Research Article

Received: 23 February 2012,

Revised: 1 June 2012,

Accepted: 2 June 2012

Published online in Wiley Online Library: 9 August 2012

(wileyonlinelibrary.com) DOI 10.1002/jat.2794

Sub-chronic exposure to paraoxon neither

induces nor exacerbates diabetes mellitus
in Wistar rat
Syed M. Nurulain,a Georg Petroianu,b Mohamed Shaullah,a Huba Kalsz,c
Murat Oz,a Tariq Saeed,d Abdu Adema and Ernest Adeghated*
ABSTRACT: There is an increasing belief that organophosphorus compounds (OPCs) impair glucose homeostasis and cause
hyperglycemia and diabetes mellitus. The present study was undertaken to investigate the putative diabetogenic effect of
sub-lethal and sub-chronic exposure to paraoxon (POX), an extremely hazardous OPC used in pesticides. The effect of paraoxon on streptozotocin-induced diabetic rats was also examined. Each rat was injected with 100 nmol of POX 5 days per week
for 6 weeks. Blood glucose levels and red blood cell acetylcholinesterase activity were measured weekly. Biochemical analysis
and morphological studies were performed at the end of the experiment. The results revealed that POX neither induces nor
exacerbates diabetes mellitus in experimental rats. Liver and kidney/body weight ratios revealed statistically insignicant
differences when compared with controls. Biochemical analysis of urine samples showed a small but not signicant increase
in protein level in all groups. Urine bilirubin was signicantly higher in the diabetes + POX group when compared with the
control group. The number of blood cells in urine was signicantly higher in the POX-treated group compared with the
control group. Hyperglycemia was noted in the diabetes and diabetes + POX groups, but neither in the saline control
nor in POX-treated normal rats. Electron microscopy of POX-treated pancreas did not show any morphological changes
in beta cells. These results suggest that POX does not cause diabetes mellitus at sub-lethal sub-chronic exposure.
Copyright 2012 John Wiley & Sons, Ltd.
Keywords: paraoxon; diabetes; acetylcholinesterase; hyperglycemia; sub-lethal dose; diabetogenic

Introduction

1036

Organophosphorus anticholinesterase compounds are esters,

amides or thiol derivatives of phosphoric, phosphonic, phosphinic acids, and phosphorothioic or phosphonothioic acids. The
phosphonic acids derivatives are more toxic than the phosphoric acids, whose oxygen atom can be substituted by sulphur or
nitrogen atoms (Bosak, 2006). The rst synthesized organophosphorus compound (OPCs) was a monoester, named tetraethyl
pyrophosphate (TEPP). The compound was synthesized in the
early 1800s by Moschnine (Petroianu, 2008). The process was
rst published in 1854 by de Clermont (Kenneth et al., 2008).
OPCs were primarily synthesized for crop protection against
insect pests but were later used to produce deadly poison such
as nerve agents. OPCs are classied into many groups which are
structurally and toxicologically different. However, a common
mechanism of action is found in all of the different groups of
OPCs: the irreversible inhibition of acetylcholinesterase (EC 3.1.1.7)
with the active centre being the serine hydroxyl group (Delno
et al., 2009). Inhibition of acetylcholinesterase results in the
accumulation of acetylcholine at nerve endings leading to overstimulation of neurons. Each OPC has a unique structural prole
for toxicity and behavior, ranging from extremely toxic nerve
agents to a moderate or slightly toxic OPCs used in pesticides. It
is well known that heavy exposure to OPC causes acute cholinergic
syndromes, which may lead to high mortality and morbidity.
Moreover, chronic or sub-chronic exposure to low doses of
OPC over a period of time may cause a variety of health problems

J. Appl. Toxicol. 2013; 33: 10361043

including neuropathy, pancreatitis, metabolic disruption and

reproductive impairment (Slotkin, 2011; Washam, 2008).
OPCs are one of the most widely used pesticides worldwide.
According to a previous report, 50% of all insecticides used in the
world belong to the OPC group (Casida and Quistad, 2004). These
pesticides are a source of occupational hazards, especially for farmers and pesticides applicators. Its domestic exposure is also common
because of the use of insecticides for roaches, ies, ticks, mites and
other insects. The unintentional or accidental exposure as well as
suicidal use is very common, particularly in third world countries.
Other sources of exposure include food chain, contaminated drinking water and air. OPCs accounts for several hundreds of thousands
of deaths worldwide every year (Karalliedde and Senanayake, 1999).

