Ceftriaxone reportedly well tolerated locally and systemically even after repeated administration. This drug is safe to use during pregnancy and there is no sign of visceral or skeletal abnormalities are found in fetus (Teelman et al., 1982; Blaise et al., 1985). It has high antimicrobial activity against a broad spectrum of Gramnegative and Gram-positive bacteria. The potential antimicrobial properties and favorable pharmacokinetic properties of ceftriaxone, including its excellent tissue penetration make this drug a suitable antibacterial. The marked species variation in the pharmacokinetics of this drug limits the extrapolation of data from other species to goat. The present study was undertaken to determine the plasma concentrations and pharmacokinetic data for ceftriaxone after single intramuscular administration in Black Bengal goat. MATERIALS AND METHODS Five adult healthy female Black Bengal goats (weighing 10-12 kg) were procured from Instructional Small Ruminant Farm, College of Veterinary Sciences and A.H., Kanke, Ranchi. They were reared under routine grazing and were provided fodder. Water was given ad libitum. The goats were kept under constant observation before commencement of experiment.
Ceftriaxone was obtained from M/S Wockhardt Pvt. Ltd. Mumbai. It was dissolved in pyrogen free sterile distilled water as a solution for intramuscular injection into goats. The drug was given as a single intramuscular at a dose rate of 50 mg/kg body weight. The quantitative analysis of drug in plasma and urine were done as per as modified method of United State Pharmacopeia (USP, 2007). The samples were stored at -200C till the time of processing. The CTA concentrations in plasma and urine were estimated by high performance liquid chromatography (HPLC) method. Chromatographic quantification The standard acetonitrile (HPLC grade) and all chemicals and solvents of analytical grades and ultrapure water (Millipore) were used for this investigation. The concentration of CTA was determined using an HPLC system (Cecil 4100), equipped with a reverse-phase and a UV/vis detector, and operated at 280 nm. The samples were separated on an RP-C18 column (4.6 X 250 mm, 5m) and eluted with a mobile phase consisting of a mixture of acetonitrile, tetraheptyl ammonium bromide, phosphate buffer (pH 7.0) and citrate (pH 5.0). A flow rate of 1 ml.min-1 was used and column oven temperature was kept at 350C ceftriaxone was quantified from the peak areas and the concentrations in plasma samples were determined by means of calibration curve obtained on analysis of blank goat plasma sample fortified with ceftriaxone (external standard) and assayed as described for the experimental samples. The recovery of ceftriaxone was >90%. Drug extraction Blood samples were collected by puncturing
Kumar et al.
jugular vein into EDTA containing vial at 5, 10, 15, 20, 30, 45 minutes and 1, 2, 3, 4, 5, 6, 8 and 12 h. In brief, the plasma was separated from blood by centrifugation at 3000 rpm for 20 minutes. The clear supernatant was separated gently. To 0.5 ml of plasma, 0.5 ml distill water and 1 ml acetonitrile was added in the ratio of 1:2 and after vortex mixing at high speed for 60 sec the tube was subjected to centrifugation at 5000 rpm for 30 min. The supernatant was collected and transferred to a tube after passing through a filter paper (Whatman No-1). The whole aliquot was vacuum filtered through a 0.45 m cellulose acetate membrane filter and 20 l of fluid was injected in HPLC system. RESULTS The disposition of CTA following single dose intramuscular administration in goat is depicted in Figure1. The semi-logrithmic plot of the plasma drug concentration following intramuscular administration of CTA exhibited biexponential decline in the plasma could be best fitted to two-compartment open model. The drug was detected in plasma up to 8 h following intramuscular administration. The plasma concentration of CTA increases gradually from 0.08 h to 0.75 h. After that the plasma level diminished up to 8 h and peak plasma concentration at 0.75 h. The pharmacokinetic determinants which describe disposition kinetics of ceftriaxone in goats following intramuscular administration are presented in table. The elimination half-life (t1/2) of ceftriaxone after intramuscular administration was 2.300.19h with an elimination rate constant 0.300.02h-1, while distribution half-life (t1/2) was 0.14 h with a distribution rate constant () of 0.300.02 h1. The drug was calculated to have an apparent volume of distribution (Vdarea) of 0.150.07 L/kg. The total body clearance which represents the sum of metabolic and excretory clearance processes was 5.080.14 ml/kg/min.
TABLE 1: Pharmacokinetic profile of ceftriaxone in plasma following single dose (50 mg/kg) i.m. administration in healthy goats (n=5). Kinetic Parameters A (g/ml) B (g/ml) Cop (g/ml) (h-1) (h-1) t1/2 (h) t1/2 (h) AUC(mg/L.h) MRT (h) ClB(ml/kg/min) Vdarea (L/kg) MeanS.E. 48.180.40 50.545.60 43.810.75 4.960.22 0.300.02 0.1400 2.300.19 163.405.43 3.270.10 5.080.14 0.150.07
A= zero time intercept of distribution phase; B = zero time intercept of elimination phase; = Distribution/absorption rate constant; = Elimination rate constant; t = Distribution/absorption half life; t=Elimination half life; Vd area = Apparent volume of distribution; ClB= Total body clearance, AUC = Total area under the time concentration curve, C0P = Zero time plasma concentration, MRT = Mean residence time.
Fig 1: Semilogarithmic plot of CTA concentration in plasma versus time in healthy goats after intramuscular administration of CTA. Each point represents mean SE of five goats.
DISCUSSION In the present study, HPLC assay was used to determine the concentrations of CTA and its active metabolite in plasma of goats. The commonly used antimicrobial assay does not distinguish parent drug from the active metabolite. The pharmacokinetics of CTA in goats was best described by a two-compartment open model of drug with passage of time following intramuscular administration. The mean residence time (MRT) obtained for CTA in goats in present study was 3.270.10 h, which is slightly higher than the value (2.630.20 h) reported by Gohil et al. (2009) following intramuscular dose of CTA in buffalo calves. However, lower AUC (26.672.2 g.h/ml) values have been reported after intramuscular administration of CTA. Following intramuscular administration of CTA in goats, the peak plasma concentration was achieved at 0.75 h. It is in agreement with the peak plasma concentration of 15.32.4 g/ml observed in cow calves (Maradiya, 2004). Short absorption half-life of CTA showed that the drug absorbed faster from the injection site. Rapid absorption following intramuscular injection has also been reported for cefotaxime (Sharma et al., 2004) and cefoperazone (Goyal et al., 2005) in buffalo calves. The elimination of CTA was rapid followed by fast clearance (5.080.14 ml/min.kg) of drug in the present study. It has been in agreement to the earlier report in cow calves (Johal and Srivastava, 1998). Rapid elimination of cefotaxime (Sharma et al., 2004) and cefoperazone (Goyal et al., 2005) following intramuscular administration in buffalo calves justifies the observations made in the present study. The drug is extensively distributed following intramuscular injection as evidenced by high Vd area
86
Kinetics of ceftriaxone in goats
(0.150.07 L/kg). High apparent volume of distribution of CTA (1.400.07 L/kg; Dardi et al., 2004) and cefotaxime (1.30 L/kg; Sharma et al., 2004) in buffalo calves supports our observation. However, moderate distribution of the drug (Vdarea; 0.55 L/kg) has also been reported following intramuscular administration of CTA in cow calves (Soback and Ziv, 1988). In the present study a single dose (50 mg/kg) i.m. administration of CTA produced therapeutically effective concentration within 0.08 h, indicative of quick rate of absorption by this route and drug concentration was higher than minimum inhibitory concentrations (MIC) remain maintained up to 8 h. The MIC of CTA against susceptible bacteria ranges between 0.06 and 0.25g/ml (Beam, 1985). The elimination half-life (2.300.19 h) following i.m administration obtained in goats in present study was slightly longer than cattle calves (116.8 min, Soback and Ziv. 1988), however much lower t1/2 values have been reported in dogs (1.17 h, Rebuelto et al., 2002). The plasma concentration and pharmacokinetic characteristics of CTA in goats following intramuscular administration indicates favorable pharmacokinetic profile, therefore, the drug by this route may be used to treat susceptible bacterial infections in goats. ACKNOWLEDGEMENT The authors express their gratitude to the Dean, College of Veterinary Science & A.H, Kanke and the Vicechancellor, BAU, Ranchi (Jharkhand), for providing necessary facilities during this study. REFERENCES Beam Jr., T.R.(1985). Ceftriaxone: a beta-lactamasestable, broad-spectrum cephalosporin pharmacology. Br. J. Clin. Pharmac. 8:237-253. Blaise, L., Tasmee, C., Tamara, A. R. and Quellant, T. B. (1985). Once daily ceftriaxone therapy for serious bacterial infection in children. Antimicrob. Agent Chemother. 27(2): 181-1 83. Dardi, M.S., Sharma, S.K. and Srivastava, A.K. (2004). Pharmacokinetics and dosage regimen of ceftriaxone in buffalo calves. Vet. Res. Commun., 28: 331-338. Gohil, P.V., Patel, U.D., Bhavsar, S.K. and Thakur, A. M. (2009). Pharmacokinetics of ceftriaxone in buffalo calves (Bubbalus bubalis) following intravenous and intramuscular administration: Irani. J. Vet. Res. 10: 33-37.
Goyal, S., Chaudhary, R.K. and Srivastava, A.K. (2005). Pharmacokinetics following intramuscular administration and dosage regimen for cefoperazone in buffalo calves. Indian J. Anim. Sci. 75: 31-32. Johal, B. and Srivastava, A.K. (1998). Pharmacokinetics, urinary excretion and dosage regimen of ceftriaxone in crossbred cow calves following single intramuscular administration. Indian J. Anim. Sci. 68:1017-1019. Mardiya, J. J., Gohil, P. V., Goriya, H. V., Bhavsar, S. K. and Thakur, A. M. (2004). Ceftriaxone pharmacokinetics following single dose intravenous administration in crossbred cow calves. Indian Soc.Vet. Pharmacol. Toxicol. 4(IV): 50-51. Rebuelto, M., Albarellos, G., Ambros, L., Kreil, V., Montoya, L., Bonafiner, R., Otero, P. and Hallu, R. (2002). Pharmacokinetics of ceftriaxone administered by the intravenous, intramuscular or subcutaneous routes to dogs. J. Vet. Pharmacol. Therap. 25(1): 73-76. Sharma, S.K., Srivastava, A.K. and Deore, M.D. (2004). Pharmacokinetic disposition of cefotaxime in buffalo calves (Bubalus bubalis) following single intramuscular administration. Indian J. Anim. Sci. 74: 590-593. Soback, S. and Ziv, G. (1988). Pharmacokinetics and bioavailability of ceftriaxone administered intravenously and intramuscularly to calves. Am. J. Vet. Res. 49: 535-538. Stoeckel, K., McNamara. P. J., Brandt, R. and Ziegler, W. H. (1981). The influence of protein binding on the pharmacokinetics of Rocephin (Roche). Paper 12th International Congress of Chemotherapy, Florence,19-24.7. Teelman, K., Scharer, K., and Udaka, K. (1982). Experimentall toxicologic von ceftriaxone. In ceftriaxone (Rocephin) ein neves parenteral cephalosporins. Hapmenklee Symposium (1981). L. Detti (ed.) Basel. Grenzach; Editione Roche 91-111 . United States Pharmacopoeial Convention (2007). British Pharmacopoeia: Ceftriaxone sodium injection, Vol. 1: 423-424.
Research Article
HAEMATO-BIOCHEMICAL PROFILE FOLLOWING MULTIPLE ORAL DOSE ADMINISTRATION OF CHLORPYRIFOS IN POULTRY