*Correspondence to: E. Adeghate, Department of Anatomy, Faculty of Medicine

& Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
E-mail: eadeghate@uaeu.ac.ae
a

Department of Pharmacology and Therapeutics, Faculty of Medicine and

Health Sciences, United Arab Emirates University, UAE

Department of Cellular Biology & Pharmacology Herbert Wertheim College of

Medicine, Florida International University, USA

Department of Pharmacology and Pharmacotherapy, Semmelweis University,

Budapest, Hungary

Department of Anatomy, Faculty of Medicine & Health Sciences, United Arab

Emirates University, Al Ain, United Arab Emirates

Copyright 2012 John Wiley & Sons, Ltd.

Paraoxon and diabetes mellitus

Although, the main target of OPC action is the central and
peripheral nervous systems, there is growing evidence that
OPC may cause a lot more disorders outside of the central and
peripheral nervous systems (Lukaszewicz-Hussain, 2010; Washam,
2008). For example, clinical and animal model studies have shown
that some OPCs cause hyperglycemia after acute exposure
(Husain and Ansari, 1988; Matin and Hussain, 1987; Meller et al.,
1981; Shobha and Prakash, 2000). A summary of studies that
showed a link between OPCs and hyperglycemia is given in
Table 1AD. According to Saldana et al. (2007) and Rezg et al.
(2010) there is a link between insecticide exposure and diabetes
mellitus. Montgomery et al. (2008) identied 10 OPC insecticides
that cause diabetes in humans, especially after long-term exposure. These agents include chlorpyrifos, diazinon and trichlorfon.
The exact mechanism responsible for the OPC-induced type 2
diabetes is unclear but the proposed mechanisms include oxidative stress, nitrosative stress, inhibition of paraoxonase, induction
of inammatory cytokines, stimulation of the adrenal gland, disturbed metabolism of the liver and inhibition of cholinesterases
(Rahimi and Abdollahi, 2007; Li and JianShe, 2009; Everett
and Matheson, 2011) and disturbance in glucose homeostasis
(Abdollahi et al., 2004; Rezg et al., 2010; Kamath and Rajini, 2007).
The majority of investigators reported that exposure to OPC
induces hyperglycemia (Ambali et al., 2011; Rahimi and Abdollahi,
2007; Seifert, 2001). According to Abdollahi et al. (2004) and
Slotkin (2011) OPCs are predisposing factor of diabetes mellitus.
Type 2 diabetes is characterized by insulin resistance, which is
compensated initially by increased insulin production. Over the
course of the disease, the pancreas fails to produce sufcient
insulin to stimulate adequate glucose uptake in adipose and
muscle tissues leading to hyperglycemia and type 2 diabetes.
Animal studies have demonstrated that tolerance to OPCs exposure develops over time, most probably as a result of decreased
expression of muscarinic receptors (Costa et al., 1982). This may
potentially lead to a decrease in insulin production. Moreover,
prolonged acetylcholine release may reduce cells sensitivity
to glucose (Gilon and Henquin, 2001).
The objective of this study was to investigate the possible
diabetogenic effect of a sub-lethal dose and sub-chronic exposure
to paraoxon (POX), an OPC compound, in adult male Wistar rats.
With the exception of azinfos-methyl, the majority of the tested
compounds belong to class III i.e. slightly hazardous, according to
WHO classication of pesticides (World Health Organisation,
2009). Therefore, it is important to determine whether POX, an
extremely hazardous OPC, is diabetogenic or not.

Material and Methods

Experimental Animals

J. Appl. Toxicol. 2013; 33: 10361043

Model of Diabetes Mellitus

After an overnight fast, rats for the model of type 1 diabetes
were rendered diabetic by a single intra-peritoneal (i.p.) injection
of streptozotocin (60 mg kg1 body weight) in a freshly prepared
citrate buffer (0.1 M pH 4.5) solution. The streptozotocin-injected
animals exhibited hyperglycemia within 24 h. Diabetes in
streptozotocin-treated rats was conrmed using a One-touch
Glucometer (Lifescan Inc., Milpitas, CA, USA). Animals with blood
glucose above 235 mg dl1 were considered diabetic and were
used for the experiments.
Experimental Design
There were four groups of 610 rats. POX was given i.p. at a
dose of 100 nmol per rat. The dose of POX was the maximum
tolerable for chronic study. It was used by Hasan et al. (2004)
in a 6-week treatment protocol.
Group 1 (G1): Saline control
Group 2 (G2): POX only treated group.
Group 3 (G3): Diabetes control group (No treatment with POX)
Group 4 (G4): Diabetes group treated with 100 nmol per rat
of POX.
The animals were treated daily for 5 days followed by a 2-day
break in a 7-day cycle, for 6 weeks.
Chemicals
POX stock solution (100 mM) was prepared in dry acetone. The
working solution for i.p. application was prepared ex tempore
by diluting the stock solution with saline. Paraoxon-ethyl and
streptozotocin were purchased from Sigma-Aldrich Chemie
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Red Blood Cell-Acetyl-Cholinesterase Activity
The blood samples for red blood cell-acetyl-cholinesterase
(RBC-AChE) measurement were collected from the tail vein.
The RBC-AChE activity was measured in diluted whole blood
samples in the presence of the selective butyryl-cholinesterase
inhibitor, ethoproprazine (Sigma-Aldrich Chemie, Steinheim,
Germany) as described previously (Worek et al., 1999). The
freshly drawn venous blood was diluted 1:100 in a solution
(0.1 M phosphate buffer + Triton-X) and immediately frozen
(Worek et al., 1999). The enzyme activity was measured using
a Milton Roy Spectronics 301 spectrophotometer (Milton Roy,
Ivyland, PA, USA). Samples were kept at 20  C until analysis.
The assay, which is based on Ellmans method, measures the
reduction of dithiobis-nitrobenzoic acid (DTNB) to nitrobenzoate
(TNB-) by thiocholine, the product of acetylthiocholine hydrolysis
(Ellman et al., 1961). The samples were diluted in 0.1 M phosphate
buffer (pH 7.4) and incubated with DTNB (10 mM) and ethopropazine (6 mM) for 20 min at 37  C prior to the addition of
acetylthiocholine. The change in the absorbance of DTNB
was measured at 436 nm. The RBC-AChE activity was calculated using an absorption coefcient of TNB at 436 nm
(e = 10.6 mM 1 cm1). All enzyme activities were expressed as

Copyright 2012 John Wiley & Sons, Ltd.

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1037

The original stock of Wistar rats was purchased from Harlan

Laboratories (Harlan Laboratories, Oxon, England). The animals
used in the actual experiments were bred at our own Animal
Facility from the original stock. Male adult rats, weighing
between 200 and 250 g were used in this study. The animals
were housed in polypropylene cages (43  22.5  20.5 cm; six
rats per cage) in climate and access controlled rooms (23  1  C;
50  4% humidity). The day/night cycle was 12/12 h. Food and
water were available ad libitum. The food was a standard maintenance diet for rats and was purchased from Emirates Feed Factory
(Abu Dhabi, UAE). All the animals procedures were carried out

with strict compliance to the Ethical Committee for the care and
use of laboratory animals.