LATA GAYAL1, S.P. SINGH2, A.H. AHMAD3 AND WASIF AHMAD1
1
PhD Scholar, 2Professor and Head, 3Professor; Department of Veterinary Pharmacology and Toxicology, C.V.A.Sc., Pantnagar-263145, Uttarakhand (India). 1 Corresponding author : E-mail-vetdoclata@gmail.com
ABSTRACT The present study was carried out to evaluate haemato-biochemical profile following multiple oral dose @15 mg/ kg bwt (1/3rd of LD50) administration of chlorpyrifos (CPF) at an interval of 24h for seven days in poultry birds. A significant (P<0.05) reduction in haematological values up to 24h post administration and thereafter gradual increase observed approaching normal values 96h post administration of chlorpyrifos.The total serum proteins, albumin and globulin significantly (P<0.05) decreased upto 48h post administration gradually returned to normal value after 96h. The serum AST and ALT activities increased upto 24h gradually decreased to normal value after 96h post administration. No significant difference was observed in the value of serum urea and uric acid whereas significant (P<0.05) increase in the values of serum creatinine and cholesterol upto 24h post administration and gradual decrease upto 96h post administration of chlorpyrifos was observed. A significant reduction in serum glucose level upto 24h post administration was observed, however , the level increased gradually reaching to normal value upto 96 h post administration of the pesticide. It is concluded from this study that chlorpyrifos produced mild to moderate haemotoxic, hepatotoxic and nephrotoxic effects in poultry.
Keywords: Biochemical, chlorpyrifos, haematological, hepatotoxic, nephrotoxic, poultry. INTRODUCTION Chlorpyrifos is a broadspectrum insecticide widely used in agriculture and animal husbandary. It is widely used to control vector responsible for spreading diseases in domestic animals and plants. It is also found to be effective against pests, insects, termites and mosquitoes. In animal husbandry, it is used against external parasites of domestic animals and poultry birds. However, very meager information is available on toxicological aspects of chlorpyrifos in poultry birds. This study was, therefore, undertaken to evaluate the haematological and biochemical effects of chlorpyrifos after 7 days multiple oral dose administration of chlorpyrifos. MATERIALS AND METHODS Experimental design 20 male RIR birds weighing 10.5kg were divided into five groups namely Group I, II, III, IV and V comprising 4 birds in each group. Group I served as control free from pesticide. Birds of Group II, III, IV and V were administered 0.08 ml of chlorpyrifos (prepared by mixing 1ml of 20% EC chlorpyrifos (CPF) with 1ml of olive oil) as multiple oral (the formulation was administered using thin plastic tube attached to a syringe) dose@15mg/kg bwt (1/3rd of LD50; 40mg/kg) for seven days at an interval of 24h. The birds were reared under uniform management and husbandry conditions, maintained on poultry feed (as per NRC). The birds were fed commercial broiler ration free from any antibiotic. Fresh water was provided ad-libitum during the entire period of experiment. The birds were observed daily morning and evening for assessing their health status and
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other signs of clinical toxicity. Following multiple (7) oral dose administration of chlorpyrifos@15 mg/kg bwt in poultry birds, blood samples were collected at 24, 48, 72 and 96 h after last dose from the left brachial vein or jugular vein. The blood samples were divided into two parts, one part was collected in heparinized tubes for hematological studies while second part in sterilized vials for collection of serum for analysis of biochemical parameters. Blood was collected from each bird in clean heparinized microcenrifuge tube (eppendorf) and hematological parameters such as packed cell volume (Jain, 1986), haemoglobin (Jain, 1986), total erythrocyte count (Natt and Herric, 1952) and total leucocyte count (Natt and Herric, 1952) were estimated immediately after the collection of blood samples. 0.1N-HCl was used for estimating the blood haemoglobin concentration while, Natt-Herric diluting fluid was used for TEC and TLC estimation. Statistical analysis Statistical analysis of data was done by using one way ANOVA technique followed by Tukeys Multiple Comparison Test by using Graph pad prism statistical software. Statistically significant difference was considered at 5 and 1percent level. RESULTS A significant (p<0.05) decrease in Hb (g/dl) and PCV (%) was observed in Group II as compared to control. Other groups, however, did not reveal any significant change in comparison to control. A significant (p<0.05) decrease in TEC (X106/l) value was observed in Group II,
Chlorpyrifos toxicity in poultry
TABLE 1: Effect on hematological parameters at different intervals following multiple (7) oral dose of CPF @ 15mg/kg in poultry. Groups I (Control) II (24 h) III (48 h) IV (72 h) V (96 h) One Way ANOVA Dose (mg/kg) 15 15 15 15 CD d.f F Hb (g/dl) 12.150.33 10.50.24a 11.20.22 11.650.25 11.70.35 0.85 15 4.89 PCV (%) 36.850.74 31.71.03a 33.350.95 33.81.14 34.851.18 3.09 15 3.49 TEC (106/l) 2.400.08 1.640.09a 1.820.07a 2.020.07ab 2.290.09bc 0.25 15 15.06 TLC (103/l) 14.510.65 10.420.32a 12.450.30 13.730.76b 14.710.48b 1.61 15 11.05
TABLE 2: Effect on serum enzymatic and protein profiles at different intervals following multiple (7) oral dose of CPF @ 15mg/kg in poultry. Groups I (Control) II (24 h) III (48 h) IV (72 h) V (96 h) One Way ANOVA Dose (mg/kg) 15 15 15 15 CD d.f F Total protein (g/dl) 3.700.19 3.170.15 2.740.20a 3.020.20 3.420.21 0.58 15 3.65 Albumin (g/dl) 2.420.16 1.760.09a 1.410.06a 1.680.14a 1.870.10a 0.35 15 10.2 Globulin(g/dl) 1.280.06 1.410.08 1.330.15 1.340.10 1.550.18 0.37 15 0.76 ALT (U/L) 5.01 0.37 6.87 0.87 5.8 0.62 5.87 0.63 5.55 0.41 1.83 15 1.25 AST (U/L) 128.3 3.04 139.1 4.92 125 4.78 116.2 7.29b 100.3 3.85abc 15.02 15 8.49
TABLE 3: Effect on biochemical parameters at different intervals following multiple (7) oral dose of chlorpyrifos @15mg/kg in poultry. Groups I (Control) II (24 h) III (48 h) IV (72 h) V (96 h) One WAY ANOVA Dose (mg/kg) 15 15 15 15 CD d.f F Cholesterol (mg/dl) 1348.69 121.59.22 128.33.64 119.34.67 99.93.40ac 19.40 15 4.04 Glucose (mg/dl) 24512.42 182.77.58a 187.45.51a 210.313.79 229.616.94 36.14 15 4.97
b
Uric acid (mg/dl) 5.810.21 7.010.57 6.470.22 6.210.22 5.980.21 0.96 15 2.19
Urea (mg/dl) 6.980.38 6.460.27 6.210.24 6.320.21 6.610.23 0.82 15 1.21
Creatinine (mg/dl) 0.480.03 0.920.12a 0.790.08 0.620.05 0.540.02b 0.21 15 6.39
Values in Table 1, 2 and 3 are Means SE (n=4), a significant (P<0.05) Vs group I, group III.
significant (P<0.05) Vs group II, c significant (P<0.05) Vs
III and IV as compared to control whereas a significant (p<0.05) increase in the values of TEC in Group IV and V was observed as compared to Group II. A significant (p<0.05) increase in the values of TEC in Group V was also observed in comparison to Group III. A significant (p<0.05) decrease in the values of TLC (X103/l) in Group II was observed as compared to control whereas a significant (p<0.05) increase in the values of TLC in Group IV and V was observed as compared to Group II. There is a time dependent increase in the hematological parameters in chlorpyrifos treated groups (Table 1). As shown in Table 2, no significant difference was observed in the values of serum proteins (g/dl) of control as well as test groups except Group III in which significant (p<0.05) decrease was observed in comparison to control. A significant (p<0.05) decrease in value of serum albumin level and A:G ratio was observed in Group II, III, IV and V as compared to control whereas no significant difference
was observed in serum globulin level in comparison to control. A significant (p<0.05) decrease in AST activity (U/ L) was observed in Group V as compared to Group I, II and Group III. Activity of AST declined significantly (p<0.05) in Group IV as compared to Group II. No significant difference was observed in the value of ALT of control as well as test groups. A significant (p<0.05) decrease in the value of glucose (mg/dl) in Group II and III was observed as compared to control. There was no significant difference in the values of urea (mg/dl) and uric acid (mg/dl) in control as well as test groups. A significant (p<0.05) increase in the value of creatinine (mg/dl) in Group II was observed as compared to control. However, significant (p<0.05) decrease in the value of creatinine in Group V was observed as compared to Group II. No significant (p<0.05) difference was observed in value of cholesterol (mg/dl) in control as well as test groups except Group V in which significant (p<0.05) decrease in the value of cholesterol was observed
Gayal et al.
as compared to control as well as Group III (Table 3). DISCUSSION Hematological parameters Hb, PCV, TEC and TLC significantly decreased in chlorpyrifos treated groups after 24 h post administration and gradually increased after 48, 72 and 96 h post administration of chlorpyrifos as the residue concentration of pesticide gradually declined in the body in the present study. Anemia may be the major reason for decline in the Hb and PCV values. Krishnamoorthy et al. (2006) also studied the effect of chlorpyrifos in broiler chicken and observed significant decrease in PCV and Hb levels indicating anaemia. A significant (p<0.05) decrease in the values of TEC and TLC was observed in the present study in chlorpyrifos fed birds initially after 24 h post administration which increased gradually as chlorpyrifos residues decreased gradually. Rawat (2002) has also reported a decrease in TEC and PCV values after single oral dose of 10 mg/kg of chlorpyrifos in birds. In another study, hematological picture of cockerels treated with 25 mg/kg of chlorpyrifos for 24 weeks revealed a significant decrease in TEC and TLC values (Kumar et al., 2010). Non significant augmentaion was observed in the value of AST and ALT of birds after 24h post administration of chlorpyrifos which indicated mild degree of damage to liver as compared to endosulfan in this study. The susceptibility of the liver to toxic compounds is due to the central role it plays in the biotransformation and disposition of xenobiotics (Murray et al., 1999). Krastav et al. (1995) also reported an increase in the activities of AST and ALT following chlorpyrifos toxicity. No significant difference was observed in the values of serum proteins whereas a significant (p<0.05) decrease in values of serum albumin level occurred at 24, 48, 72 and 96 h post administration of chlorpyrifos. Krishnamoorthy et al. (2006) also observed similar effect of chlorpyrifos and observed significant decrease in serum albumin level in broiler chickens. Oral exposure to chlorpyrifos (25 mg/kg) in feed for 24 week also declined levels of total serum proteins in cockerels (Kumar et al., 2010). There was a significant (p<0.05) increase in the value of creatinine after 24 h post administration of chlorpyrifos where as non significant difference observed in value of urea and uric acid. It might have occurred due to mild nephrotoxic effect of chlorpyrifos in birds. Ambali et al. (2007) evaluated the effect of subchronic chlorpyrifos poisoning and also observed a significant increase in creatinine values in mice. There was no significant difference in cholesterol value after 24 h post administration of chlorpyrifos in poultry
birds whereas significant (p<0.05) decrease in the value of cholesterol after 96 h was observed in comparison to control. Similar effects showing significant decrease in cholesterol values were also reported in chlorpyrifos intoxicated broiler chicken by Krishnamoorthy et al. (2006). A significant (p<0.05) decrease in the value of glucose after 24 and 48 h post administration and a non significant difference was observed at 72 and 96 h post administration of chlorpyrifos due to gradual decline in the level of pesticide residues. Similar results were reported by Kumar et al., (2010) after 24 week of oral exposure of chlorpyrifos (25 mg/kg) where serum glucose levels were decreased in chicks. It is concluded from this investigation that chlorpyrifos following multiple (7) oral dose @ 15 mg/ kg administration produce mild to moderate degree of hepatotoxic and nephrotoxic effects in poultry birds. REFERENCES Ambali, S., Dayo, A., Igbokwe, N., Shittu, M., Kawu M., Ayo, J. (2007). Evaluation of subchronic chlorpyrifos poisoning on haematological and serum biochemical changes in mice and protective effect of Vit C. J. Toxicol. Sci. 32: 111-120. Jain, N.C. (1986). Schalms Veterinary Haematology. 4th Edn. Lea and Febringer, Philadeiphia. Krishnamoorthy, P., Vairamuthu S., Balachandran2, C. and Muralimanohar, B. (2006). Chlorpyriphos and T-2 Toxin Induced Haemato-Biochemical Alterations in Broiler Chicken. International J. Poultry Sci. 5: 173-177. Kumar, A.; Singh, S.P. and Sharma, L.D. (2010). Immunotoxicological evaluation of chlorpyrifos following medication with Withania Somnifera in cockerels. J. Vety. Pharmacol. Toxicol. 9: 3437. Krastev, A.R., Sengalevic, G., Kuzmanov, N. and Abdulaziz, M. (1995). Study on the effect of some insecticides on forage cultures and their influences in the homeostasis in rumen animals. Selekeija-ISeenaralvo. 2: 353-358. Murray, R. K., Granner, D. K., Mayes, P. A., and Rodwell, V. W. (1999). Harpers Biochemistry, 22nd ed. USA, Prentice Hall. Natt, M.P. and Herrick, C.A. 1952. A new blood diluent for counting the erythrocytes and leucocytes of the chicken. Poult. Sci. 31: 735-778. Rawat, D. (2002). Survey on residues of different pesticides in Garhwal region of Uttaranchal and their toxicological evaluation in poultry. M.V.Sc. thesis submitted to G.B.P.U.A.&T. Pantnagar, India.
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Research Article
TOXICO-PATHOLOGICAL STUDY OF ALPHACYPERMETHRIN IN DAY OLD WLH CHICKS

NAVNEET KUMAR PANDEY1 AND A.M.THAKER2
1 Assistant Director, Division of Toxicology, Krish Biotech Research Pvt Ltd, Kalyani, Nadia, West Bengal-741235, Professor & Head, Department of Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, A.A.U. Anand, Gujarat-388001 *Corresponding author: E-mail: neetvet@gmail.com; navneet.p@krishbiotech.com
ABSTRACT The present study was conducted in day old White Leghorn chicks. An approximate medium lethal dose (ALD50) of alphacypermethrin used for the study was 562.5 mg/kg body weight. One hundred twenty five birds were randomly divided into five different groups, C1, C2, T1, T2 and T3. Alphacypermethrin was administered at dose rates of 14.0625 mg/kg, 18.725 mg/kg and 28.125 mg/kg for 28 days daily in corn oil. Blood sample was collected on day 15 and day 29 to study effect on various hematological and biochemical parameters. Alphacypermethrin, a synthetic pyrethroid insecticide increased the activities of serum transaminases (ALT, AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine (CRT) and serum glucose level, while it decreased the total serum protein (TP), albumin (ALB) and globulin (GLB) level, but no alteration on hematology in white leghorn (WLH) chicks. The insecticide also produced adverse effects on kidney, lung, liver, thymus, spleen and bursa. It is concluded that alphacypermethrin altered biochemical parameters and histopathology of vital organ tissues in white leghorn chicks after repeated oral exposure. Key words: Alphacypermethrin, biochemical, hematological, transaminases, white leghorn chicks,
INTRODUCTION Pesticide plays one of the important role in agricultural breakthrough. All the pesticides are good insecticides and are also randomly used for the control of ectoparasite infestataions in domesticated animals. At persent, specially synthetic pyrethroids replaces all other pesticides. Alphacypermethrin a synthetic pyrethroid type II insecticide is extensively used as ectoparasiticide in animals, agriculture crop production and public health programme. Though some of the toxic action of alphacypermethrin has been described in EHC-91, but the report on effect after oral administration of alphacypermethrin on hematology, serum biochemistry parameters and histopathology of tissue in WLH chicks is scarcely available. Therefore, the present study has been undertaken to study the effect on hematology, biochemical parameters and histopathology following repeated oral dosing of alphacypermethrin. MATERIALS AND METHODS Experimental design A total of 125, day-old WLH chicks were procured and housed at Central Poultry Research Station (CPRS), Anand Agricultural University, Anand. Chicks were randomly divided into five groups (C1, C2, T1, T2 and T3) of 25 each. Chicks were maintained on standard chick ration and husbandry practices as followed by the research station. Water was provided ad libitum. All standard managemental procedures were adopted to keep the birds free from stress. Technical grade of alphacypermethrin was
procured from Meghmani Corporation Pvt Ltd, Ahmedabad, Gujarat. LD50 of alphacypermethrin taken in to consideration for this study was 562.5 mg/kg body weight (Singh and Sharma, 2003). Group C1 and group C2 were untreated control and vehicle (corn oil) control respectively. Three different dose levels of alphacypermethrin in ascending order, i.e., 14.0625, 18.725 and 28.125 mg/kg body weight were formulated in corn oil and administered daily to group T1, T2 and T3 for a period of 28 days through oral gavaging. Clinical signs Birds were observed for any signs of toxicological significance during entire period of experimentation and weekly body weight was also recorded. Hemato-biochemical paratmeter Six birds from each group were sacrificed after 14 days and 28 days of experimentation. Blood sample was collected in 1% EDTA coated volex tubes for estimation of Hemoglobin, Packed cell volume, Total erythrocyte count and Total leukocyte count. Blood samples were also collected in glass test tubes for serum separation, which was utilized for estimation of serum amino transaminase (AST and ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine (CRT), total protein (TP), albumin (ALB), globulin (GLB) and serum glucose by diagonistic kits. Histopathology At the end of the study tissue of liver, kidney, lung, thymus, spleen and bursa were collected for histopathological examination. Tissue were trimmed in to
Pandey and Thaker
TABLE 1: Effect of daily oral administration of alphacypermethrin on body weight (gm) in WLH chicks Group& Dose 0 day C 1 (Control) C 2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) 35.200.76 35.480.57 a 34.600.60 a 35.080.79 a 35.480.58 a
a
Body Weight (gm) 7day 54.601.57 53.241.50 a 51.321.19 a 52.241.30 a 50.761.35 a

a
14day 78.632.811 77.573.42 a 72.603.24 ab 69.204.09 ab 65.933.09 a

a
21 day 124.635.70 116.06.74 ab 105.55.54ab 104.36.09b 98.065.54 b

a
28 day 175.688.01 a 159.8911.08 ab 140.208.07 bc 148.338.91 b 121.535.95 c
Note: Data in the column having different superscript differ significantly (p< 0.05). TABLE 2a: Effect on hematological parameters after 14 days of oral exposure of alphacypermethrin in WLH chicks (n=6) Group& Dose Hb (gm/dl) C 1 (Control) C 2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) 9.660.42 9.46 0.49a 8.83 0.33a 8.830.16a 9.330.42a
a
Hematological parameters (Mean SE ) (n=6) PCV (%) 29.50 1.54 29.00 1.32 a 29.50 0 .99 a 30.66 1.23 a 30.00 1.24 a
a
TLC (x10 3 /lit) 1946.17 34.29 1959.6737.01 a 1918.0022.11 a 1939.1720.78 a 1908.8344.52 a

a
TEC (million / lit) 1.940.03a 1.80 0.03a 1.88 0.06a 1.85 0.05a 1.86 0.04a
Note: Data in the column having different superscript differ significantly (p< 0.05). TABLE 2b: Effect on hematological parameters after 28 days of oral exposure of alphacypermethrin in WLH chicks (n=6) Group& Dose C 1 (Control) C 2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) Hematological parameters (Mean SE ) (n=6) Hb (gm/dl) 9.40 0.36 9.73 0.35a 10.00 0.23a 10.160.18a 10.000.14a
a
PCV (%) 28.00 0 .57 27.50 0 .76 a 30.16 1.19 a 29.00 0 .96 a 28.66 0 .88 a
a
TLC (x10 3 /lit) 1938.5016.99 1982.509.50 a 1945.0020.54 a 1956.33325.65a 1925.0029.25 a