S. M. Nurulain et al.
Table 1. A. Studies on rats that show the effect of organophosphorus compounds (OPCs) on blood glucose homeostasis in acute application
Study

Type of OPC

Kuzminskaia et al., 1978

Rodrigues et al., 1986
Matin and Hussain, 1987
Husain and Ansari, 1988
Matin et al., 1990
Shih and Scremin, 1992
Ikizceli et al., 2005
Panahi et al., 2006
Romero-Navarro et al., 2006
Lasram et al., 2008
Joshi and Rajini, 2009
Krishna and Ramachandran, 2009

Valexon (Class III: slightly hazardous)

Malathion (Class III: slightly hazardous)
Malathion (Class III: slightly hazardous)
Diazinon Class II: Moderately hazardous
Diazinon (Class II: Moderately hazardous)
Soman (Deadly poison nerve agent)
Fenthion Class II: Moderately hazardous)
Malathion (Class III: slightly hazardous)
Dichlorvos (Class IB: Highly hazardous)
Malathion (Class III: slightly hazardous)
Acephate (Class III: slightly hazardous)
Chlorpyriphos (Class II: Moderately hazardous)

Joshi and Rajini, 2012

Ruckmani et al., 2011

Monocrotophos (Class IB: Highly hazardous)

Malathion (Class III: slightly hazardous)

Reported result on glycemia

Disturbance in glucose homeostasis
Transient hyperglycemia
Hyperglycemia
Hyperglycemia
Hyperglycemia
Increased blood glucose
Hyperglycemia
Increased blood glucose
No effect on glucose metabolism.
Transient hyperglycemia
Reversible hyperglycemia
Co-administration of Chlorpyrifos
and lead causes hyperglycemia
Reversible hyperglycemia
Transient hyperglycemia

B. Studies on rats that show the effect of organophosphorus compounds (OPCs) on blood glucose homeostasis in chronic/
sub-chronic application
Study
Gupta, 1974
Deotare and Chakrabarti, 1981
Reena et al., 1989
Sarin and Gill, 1999
Seifert, 2001
Hagar and Fahmy, 2002
Abdollahi et al., 2004
Pournourmohammadi et al., 2005
Rezg et al., 2006
Panahi et al., 2006
Pournourmohammadi et al., 2007
Kamath and Rajini, 2007
Sadeghi-Hashjin et al., 2008
Ambali et al., 2011
Wang et al., 2009
Slotkin, 2011
Begum and Rajini, 2011
Ruckmani et al., 2011

Type of OPC
Malathion ( Class III: slightly hazardous)
Acephate (Class III: slightly hazardous)
Dimethoate (Class II: Moderately hazardous)
Dichlorvos (Class IB: Highly hazardous)
Diazinon (Class II: Moderately hazardous)
Dimethoate (Class II: Moderately hazardous)
Malathion (Class III: slightly hazardous)
Malathion (Class III: slightly hazardous)
Malathion (Class III: slightly hazardous)
Malathion (Class III: slightly hazardous)
Malathion (Class III: slightly hazardous)
Dimethoate (ClassII: Moderately hazardous)
Azinfos methyl, Malathion (Class IB , III)
Chlorpyrifos (ClassII: Moderately hazardous)
Chlorpyrifos (Moderately hazardous)
Chlorpyrifos (Moderately hazardous)
Monocrotophos (Class IB: Highly hazardous)
Malathion (Class III: slightly hazardous)

Reported result on glycemia

No effect
Increased blood glucose
Hyperglycemia
Hyperglycemia
Hyperglycemia
Increased blood glucose
Hyperglycemia
Hyperglycemia
No change in blood glucose
Hyperglycemia
Hyperglycemia
Increased blood glucose
No diabetogenic effect
Hyperglycemia
Increased blood glucose
Normal serum glucose
Marginal increase
Progressive hyperglycemia

C. Clinical studies/case reports that show organophosphorus compounds (OPCs)-induced hyperglycemia in patients.
Study
Martn et al., 1996
Meller et al., 1981

OP compounds
OP poisoning

Type of OPC

Moore and James, 1981

Shobha and Prakash, 2000
Wu et al., 2001
Yanagisawa et al., 2006
Akyildiz et al., 2009
Kumar and Nayak, 2011

OP poisoning
OP poisoning
methamidophos
Sarin (Deadly toxic/nerve agent)
OP poisoning
OP poisoning

Reported result on glycemia

Pancreatitis and central diabetes insipedus
Diabetes mellitus and non ketotic
hyperglycemia
Hyperglycemia, glycosuria, ketonuria
Transient glycosuria in patients.
Hyperglycemia
Hyperglycemia, ketonuria
Diabetic ketoacidosis.
Diabetic ketoacidosis.