a
TEC (million / lit) 1.900.048a 1.880.04 a 1.910.05 a 1.880.04a 1.840.05a
Note: Data in the column having different superscript differ significantly (p< 0.05). TABLE 3a: Effect of daily oral administration after 14 days exposure of alphacypermethrin on biochemical parameters in WLH chicks (n=6) Group& Dose ALT (U/Lit) C 1 (Control) C 2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) AST (U/Lit) 17.57 1.26a 119.365.12 a 15.58 1.02a 124.434.94 a 23.63 1.72b 163.9010.35 b 27.35 0.84c 194.154.75 b d 31.07 0.95 203.416.58 b 37.16 36.95 53.26 57.32 62.73 Biochemical parameters ALP (U/Lit) 2.87a 2.45a 1.22b 3.52b 4.74b LDH (U/Lit) 949.33 50.89a 908.66 89.29a 932.66 63.36a 1069.10 77.62 a 1329.60 85.03 b CRT (mg/dl) 0.230.02 a 0.240.03 a 0.240.03 a 0.320.03 ab 0.360.03 b GLU (mg/dl) 134.904.07 a 138.7013.36 a 150.3310.66 a 196.6016.48 b 274.6622.84 c
Note: Data in the column having different superscript differ significantly (p< 0.05). TABLE 3b: Effect of daily oral administration after 28 days exposure of alphacypermethrin on biochemical parameters in WLH chicks (n=6) Group& Dose ALT (U/Lit) C 1 (Control) C 2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) 17.89 16.16 27.22 29.78 41.99 0.80a 0.99a 2.15b 1.09b 1.90c AST (U/Lit) 121.55 8.27 a 123.65 4.08 a 157.35 14.93 b 200.65 6.88 c 262.24 13.25 d 37.46 38.46 56.84 62.00 70.01 Biochemical parameters ALP (U/Lit) 3.25a 2.69a 2.96b 1.27b 2.03c LDH (U/Lit) 1084.1577.82a 947.66177.15a 1252.65186.75a 1250.73119.88a 1834.00142.13b CRT (mg/dl) 0.280.03 a 0.330.04 a 0.580.09 ab 0.890.15 bc 1.280.35 c GLU (mg/dl) 153.5310.52 a 150.8311.66 a 179.718.14 a 211.405.58 b 153.5310.52 a
Note: Data in the column having different superscript differ significantly (p< 0.05).
10-15 mm thickness and fixed in 10% neutral buffered formalin and processed by standard methods and
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embedded in paraffin wax. Section of 3-5 micron thickness were taken and stained with haematoxylin and eosin (H&E)
Alphacypermethrin toxicity in chicks
TABLE 3c: Effect of daily oral administration after 14 and 28 days exposure of Alphacypermethrin on Biochemical parameters in WLH cockerel (n=6) Group& Dose Total Protein (gm/dl) C1 (Control) C2 (Vehicle Control) T 1 (14.0625 mg/kg) T2 (18.725 mg/kg) T 3 (28.125mg/kg) 2.480.04 a 2.470.04 a 2.180.14 ab 2.140.09 ab 2.090.11 b Biochemical parameters After 14 Days of exposure Albumin (gm/dl) 1.300.03 a 1.310.09 a 1.150.07 b 1.190.03 ab 1.210.04 ab Globulin (gm/dl) 1.1830.061a 1.1640.117a 1.043 0.087a 0.978 0.191a 0.876 0.108a After 28 Days of exposure Total Protein (gm/dl) 2.860.19 ab 2.640.07 a 2.380.18 b 2.300.12 ab 2.240.06 b Albumin (gm/dl) 1.380.08ab 1.590.13 a 1.160.11 bc 1.210.15 ab 1.280.08 c Globulin (gm/dl) 1.3810.085ab 1.5970.130a 1.2080.155 ab 1.1020.109 bc 0.9780.085c
Note: Data in the column having different superscript differ significantly (p< 0.05)
for histopathological evaluation (Luna, 1968). Statistical analysis All the values were expressed as mean S.E.M (Standard error of mean). Statistical analysis was done by using SPSS 7.5. Statistical significance of differences between two mean was assessed by one way ANOVA test. Means having different superscripts at a particular period of treatment differs significantly (P<0.05). RESULTS AND DISCUSSION Alphacypermethrin did not produce any gross effect at dose rate of 14.0625 mg/kg body weight. However at higher doses ranging from 18.725 and 28.125 mg/kg body weight it exerts sign of toxicity followed by prolonged depression, licking and salivation. Significant decrease in body weight (Table-1) was observed in treatment groups as compared to control group on day 14 onwards till the end of the observation period. There was no significant alteration noticed in hematological parameters in treatment group as compared to concurrent control birds as it is summarized in Table2a and 2b. The effect of alphacypermethrin on biochemical parameters is summarized in Table-3a, 3b & 3c. Alphacypermethrin significantly increased the levels of serum AST, ALT, ALP, LDH, CRT and glucose in treated birds as compared to control. On the other hand significant decrease in serum ALB, GLB and TP was observed in alphacypermethrin treated birds as compared to control groups. It was evident that alphacypermethrin produced hemorrhages and odema in lungs; necrosis, hemorrhages and fattty changes in liver; intertubular hemorrhages and necrosis in kidney; condensation and vacuolations in spleen; reticuloendothelial cell hyperplasia in thymus and atrophy of bursal follicles. In present investigation, alphacypermethrin administration did not influence hemoglobin, packed cell volume, total erythrocyte count and total leukocyte count. Similarly, no changes were observed in hemoglobin and PCV on administration of different insecticides in poultry
(Gupta and Paul, 1972; Mohiuddin and Ahmed, 1986; Thaker and Garg, 1993). The acivities of serum enzymes like AST, ALT, ALP, LDH and CRT were increased significantly in alphacypermethrin treated chicks. Chemically induced cellular alterations varies from simple increase of metabolism to death of cell and the increase or decrease of enzyme activities correlated with the intensity of cellular damage. The increase of transaminase activity was the consequence of alphacypermethrin induced pathological changes of tissues. Similar findings were also reported by Manna et al., 2004. Alphacyno group of pyrethroids increases the glucose utilization in the brain tissues (Lock and Berry, 1981). Inorganic phosphorus, thus required for the phosphorylation may be met from increased alkaline phosphatase (ALP). Alphacypermethrin causes hyperglycemia in the present experiment which was also reported by Kaur and Sandhu, 2006. Hyperglycemia may be due to involvement of adrenal medulla in treated birds. Insecticides stimulate catecholamines that are known to inhibit the insulin secretion by activation of alpha-receptor of pancreas (Weiner, 1985). The decreased insulin level has been reported in certain insecticide poisoning where hyperglycemia was observed (Coore and Randle, 1964; Tyler et al., 1972; Giri et al.,1983). Decreased TP, ALB and GLB were observed in the present study. Similar findings of decrease in total protein and albumin has also been observed in birds by different insecticides (Bhushan, 1993; Khurana et al., 1996; Singh et al., 2001). Histopathological findings reported in this study were similar to histopathological changes of liver, kidney, lungs, thymus and spleen reported in mice treated with alphamethrin (Luty et al., 2000). ACKNOWLEDGEMENT Meghmani Corporation Pvt Ltd, Ahmedabad, for providing technical grade of alphacypermethrin for conduct of the experiment. REFERENCES Bhushan, B. (1993). Study on immunotoxicological effects
Pandey and Thaker
of malathion and carbaryl in chickens. M. V. Sc. Thesis submitted to CCS Haryana Agriculture University, Hisar, India. Coore, H.C. and Randle, P.J. (1964). Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro. Biochem. J. 93: 66-78. Giri, S.N., Curry, D.L., Stabenfeldt, Q., Spangler, W. L., Chandler, D. B. and Schiedt, M. J. (1983). Effects of parquet on plasma glucose, cortisol, catecholamines and insulin in the beagle. Environ. Res. 30: 80-88. Gupta, P.K. and Paul, B.S. (1972). Influence of dietary intake of malathion on the haematology and plasma electrolytes in chickens. Poult. Sci. 51: 1574-1577. Khurana, R., Chauhan, R. S. and Mahipal, S. K (1996). Immunopathology of chronic cypermethrin toxicity in poultry. In: Proceedings of XIII Annual conference of IAVR and National Syposium on Impact of Environmental Pollution on Health and production of livestock, Poultry and Wild Life. Pantanagar, pp-71(body weight). Kaur, A. and Sandhu, H.S. (2006). Dermal toxicity of alphamethrin and fenvalerate in cross bred cow calves. Indian Vet. J. 83: 25-28. Luna, L.G. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed., McGraw Hill Book Co., New York. Luty, S., Latuszynska, J., Obuchowska-Przebirowska, D., Tokarska, M. and Haratym-Maj, A. (2000). Sub acute toxicity of orally applied alpha-cypermethrin in Swiss mice. Ann. Agric. Environ. Med. 7(1):3341.
Lock, A. and Berry, N. (1981). Biochemical changes in rat cerebellum following cypermethrin administration, Toxicol. Appl.Pharmacol. 59: 508-514. Mohiuddin, S.M. and Ahmed, M.N. (1986). Effect of feeding ekalux (Quinalphos) pesticide in poultry. Indian Vet. J. 63: 796-798. Manna, S., Bhattacharya, D., Mandal, T.K. and Das, S. (2004). Repeated dose toxicity of deltamethirn in rats. Indian J. Pharmacol. 37(3): 161-164. Singh, B.P., Singhal, L.K. and Chauhan, R.S. (2001). Fenvalerate induced cell-mediated and humoral immune alterations in chickens. J. Immunol. Immunopathol. 3: 59-62 Singh, S.P and Sharma, L.D. (2003). Evaluation of median lethal dose alphacypermethrin in 8 week old WLH chicks. J .Vet. Pharmacol. Toxicol. 3: 90-93. Thaker, A.M. and Garg, B.D. (1993). Biochemical alteration in chicks following long term exposure to endosulfan and malathion. Indian J. Poult. Sci. 28(1): 51-55. Tyler, J.M., Kajinuma, H. and Mialhe, P. (1972). Stimulation of pancreatic glucagon secretion by epinephrine in vivo. Fed. Proc. 31: 26-30. Weiner, N. (1985). Norepinephrine, epinephrine and the sympathomimetic amines. In: The Pharmacological Basis of Therapeutics. 9th Edn. (A.G. Goodman, L.S.Goodman, T.W.Rall and F.Murad). MacMillan Publishing Co. Inc. New York. pp.145-180.
94
Research Article
CHRONIC TOXICITY STUDY OF ZINC SULPHATE IN COCKERELS

MIR MUDASIR1, A.K. THATHOO2 AND S.P. SINGH3
3 1, 2 Department of Veterinary Pathology; Department of Veterinary Pharmacology and toxicology, CVASc, Pantnagar. 1 Corrosponding Author: Email: drmirmudasir@gmail.com
ABSTRACT
The present experiment was carried out to assess the toxico-pathological effects of zinc sulphate in cockerels. A total of 80 WLH cockerels (one month old) were randomly divided into four groups (C, G1, G2 and G3) of 20 birds each. Zinc sulphate was incorporated via drinking water @ 15000 ppm, 25000ppm and 35000ppm to group G1, G2 and G3 respectively, for a period of 12 weeks. The birds of group C served as control. Birds were sacrificed for study of gross (macroscopic) lesions if any. In each slaughter, blood and serum samples were collected to assess the haematological and biochemical parameters. A significant decrease in haemoglobin (Hb), packed cell volume(PCV), total erythrocyte count(TEC) and total leucocyte count (TLC) were observed. However, a significant increase in haemoglobin and PCV in group G2 on 90th day and group G3 on 60th and 90th day of experiment were observed. The results of biochemical tests presented a significant decrease (P<0.01) in glucose and total cholesterol in all the experimental groups as compared to control. The levels of total protein, albumin and globulin were significantly decreased in group G1 from 20th to 90th day of experiment, while in group G2 and G3 a significant linear decrease in total protein, albumin and globulin were observed from 20th to 60th day of experiment while on 90th day a significant (P<0.05) increase was observed in group G2. In group G3 a significant (P<0.05) increase was observed on 60th and 90th day of experiment as compared to 20th day of experiment. A significant (P<0.05) increase in creatinine, alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase(ALP) levels were found in all the experimental birds as compared to control. It is concluded from this study that zinc sulphate above 15000 ppm produced hepatotoxic and nephrotoxic effects after oral administration for 90 days in drinking water in cockerels. Key words: Chronic toxicity, cockerels, haemaoto-biochemical, toxicity, zinc sulphate.
INTRODUCTION Amongst various trace minerals, zinc (Zn) is an essential element by being an integral part of more than 200 enzymes and participates in many metabolic and enzymatic functions in the body. Zn is also essential in the proper functioning of the immune system by virtue of its role in the functioning of B and T-cells (Sahin et al., 2005).One of the most significant functions of Zn is related to its antioxidant role and its participation in the antioxidant defense system (Powell, 2000). Proper concentration is of key importance to the proper functioning of heterophils, mononuclear phagocytes and T lymphocytes (Hengmin et al., 2004). As Zn has high bioavailability from zinc sulfate (ZnSO4), this study was undertaken to evaluate its chronic toxicity following its administration at high levels in drinking water in cockerels. MATERIALS AND METHODS Experimental design The experiment was carried out on eighty, one month old white leghorn cockerels. After one week of acclimatization, the chicks were randomly divided into four groups viz control (C), G1, G2 and G3 with 20 birds each. Zinc sulphate was added in drinking water @ 15000, 25000 and 35000 ppm to group G1, G2 and G3 for a period of 12 weeks. Feed and water was provided in ad libitum throughout the study. Birds were sacrificed to examine gross lesions, if any. On each slaughter blood and serum samples were collected to
record haematological and biochemical parameters. Haemato-biochemical examination Total erythrocyte count (TEC, 106/mm3) and total leucocyte count (TLC, 103/ mm3) were done according to the method of Natt and Herric (1952) using a diluents containing 0.1M Nacl, 0.008M Na2HPO4..12 H2O, 0.002M KH2PO4, 0.017M Na2SO4, 7.5ml formaldehyde and 0.1 g methyl violet 2 B. Packed cell volume (PCV, %), haemoglobulin (Hb, g/dl) (Jain, 1986) and differential leucocyte count (DLC, %) were done by preparing thin blood smears from a drop of blood without anticoagulant (Lucas and Jamroz,1961). Blood samples were collected from the wing vein of each bird at 20, 40, 60 and 90th day for haematological parameters like total protein, albumin, globulin and total cholesterol, glucose by and creatinine were estimated by diagonistic kits. Serum enzymes AST and ALT were estimated by method of Moss and Henderson (1994). The data were expressed as MeanS.E and statistically analyzed by two way ANOVA described by Snedecor and Cochran (1989). RESULTS AND DISCUSSION In the present study, a significant decrease (p<0.05) in Hb and PCV was observed in all the experimental groups (G1, G2 and G3) in comparison to control. In group G2 a significant increase (P<0.05) in PCV and Hb level was observed on 90th day in comparison to 60th day of experiment. In group G3 a significant increase
Mudasir et al.
TABLE 1: Haematological parameters following oral administration of zinc sulphate @ 15000 , 25000 and 35000 ppm for 90 days (Mean SE) in cockerels.
Groups/ Time Interval (days)
20 40 60 90
96
20 40 60 90
(p<0.05) in PCV concentration was observed on 60th and 90th day in comparison to 20th and 40th day of experiment (Table 1).The decrease in Hb and PCV might be due to the interference of high zinc levels with iron and copper absorption leading to anemia..Smith and Larson (1946) reported that high levels of dietary zinc results in copper deficiency which leads to macrocytic hypochromic anemia. This may be a cause of decreased Hb and PCV levels in this study. Mulhern et al. (1956) reported decrease in hematocrit in second generation mice on administration of excess dietary zinc @ 2000 ppm in maternal diet for 8 weeks. Ott et al. (1966) observed decrease in Hb and PCV in cattle following higher doses of ZnO @ 2.1g and 3.1 g. Hilmy et al. (1987) also reported decrease in hemoglobin and haematocrit values at high level of zinc in Catfish. Increase in PCV and Hb concentration in group G2 and G3 on 60th and 90th day of experiment may be due to decrease in water intake by experimental birds that might have led to haemoconcentration. A significant decrease (P<0.05) in TEC was observed in experimental groups (G2 and G3) throughout the study in comparison to control group. However, in group G1 a significant decrease on 60th day and 90th day was observed as compared to 20th and 40th day of experiment (Table 1).The decrease of TEC might be due to either altered erythropoiesis or hemolysis. In the present study, a significant decrease (P<0.05) in TLC was observed in all the experimental groups (G1, G2 and G3) in comparison to control group (C).Group G3 had significant decrease (P<0.05) as compared to G2 and G1 as shown (Table 1). In the present study, the differential leucocyte count (DLC) did not differ significantly in experimental and control birds (Table 2). Haematological findings of this study are in agreements with the findings of Maita et al. (1981) who reported a significant decrease (P<0.05) in haematocrit, Hb, PCV, TEC without a significant in DLC after administration of high doses of zinc sulphate (ZnSO4) @ 3000 ppm and 30000 ppm in a 13 week study in rats. Nurcan Donmez et al. (2001) also reported insignificant variation in DLC of broiler chicks fed 1000 ppm in drinking water for a period of 8 weeks. In the present experiment a linear significant decrease (P<0.05) in serum glucose and cholesterol concentration was observed throughout the experiment in comparison to control (Table 3).Similar findings were also reported by Levengood et al. (2000) and Maita et al. (1981) who also found a significant decrease in glucose concentration in zinc dosed ducks and rats. During the present study a significant decrease in total protein , albumin and globulin was observed in all the experimental groups G1, G2 and G3 after 90 days, however, in group G2 and G3 a significant increase was observed on 60th and 90th day as compared to 20th and 40th day of experiment (Table 3) which could be attributed to less intake of water due to reduced feed intake. Decrease in plasma protein, albumin and globulin could be attributed to renal excretion or impaired protein synthesis or due to liver disorder (KoriSiakpere, 1995). Results are in accordance to the findings of other worker (Maita et al.,1981; Levengood et al.,2001). During the present study, a significant increase (P<0.05) in serum creatinine was observed on 90th day as compared to 20, 40 and 60th day of experiment in group G1 and G2. In group G3, a linear significant increase (P<0.05) in creatnine was observed from 20th to 90th day of experiment. (Table 3). An increase in the serum creatinine levels gave an indication
a,b,c,d- mean values in columns with no common superscripts differ significantly (p<0.05) , w,x,y,z - mean values in rows with no common superscripts differ significantly (p<0.05)
27.661.45a,z 22.330.881b,z 25.330.881c.y 280.577 d,x 30.000.577 a,y 270.577b,y 230.577c,z 26.330.881 d,y 36.831.01a,w 370.577 a,w 360.577 a,w 361.00a,w Hemoglobin (g/dl) 8.500.478a,z 6.930.296c,z 7.90.208b,z 8.660.120a,z 10.660.440a,w 10.510.246a,w 10.490.452a,w 10.40.321a,w 9.910.076a,x 9.530.068b,x 9.330.139b,x 9.230.033c,x PCV (%)
2.600.028a,w 2.610.02a,w 2.620.05a,w 2.610.02a,w
2.490.023a,w 2.430.005a,x 2.220.011b,x 1.890.14c,x
G1
9.430.120a,y 8.400.057b,y 8.040.05c,y 8.430.120b,x TEC X 106 cells /l 2.440.017a,x 2.320.011 b,y 2.080.094c,y 1.630.19d,y
G2
2.400.02a,x 2.270.015b,y 1.890.098c,z 1.460.097d,z
G3
25.150.596a.w 24.850.509a,w 24.500.181a,w 24.880.563a.w
33.830.6a,x 300.577 b,x 27.331.20c,x 24.330.881d,,z TLC X 103 cells /l 23.280.35a,x 21.660.33b,x 19.980.338c,x 17.760.145d,x
G1
21.950.033 a,y 18.740.211 b,y 17.450.259c,y 15.460.257 d.y
G2
18.320.196a,z 16.460.264b,z 15.590.163c,z 12.860.131d,z
G3
TABLE 2: Heterophils and monocytes count on DLC following oral administration of zinc sulphate @ 15000 , 25000 and 35000 ppm for 90 days (Mean SE) in cockerels.
G1 G2 G3 C G1 G2 G3 C G1 G2 G3
Groups Time interval (days) Heterophils (%) 2.50.5a,w 1.50.5a,w 21.5a,w 20.75a,w 21.0a,w 10.01a,w 1.50.5a,w 1.50.5a,w 1.50.5a,w 1.50.5a,w 10.25a,w 21.0a,w 20.5a,w 1.50.5a,w 2.51.5a,w 31.0a,w 2.50.5 a,w 2.50.5 a,w 3 0.5a,w 3 1.0a,w 3.51.5a,w 2.51.5a,w 42.0a,w 62.5a,w 10.06a,w 0.50.02a,w 0.50.02a,w 0.50.02a,w 00.0a,w 00.0a,w 00.0a,w 10.6a,w Monocytes (%) Basophils 10.05a,w 0.50.02a,w 10.06a,w 0.50.03a,w
20 40 60 90
30 1.0a,w 31 1.0a,w 31 1.0a,w 31.5 0.5a,w
32 1.0 a,w 31.50.5a,w 31.51.5a,w 29 1.0 a,w
10.05a,w 00.0a,w 10.05a,w 20.5a,w
20 40 60 90
64.50.5a,w 65.51.0a,w 658.0a,w 651.0a,w
32.5 1.5a,w 32.5 0.5a,w 33 1.0a,w 35.51.25a,w 32.50.5,a,w 34.5 0.5a,w 34 0.25a,w 36.51.5a,w Lymphocytes (%) 641.2a,w 62.51.5a,w 611.0a.w 651.5a,w 63.50.5a,w 60.50.5a,w 64.50.5a,w 61.50.5a,w 581.0a,w 64.50.5a,w 58.50.45a,w 52.50.5a,w
31.0a,w 2.50.5a,w 31.0a,w 4.51.5a,w Eosinophils(%) 10.02a,w 10.2a,w 20.45a,w 2.50.5a,w
a,b,c,d- mean values in columns with no common superscripts differ significantly (p<0.05), w,x,y,z - mean values in rows with no common superscripts differ significantly (p<0.05)
TABLE 3: Biochemical parameters following oral administration of zinc sulphate @ 15000 , 25000 and 35000 ppm for 90 days (Mean SE) in cockerels. G2 G3 C G1 G2 G3 C G1 G2 G3
Groups Time interval (days) Glucose (mg/dl) 175.511.21a,z 171.281.36b,z 163.331.47c,z 156.610.60d,z 1.870.043a,y 0.870.026d,z 1.110.065c,y 1.290.068b,x 0.770.007a,z 0.770.006a,z 0.780.001a,z 0.780.008a,z 154.390.42a,w 152.690.65a,w 151.941.10a,w 151.824.24a,w Cholesterol (mg/dl)
G1
Albumin (g/dl) 1.580.008a,w 1.610.008a,w 1.590.023a,w 1.650.01a,w 3.830.063a,w 3.790.046a,w 3.720.035a,w 3.690.018 a,w
20 40 60 90
2131.52a,w 211.160.78a,w 214.020.96a,w 212.920.14a,w
198.01.52a,x 187.661.45b,x 183.490.62c,x 178.810.96d,x
20 40 60 90
2.240.071a,w 2.150.057a,w 2.120.051a,w 2.040.017a,w
2.180.075a,w 1.590.073b,x 1.300.063c,x 1.180.023d,x
186.01.52a,y 180.911.86b,y 177.421.40c,y 170.370.91d,y Globulin (g/dl) 2.080.063a,x 1.00.055d,y 1.270.650b,x 1.110.092c,x
152.331.25a,x 150.671.67a,x 148.931.06a,y 148.201.21b,x 145.480.75b,y 141.640.84b,z 146.241.02c,x 141.930.41c,y 139.070.87c,z 142.281.15d,x 136.841.32d,y 132.200.13d,z Creatinine (mg/dl) 0.810.007c,y 0.840.003c,x 0.860.004d,w 0.830.002c,y 0.850.007c,x 0.880.009c,w 0.840.002b,y 0.890.005b,x 0.920.014b,w 0.890.002a,y 0.970.011a,x 0.980.003a,w
1.410.035a,x 1.290.037a.y 1.080.066b,z 1.250.035b,x 1.050.033c,y 0.900.041c,z 1.00.008c,x 0.770.060d,y 1.090.022b,z 0.80.0.031d,z 1.140.036b,y 1.290.039a,x Total protein (g/dl) 3.590.09 a,w 3.380.077a,w 2.950.031a,x 2.840.066a,x 2.050.023b,x 1.780.058a,y 2.310.065b,x 1.880.032b,x 2.180.043a,x 1.980.014b,x 2.230.064b,x 2.580.102a,x
a,b,c,d- mean values in columns with no common superscripts differ significantly (p<0.05) , w,x,y,z - mean values in rows with no common superscripts differ significantly (p<0.05)
TABLE 4: Activities of serum enzymes following oral administration of zinc sulphate @ 15000 , 25000 and 35000 ppm for 90 days (Mean SE) in cockerels. G2 ALT (U/L) G3 C G1 G2 AST (U/L) 37.980.52a,w 37.870.35a,w 37.690.26a,w 38.060.16a,w 41.790.42d,x 48.550.56b,x 44.790.45c,x 53.210.34a,x 44.780.43d,y 52.290.83b,y 48.500.36c.y 55.470.46a,y G3 C G1 G2 ALP (U/L) 47.670.61d,z 55.300.49b,z 52.040.7c,z 59.070.35a,z 112.171.30a,z 110.760.86a,z 114.331.20a,z 1131.56a,z 119.031.61c,y 125.992.30b,y 129.472.28a,y 1311.56a,y 123.221.70c,x 131.531.73b,x 134.941.04a,x 135.840.64a,x G3
Groups Time Interval (days)
G1
20 40 60 90
90.372.01a,z 90.631.16a,z 90.910.89a,z 91.581.34a,z
94.210.4d,y 96.930.68d,x 99.811.22d,w 95.600.66c,y 98.420.16c,x 101.700.94c,w 99.380.37b,y 104.950.61b,x 110.470.31b,w 105.501.73a,y 111.560.71a,x 116.620.43a,w
125.131.22c,w 136.001.41b,w 136.660.92b,w 139.030.67a,w
Zinc sulphate toxicity in cockerels
a,b,c,d- mean values in columns with no common superscripts differ significantly (p<0.05) ,w,x,y,z - mean values in rows with no common superscripts differ significantly (p<0.05)
Mudasir et al.
of probable kidney damage. Histopathological changes in kidneys were also reported by authors which could be correlated with the above biochemical findings. Similar results were observed by Llobet et al. (1988) with a significantly increased creatnine in rats on administration of high levels of dietary Zn @ 640mg/kg-day for a period of 12 weeks. There was a significant increase in ALT and AST activities in all the experimental groups G1, G2 and G3 at all intervals as compared to control (Table 4). Similarly, ALP had a significant increase in group G1 and G2 on 20, 40 and 60th day of experiment with a non-significant increase on 90th day as compared to 60th day of experiment. In group G3, a significantly increased ALP was observed from 20th to 90th day of experiment (Table 4).Increased activities of serum AST and ALT enzymes indicate possible hepatic damage. These enzymes being cytoplasmic in location are released into general circulation after hepatocytes degeneration (Ahmed and Khater, 2001). This finding was further supported by histopathological changes of liver reported earlier by the authors. Change in the enzymic activities is in agreements with the findings of other repots by Maita et al. (1981) in rats and Levengood et al. (2000) in Zn intoxicated mallards. It is concluded from this investigation that oral administration of zinc sulphate above 15000 ppm for 90 days in drinking water produced hepatotoxic and nephrotoxic effects in cockerels ACKNOWLEDGEMENT The authors are thankful to Span Diagnostic Limited and ERBA Limited for providing kits for this study and Director of Instructional Poultry Farm Nagla for providing the experimental birds. REFERENCES Ahmed, M.B. and Khater, M.R. (2001).The evaluation of the protective potential of Ambrosia maritima extract on acetaminophen-induced liver damage.J. Ethnopharmacol. 75: 69-174. Hengmin Cui, Peng Xi, Deng Junliang, Li Debing and Yang Guang. (2004).Pathology of Lymphoid organs in chickens fed a diet deficient in zinc. Avian Pathol. 33 (5):519-524. Hilmy, A.M., El-Domiaty, N.A., Dadbees, A.Y. and Latife. H. A.A. (1987). Toxicity in Tilapia zilli and Clariaslazera (Pisces) induced by zinc, seasonally. Comparative Biochemistry and Physiol. 86: 263-265. Jain, N.C. (1986). Schalms Veterinary Haematology. 4th Ed. Philadeiphia, Lea and Febringer. Kori- Siakpere, O., (1995). Some alterations in haematological parameters in Clariasisheriensis (Sydenham) exposed to sublethal concentration of water borne lead. Bioscience Res. Commun.8(2):
93-98. Leavengood, J.M., Sanderson, G.C., Anderson, W.L., Foley, G.L., Brown, P.W. and Seets, J.W. (2000).Influence of diet on the hematology and serum biochemistry of zinc-intoxicated mallards.Journal of Wildlife Diseases. 36: 111 123. Llobet, J.M., Domingo, J.L. and Colomina, M.T. (1988).Subchronic oral toxicity of zinc in rats. Bull. Environ. Contam.Toxicol.,41: 36-43. Lucas,A.M. and Jamroz, C. (1961).Atlas of Avian Hematology, Govt. Printer, Washington, D.C. Maita, K., Hirano, M., Mitsumori, K., Takahashi, K. and Shirasu, Y.(1981).Subacute toxicity studies with zinc sulfate in mice and rats. J. Pestic. Sci.6: 327-336. Moss, D.W. and Henderson, A.K. (1994). Clinical enzymology, In Tietz Textbook of clinical chemistry, 3rd ed., C.A. Burtis and E.R. Ashwood, Eds. W.B. Saunders, Philadelphia. pp 617-721. Mulhern, S.A.,Stroube, W.B. and Jacobs.R.M. (1956). Alopecia induced in young mice by exposure to excess dietary zinc. Biomedical and Life Sciences Cellular and Molecular Life Sc. 42(5): 551-553 Natt, M.P. and Herrick, C.A. (1952).A new blood diluent for counting the erythrocytes and leucocytes in the chicks.Poult. Sci.31: 735-778. NurcanDonmez, HseyinDnmez, H., Ercankeskin and ilhamiCelik, (2001).Effects of Zinc Supplementation to Ration on Some Hematological Parameters in Broiler Chicks. Biol. Trace Elem. Res. 87:125-131. Ott, E. A., Smith, W.H., Harrington, R.B. and Beeson, W.M. (1966).zinc toxicity in ruminants I. Effect of high level of dietary zinc on gains, feed consumption and feed efficiency of lambs.J. Ani. Sci. 25: 414-418. Powell S.R.( 2000). The antioxidant properties of zinc. J. Nutr. 130: 1447S-1454S. Prasad, A.S. and Kucuk, O. (2002).Zinc in cancer prevention. Cancer Metastasis Rev. 21:291295. Sahin, K., Smith, M.O., Onderci, M., Sahin, N., Gursu, M.F. and Kucuk, O., (2005).Supplementation of zinc from organic or inorganic source improves performance and antioxidant status of heatdistressed quail. Poult. Sci.84: 882-887. Smith, S.E. and Larson, E.J. (1946). Zinc toxicity in rats: antagonistic effects of copper and liver. J. Biol Chem.163: 29-38. Snedecor, G.W. and Cochran, W.G. (1989).Statistical Methods.8th ed. Ames, Iowa State University Press. Received on : 22- 09-2011 Accepted on: 12-02-2012
98
Short Communication
ANTIBACTERIAL ACTIVITIES OF MEDICINAL PLANTS OF NORTH-WEST HIMALAYAN REGION

D.K. SHARMA1, C. VARSHNEYA2 AND C. SHEKHAR3
1
Asstt.Prof., 2Professor and Head, Department of Pharmacology & Toxicology, 2Dean, DGCNCOVAS, CSKHPKVV, Palampur-176062 (H.P.); 3Veterinary officer Central research institute (CRI) Kasauli (H.P) 1 Corresponding author: Email: dineshcovas79@gmail.com
ABSTRACT
The present study was designed to screen the antibacterial activities of methanolic extracts of Bauhinia Variegata (Bark), Glycyrrhiza glabra (stem), Murraya Koenigii (leaves) and Hippophae rhamnoides (leaves) by invitro disc diffusion method. The antibacterial activity of the medicinal plants was tested against Staphylococcus aureus (S.aureus), Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa), Bacillus subtilis (B.subtilis), Proteus mirabilis (P.mirabilis) and Klebsiella pneumonia (K.pneumonia). The results revealed that methanolic extract of H. rhamnoides (leaves) showed maximum zone of inhibition against all the bacterial strains. The maximum zone of inhibitions of B. Variegata (bark) and G. glabra (stem) were observed against S. aureus). M.koenigii and H. rhamnoides (leaves) extract showed maximum activity against P. aeruginosa when compared with other bacterial species. It is concluded from this study that the plant extracts of B. variegata, G. glabra, M.Koenigii and Hippophae rhamnoides (leaves) possessd good antibacterial activity. Key words: Bauhinia Variegata , Glycyrrhiza glabra , Murraya Koenigii, Hippophae rhamnoides
The development of drug resistance as well as appearance of undesirable side effects of certain antibiotics (WHO, 2002) has led to the search of new antibacterial agents including medicinal plants. A number of reports concerning the antibacterial screening of plant extracts of medicinal plants notably have appeared in the literature (Salvat et al., 2001). The present study was designed to screen the antibacterial activities of four medicinal plant extracts; Bauhinia Variegata (B.Variegata), Glycyrrhiza glabra (G.glabra), Murraya Koenigii (M.Koenigii) and Hippophae rahmnoides (H. rahmnioides) The medicinal plant parts, Bauhinia Variegata (bark), Glycyrrhiza glabra (stem), Murraya Koenigii (leaves) were collected locally from Palampur (H.P.).The leaves of Hippophae rahmnoides were brought from Lahaul region of H.P. The medicinal plant parts were shade dried and grounded to fine powder. The powder was extracted with methanol and lyophilized and stored for further use. The antibacterial activity of the medicinal plants was tested against Staphylococcus aureus (S.aureus), Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa), Bacillus subtilis (B.subtilis) , Proteus mirabilis (P.mirabilis) and Klebsiella pneumonia (K.pneumonia). The screening of antibacterial activity was carried out by disc diffusion method (Bauer et al.,1966). McFarland 0.5 standardized suspension of bacteria (108 CFU/ml) was used to lawn Muller Hinton agar plates (MHA) evenly using a sterile swab.10l of each extract (5mg/ml) was added to separate sterile filter paper discs (6mm) and then allowed to dry. The discs were placed by sterile forceps on to the MHA plate at a distance of 24mm. The ampicillin disc (10g) was used as positive control. The
plates were incubated at 370c for 18-24h. After the incubation, the plates were examined for zone of inhibition. The inhibition zones were then measured in triplicate using vernier callipers. The antibacterial activities of all the plant extracts against the bacterial strains were examined and were assessed by the presence or absence of inhibition zones (Table1). Antibiotic Ampicillin was used as reference control. On comparison, H. rahmnoides showed maximum zone of inhibition against all the bacterial strains. (Upadhyay et al., 2010) reported that H. rahmnoides leaf extracts had growth inhibiting effect against Bacillus cereus , Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. The maximum zone of inhibitions of B. Variegata (bark) and G. glabra (stem) were observed against S. aureus (130.58 & 10.670.33, mm in diameter,respectively). M.koenigii and H . rahmnoides leaves extract showed maximum activity against P. aeruginosa.( 11.670.33 & 16.330.33,mm in diameter,respectively) when compared with other bacterial species. No activity was observed by M.koenigii against P. mirabilis. This could be due to the distinctive feature of gram-negative bacteria, which is the presence of a double membrane surrounding each bacterial cell. In case of gramnegative bacteria this outer membrane excludes certain drugs and antibiotics from penetrating the cell. The activities of all the plant extracts used were however less when compared to the standard antibiotic ampicillin(Table1). From these studies it could be concluded that extracts of B. variegata, G. glabra, M.Koenigii and H. rahmnoides have antibacterial activity.
Antibacterial activities of medicinal plants
TABLE 1. Antibacterial activity of different plant extracts against pathogenic bacterial strains. Name of Bacteria Ampicillin* Zone of inhibition (mm)
B. Variegata (bark)
130.58 12.5 0.29 10.330.88 9.330.44 90.58 9.330.33
G. glabra (stem)
10.670.33 8.50.29 9.330.33 8.830.17 9.170.17 8.50.29
M. Koenigii (leaves)
9.50.29 8.670.44 11.670.33 9.330.33 8.670.33
H.rhamnoides (leaves)
14.670.33 160.58 16.330.33 150.58 160.58 15.50.29
S.aureus(G+ve) E.coli (G-ve) P. aeruginosa (G-ve) B. subtilis (G+ve) P. mirabilis (G-ve) K. pneumonia (G-ve)
18.670.33 210.58 16.670.33 10.33 0.88 21.670.44 190.58
Values are triplicate mean Standard error; Amp* - Standard antibiotic disc of Ampicillin (10 g /disc) G +ve= Gram positive, G-ve = Gram negative
The antibacterial properties of the plant extracts may be due to their phytochemical constituents. Further studies are needed to isolate and characterize the bioactive compounds responsible for the antibacterial activity. REFERENCES Bauer, R.W., Kirby, M.D.K., Sherris, J.C. and Turck M. (1966). Antibiotic susceptibility testing by standard single disc diffusion method. American J. Clinical Pathol. 45: 493-496. Salvet, A., Antonnacci,L., Fortunado, R.H., Suarez, E.Y. and Godoy, H.M. (2001). Screening of some
plants from northern Argentina for their antimicrobial activity J. Ethnopharmacol. 32: 293297 Upadhyay, N.K., Kumar, M.S. and Gupta, A. (2010). food Chem Toxicol. Antioxidant, cytoprotective and antibacterial effects of Sea buckthorn (Hippophae rhamnoides L) leaves. 48: 3443-3448. WHO (2002) Traditional Medicine Strategy, 20022005.WHO Publication 1-6.
Executive Committee of ISVPT for 2014-15

President Vice President Secretary General Finance Secretary Joint Secretary Chief Editor, JVPT Secretary International Relations Secretary (HQ) Immediate Past Secretary General Executive Body Members : : : : : : : : : : Dr. Anil Kumar Srivastava (Karnal) Dr (Mrs) Mudasir Sultana (Jammu) Dr. N. Gopa Kumar (Thrissur) Dr. S.K. Bhavsari (Navsari) Dr. Thakur Uttam Singh (lzatnagar) Dr. S.P. Singh ( Pantnagar) Dr. A.H. Ahmad (Pantnagar) Dr. A M Thaker (Anand) Dr. C. Varshneya (Palampur) Dr. Raju Prasad (Ranchi) Dr. Nitesh Kuinar (MHOW) Dr. Shahid Prawej (Jammu) Dr. Vinod Kumar Dumka (Ludhiana) Dr. Dhirendra K.umar (Izatnagar) Dr. Jagadeesh Sanganal (Bangalore)
100
Short Communication
PREGNANCY-DEPENDENT ALTERATIONS IN SENSITIVITY OF SEROTONERGIC RECEPTORS IN BUFFALO MYOMETRIUM

ABHISHEK SHARMA, SOUMEN CHOUDHURY, UDAYRAJ P NAKADE, RAJKUMAR SINGH YADAV AND SATISH KUMAR GARG*
Department of Pharmacology and Toxicology, College of Veterinary Sciences and Animal Husbandry UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Gau Anusandhan Sansthan, Mathura, U.P. (281 001), India. *Corresponding Author: email: profsatish@gmail.com; Phone: +91-9456054995
ABSTRACT
5-HT (serotonine) is known to be an important amine and involved in regulating several physiological processes including maintenance of pregnancy. But it almost failed to produce any marked contractile effect on buffalo myometrium (pregnant) and even with highest concentration of 10-5 M, the contractile effect (Emax) was found to be only 0.25 0.03g (early pregnancy) and 0.08 0.01g (mid pregnancy). Preliminary observations revealed that buffalo myometrium was almost insensitive to 5-HT which further decreased with an increase in the stage of pregnancy and such an effect might have been either due to lack of serotonergic receptors or required sensitization by some regulator (s), however, it needs further investigations. Key words: Buffalo myometrium, serotonin, pregnancy.
Serotonin (5-hydroxytryptamine, 5HT) is a wellknown neurotransmitter in the central nervous system apart from being present in cells of many other systemsenterochromaphin cells of stomach and small intestines and intestinal neurons, uterine mast cells and uterine tubes, ovaries and uterine cervix. The function of serotonin is complex, probably due to diversity of serotonin receptors located directly on smooth muscle cells, on nerve endings and within the central nervous system. It has been discovered that serotonin, in various animal species, has stimulating or inhibiting influence on the motor activity of alimentary tract (Ford et al., 1992). It also produces differential effects on the motor activity of uterine smooth muscles in human and animal species. But despite its presence in uterine tissues of buffaloes (Sharma et al., 2003), no information is available on its effect on buffalo myometrium, therefore, the present study was undertaken. Myometrial strips from pregnant uteri of nondescript buffaloes were collected from local abattoir. Uteri were cut open to ascertain the stage of pregnancy (early or mid) based on the presence of foetus and its curved crown versus rump length (Soliman et al., 1970). Myometrial strips were prepared and mounted in thermostatically controlled (37.0 0.5 C) organ bath (Ugo Basile, Italy) of 10 ml capacity containing continuously aerated (95% O2 + 5% CO2) Ringer Locke solution (RLS, pH 7.4). Isometric tension was recorded under 2g resting tension using Chart V5.4.1 software programme (Powerlab, AD Instruments, Bella Vista, NSW, Australia). Following stabilization of tissues, myometrial strips were exposed to cumulative concentrations of 5-HT at 1.0 log dose unit and the effect of increasing concentrations of 5-HT on myometrial spontaneity of
different stages of pregnant buffaloes uteri are shown in Fig 1. 5-HT exhibited concentration-dependent contractile effect on early (0-3 months; Fig. 1A) and mid stage pregnant uteri (3-6 months; Fig. 1B). The minimum threshold concentration of 5-HT required to initiate contractile effect was 10-9 M and the maximal effect on early pregnancy was observed at 10-5 M concentration. On mid-pregnancy stage uterus (3-6 months), 5-HT initially exhibited dose-dependent excitatory effect with maximal contractile effect at 10-6 M but with further addition of higher dose, 5-HT produced slightly inhibitory effect as shown in Fig. 1B. On mid-pregnancy myometrium, there was significant (P<0.05) reduction in 5-HT-induced contraction at all the dose levels compared to that on early-pregnant uterus. Fig. 2 illustrates the cumulative concentrations response curves of 5-HT on myometrial strips from uteri of during different stages of pregnancy (early and mid). pD2 and the maximal response (Emax) values revealed that there was significant (P<0.05) reduction in the maximum contraction in mid stage (0.08 0.01 g; n=5) of pregnancy compared to that on early pregnancy (0.25 0.03 g; n=5) but without appreciably affecting the potency as the pD2 values ranged between 9.59 and 9.65. 5-HT is known to be potent spasmogen on uteri of human (Rudolph et al., 1993), rats (Helton and Colbert, 1994), and rabbit myometrium (Sohorova et al., 1991), but produces inhibitory effect on pig uterus through 5HT7 receptors (Kitazawa et al., 1998). Emax value of just 0.25 0.03 g in early pregnancy and 0.08 0.01 g in mid pregnant uteri suggested that buffalo myometrium was almost insensitive to the effect of 5-HT. Our results on buffalo myometrium are in
Sharma et al.
Fig. 1 : Physiographic recordings showing concentration-dependent effect of 5-HT (10-11 10-5 M ) on spontaneity of myometrial strips of early pregnant (A) and mid pregnant (B) buffaloes.
Fig. 2: Cumulative concentration response curves of 5-HT on myometrial strips from early pregnant (0-3 months; n=5) and mid pregnant (3-6 months; n=5) buffaloes. Vertical bars represents SEM. Data were analyzed by two-way ANOVA followed by Bonferoni post-hoc tests
agreement with the electrophysiological studies on sheep myometrium where serotonin failed to produce any contractile effect unless sensitized with stilboestrol (Zawadzki et al., 1999). Therefore, the possibility adequate serotonergic receptors or their sensitization by different regulator (s) including estrogen in buffalo myometrium cant be ruled out, but it requires further investigation. REFERENCES Ford, A.P.D.W., Baxter, R.M., Eglen, R.M. and Clarke, D.E. (1992). 5-Hydroxytryptamine stimulate cyclic AMP formation in the tunica muscularis mucose of the rat oesophagus via 5-HT4 receptors. Eur. J. Pharmacol. 211: 117. Helton, D.R. and Colbert, W.E. (1994). Alterations of in vitro 5-HT receptor pharmacology as a function of
102
multiple treatment with 5-hydroxytryptamine or 8hydroxy-2-(di-N-propylamino) tetralin in rat isolated aorta, uterus and fundus, and guinea-pig isolated trachea. J. Pharm. Pharacol. 46(11): 902. Kitazawa, T., Kubo, O., Satoh, M. and Teneike, T. (1998). Involvement of 5-hydroxytryptamine 7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine. Br. J. Pharmacol. 123: 173. Rudolph, M.I., Reinicke, K., Cruz, M.A., Gallardo, V., Gonzalez, C. and Bardisa, L. (1993). Distribution of mast cells and the effect mediators on contractility in human myometrium. Brit. J. of Obst. and Gynaecol. 100: 1125. Sharma, R. K., Pant, H.C., Sabir, M. and Garg, S. K. (2003). Concentration of 5-HT (serotonine) in placenta and uterus of buffaloes during pregnancy. Indian J. Animal Sci. 72:185-186. Sohorova, R., Mosnarova, A., Huzulikova, I. and Babel, P. (1991). Comparison of changes in uterine reactivity to endogenous substances in rabbits and the effects of in-vivo and in-vitro administration of estradiol and testosterone. Bratisl. Lek. Listy. 92: 346. Soliman, M.K. (1970). Studies on the physiological chemistry of the allantoic, amniotic fluids of buffaloes at the various periods of pregnancy. Indian Vet. J. 52: 106-112. Zawadzki, W., Zieba, D. and Dejneka, J. (1999). Changes of uterus myoelectrical activity under influence of serotonin in sheep sensitised and non-sensitised with stilboestrol, EJPAU. 2(2): 02.
Received on: 16-12-2012 Accepted on : 28-12-2012
JOURNAL OF VETERINARY PHARMACOLOGY AND TOXICOLOGY

ISSN 0972-8872
Official Publication of the Indian Society of Veterinary Pharmacology & Toxicology December 2012 Volume 11 Issue 1-2
CONTENTS
Research Review 1. G-PROTEIN COUPLED RECEPTORS: PAST AND PRESENT STATUS S.P. SINGH, A.H. AHMAD, WASIF AHMAD AND N.K. PANKAJ 1-7
Research Articles 2. CNS DEPRESSANT AND ANTI-CONVULSANT ACTIVITY OF HYDROETHANOL EXTRACT OF 8-12 DRYMARIA CORDATA WILLD C. C. BARUA, J. D. ROY, B. BURAGOHAIN, A. TALUKDAR, A. G. BARUA, P. BORAH AND MANGALA LAHKAR HOMOLOGY MODELING OF CANINE PROSTAGLANDIN G/H SYNTHASE-2 BHASKAR GANGULY, SUDHIR KUMAR, TANUJ AMBWANI AND S.P. SINGH EFFECT OF DOCOSAHEXAENOIC ACID ON CONCENTRATION-RESPONSE RELATIONSHIP OF 5-HT AND REVERSAL OF 5-HT CONTRACTION IN SHEEP CORONARY ARTERY THAKUR UTTAM SINGH, SUBHASHREE PARIDA, SATISH KUMAR GARG, SANTOSH KUMAR MISHRA PROGESTERONE PROFILE AND CONCEPTION RATE FOLLOWING INSULIN TREATMENT DURING LUTEAL PHASE OF ESTROUS CYCLE IN BUFFALOES Q. HUQ, H.P. GUPTA, SHIV PRASAD, V.S. RAJORA AND ASAD HUSSAIN PHARMACOKINETICS OF CEFEPIME IN FEBRILE BUFFALO CALVES SHWETA JAIN, NEETU RAJPUT AND Y. P. SAHNI IMPACT OF SUB-CHRONIC ORAL EXPOSURE OF IMIDACLOPRID ON BIOCHEMICAL PROFILE OF CROSSBRED CALVES BARINDERJIT KAUR, RAJDEEP KAUR AND H.S. SANDHU NITRIC OXIDE AND C-GMP-INDEPENDENT 2-ADRENOCEPTORS MEDIATED TOCOLYSIS IN BUFFALOES SOUMEN CHOUDHURY, THAKUR UTTAM SINGH, SATISH KUMAR GARG AND SANTOSH KUMAR MISHRA THRESHOLD PLASMA FLUORIDE LEVELS IN GOATS FOR SUBACUTE ORAL TOXICITY OF FLUORIDE AND EFFICACY OF ALUMINIUM SULPHATE AS AN AMELIORATIVE AGENT VINAY KANT, A. K. SRIVASTAVA, R. RAINA, P.K. VERMA, N.K. PANKAJ, P. SINGH AND S.K. UPPAL 13-15 16-18
3. 4.
5.
19-21
6. 7.
22-24 25-28
8.
29-32
9.
33-36
10. PHARMACOKINETICS OF LEVOFLOXACIN FOLLOWING SUBCUTANEOUS ADMINISTRATION IN CATTLE CALVES ARVIND KUMAR, ANU RAHAL, RAM RAGVENDRA, RAJESH MANDIL, ATUL PRAKASH AND SATISH K. GARG 11. PHARMACOKINETICS OF PEFLOXACIN IN E.COLI (K88 STRAIN) ENDOTOXIN INDUCED FEBRILE GOATS A. K. NATH, R.K.ROY, D.C.ROY AND R. GOGOI 12. ACUTE TOXICITY AND NEUROBEHAVIOURAL EFFECTS OF ACETAMIPRID IN MICE V. KARTHIKEYAN, S. K. JAIN AND J. S. PUNIA
37-40
41-42 43-49
Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2
CONTENTS
13. COMPARATIVE ANALYSIS OF PESTICIDE RESIDUES IN FODDER AND BUFFALO PLASMA COLLECTED FROM THREE DIFFERENT DISTRICTS OF PUNJAB V. K. DUMKA, H. S. SANDHU, S. RAMPAL, RAJDEEP KAUR AND KAMALPREET KAUR 14. PARALYTIC EFFECT OF PEGANUM HARMALA LINN. ALCOHOLIC EXTRACT ON FASCIOLA GIGANTICA S. W. HAJARE, M. K. LONARE AND DINESH KUMAR 15. HAEMATO-BIOCHEMICAL PROFILE AFTER REPEATED ADMINISTRATION OF PEFLOXACIN IN GOATS NIRBHAY KUMAR, BIPIN KUMAR, S.D. SINGH AND C. JAYACHANDRAN 50-52
53-55 56-58
16. EVALUATION OF TOXICITY OF ENDOSULFAN FOLLOWING MULTIPLE ORAL ADMINISTRATION IN POULTRY 59-61 LATA GAYAL, S.P. SINGH AND A.H. AHMAD 17. SUB ACUTE DERMAL TOXICITY OF BIFENTHRIN WITH SPECIAL REFERENCE TO HAEMATOBIOCHEMICAL CHANGES IN RAT MUNEER AHMAD DAR, RAJINDER RAINA, NRIP KISHORE PANKAJ, MUDASIR SULTANA , PAWAN KUMAR VERMA AND AADIL MEHRAJ 18. PREGNANCY-DEPENDENT ALTERATIONS IN FREQUENCY AND AMPLITUDE OF MYOMETRIAL SPONTANEITY IN BUFFALOES ABHISHEK SHARMA, SOUMEN CHOUDHURY, UDAYRAJ P NAKADE, RAJKUMAR SINGH YADAV AND SATISH KUMAR GARG 19. PROTECTIVE ROLE OF VITAMIN E AND PHENYTOIN IN CHLORPYRIFOS INDUCED DELAYED NEUROPATHY IN HEN K. KAVITHA, B. KALA KUMAR, S.S.Y.H. QUADRI, Y.N. REDDY AND M. ALPHA RAJ 20. STUDY ON IMMUNOTOXIC POTENTIAL OF ALPHACYPERMETHRIN IN WLH CHICKS NAVNEET KUMAR PANDEY AND A.M.THAKER 21. DEVELOPMENT OF THERAPEUTIC MODULE FOR JATROPHA CURCAS SEED AND SEED OIL TOXICITY IN GOATS AMIT SHUKLA AND S.P. SINGH 22. PROTECTIVE EFFECT OF TRIANTHEMA PORTULACASTRUM ON CADMIUM INDUCED TOXICITY IN RATS Y. PAVAN KUMAR, K. ADILAXMAMMA, U. VENKATESWARLU, T.S. CHANDRASEKHARA RAO AND M. ALPHA RAJ 23. DISPOSITION KINETICS OF CEFTRIAXONE AFTER INTRAMUSCULAR ADMINISTRATION IN GOATS NAVEEN KUMAR, B.K. ROY, VIJAY KUMAR, WASIF AHMAD AND N. K. PANKAJ 24. HAEMATO-BIOCHEMICAL PROFILE FOLLOWING MULTIPLE ORAL DOSE ADMINISTRATION OF CHLORPYRIFOS IN POULTRY LATA GAYAL, S.P. SINGH, A.H. AHMAD AND WASIF AHMAD 25. TOXICO-PATHOLOGICAL STUDY OF ALPHACYPERMETHRIN IN DAY OLD WLH CHICKS NAVNEET KUMAR PANDEY AND A.M.THAKER 26. CHRONIC TOXICITY STUDY OF ZINC SULPHATE IN COCKERELS MIR MUDASIR, A.K. THATHOO AND S.P. SINGH Short Communications 27. ANTIBACTERIAL ACTIVITIES OF MEDICINAL PLANTS OF NORTH-WEST HIMALAYAN REGION D.K. SHARMA, C. VARSHNEYA AND C. SHEKHAR 28. PREGNANCY-DEPENDENT ALTERATIONS IN SENSITIVITY OF SEROTONERGIC RECEPTORS IN BUFFALO MYOMETRIUM ABHISHEK SHARMA, SOUMEN CHOUDHURY, UDAYRAJ P. NAKADE, RAJKUMAR SINGH YADAV AND SATISH KUMAR GARG
Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2
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65-68
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72-75 76-79
80-84
85-87 88-90
91-94 95-98
99-100 101-102

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Review Article

G-PROTEIN COUPLED RECEPTORS: PAST AND PRESENT STATUS

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7

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Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7 5

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7

Received on:16-08-2012 Accepted on:26-12-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/1-7 7

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/8-12

Activity of Drymaria cordata

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/8-12 9

Before treatment 154.660.49 142.161.49* 138.500.67* 148.161.49* 185.661.25

Aftertreatment 147.500.56* 123.161.30 95.001.50 87.330.49 121.830.87

% reduction 4.62 13.30 31.30 41.02 34.36

Activity of Drymaria cordata

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/8-12 11

Received on: 21-1-2012 Accepted on: 23-4-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/8-12

HOMOLOGY MODELING OF CANINE PROSTAGLANDIN G/H SYNTHASE-2

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/13-15 13

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/13-15

Homology modeling of canine PTGS-2

Received on: 12-03-2012 Accepted on: 15-05-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/13-15 15

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/16-18

Effect of DHA on sheep coronary artery

Fig. 2. Effect of DHA on reversal of 5-HT-induced contraction in sheep coronary artery.

Fig. 3. Effect of DHA on basal tone in sheep coronary artery.

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/16-18 17

Received on: 11-04-2012 Accepted on: 22-06-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/16-18

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/19-21 19

Days of estrous cycle Day-0 Day-12 Day-14 Day-18 Day -30

Significant at 5% level of significance within group.

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/19-21

Progesterone in insulin treated buffaloes

Received on: 17-02-2012 Accepted on: 22-05-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/19-21 21

PHARMACOKINETICS OF CEFEPIME IN FEBRILE BUFFALO CALVES

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/22-24

Kinetics of cefepime in calves

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/22-24 23

Received on: 15-11-2011 Accepted on: 12-02-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/22-24

IMPACT OF SUB-CHRONIC ORAL EXPOSURE OF IMIDACLOPRID ON BIOCHEMICAL PROFILE OF CROSSBRED CALVES

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/25-28 25

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/25-28

Impact of imidacloprid in crossbred calves

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/25-28 27

Received on: 27-02-2012 Accepted on: 22-04-2012

INDIAN SOCIETY OF VETERINARY PHARMACOLOGY & TOXICOLOGY

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/25-28

NITRIC OXIDE AND C-GMP-INDEPENDENT 2-ADRENOCEPTORS MEDIATED TOCOLYSIS IN BUFFALOES

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/29-32 29

Fig. 1: Spontaneous contractility of isolated myometrial strips of buffaloes.

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/29-32

2-adrenoceptors mediated tocolysis in buffaloes

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/29-32 31

Received on : 14-03-2012 Accepted on : 20-06-2012

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/29-32

Journal of Veterinary Pharmacology and Toxicology/December 2012/Vol.11/Issue 1-2/33-36 33