D. Epidemiological studies that show the risk of developing type 2 diabetes after chronic exposure to organophosphorus
compounds (OPC).

1038

Study
Saldana et al., 2007
Montgomery et al., 2008

wileyonlinelibrary.com/journal/jat

Type of OPC
Diazinon (Class II: Moderately hazardous)
Dichlorvos (Class IB: Highly hazardous)

Copyright 2012 John Wiley & Sons, Ltd.

Reported result on glycemia

Gestational diabetes mellitus
Risk of diabetes increase with exposure days

J. Appl. Toxicol. 2013; 33: 10361043

Paraoxon and diabetes mellitus

a percentage of baseline activities (100%). The sums of enzyme
activities over time in individual animals (6 weeks) were compared using the MannWhitney rank order test using SPSS
software (SPSS Inc. Chicago, IL, USA). P 0.05 was considered
to be statistically signicant.
Blood Glucose Measurement
Blood glucose was checked with a One-touch Gucometer (Lifescan
Inc., Milpitas, CA, USA) on day 5 of each week of a 7-day cycle
before the application of the 5th injection of POX. The blood
samples were collected from the tail vein.
Biochemical Test of Urine
After 6 weeks of treatment, a semi-quantitative biochemical test of
urine samples was done using Combur10-Test strips (Boehringer,
Mannheim, Germany). The urine was collected in sterile petri-dish
and the dipstick was soaked and the color change was read
according to the information given in the instruction manual.
Statistical analysis using SPSS 11.0 was performed. The limit of
statistical signicance was set at P 0.05.
Organ and Body Weight
At the end of 6 weeks, the rats were decapitated using a guillotine
and the liver and kidney were removed, weighed and compared
with the total body weight.

Figure 1. Mean red blood cell-acetyl-cholinesterase (RBC-AChE) activity

6 weeks after treatment. Note a signicant (P < 0.05) reduction in activity
after paraoxon (Pox) treatment and diabetes with Pox treatment. Data
are mean  standard deviation (SD). *Shows the statistical signicance
when compared with the corresponding control.

POX-treated rats group was signicantly lower (Bonferroni factor

6) compared with the saline control (Fig. 1). Intraperitoneal injection of 100 nmol POX to rats in groups G2 and G4 caused
49  13 and 49  6 (mean %  SD) inhibition of RBC-AChE activity, respectively. Six weeks of STZ-induced diabetes (G3) resulted
in a 15  5 decrease in RBC-AChE enzyme activity. The trend of
POX-induced inhibition of RBC-AChE in G2 was progressive with
no signicant difference between weekly intervals. In G4, the
percentage inhibition remained almost the same throughout
the experimental period. In G3, there was a small but not significant inhibition of RBC-AChE throughout the course of the study.

Electron Microscopy
Samples of the pancreas from all four groups were cut into small
pieces and xed for 5 h in 2.5% glutaraldehyde in Karnovskys
solution (Karnovsky, 1965). The glands were washed overnight
in cacodylate buffer (4 C) and post xed with 1% osmium tetroxide for 1 h. The samples were later washed six times in cacodylate buffer and dehydrated in graded concentrations of ethyl
alcohol. They were later treated with propylene oxide; two
changes of 15 min and immersed in propylene oxide; and resin
(1:1) for 1 h followed by pure resin overnight. The samples were
embedded and polymerized at 60  C for 24 h. Semi- and ultrathin sections were cut using a diatome knife (supplied by Agar
Scientic, Essex, England). The semithin sections were placed
on glass slides and stained with toluidine blue while the ultrathin sections were mounted on 3.0 mm 200 mesh copper grids
and contrast stained with saturated aqueous uranyl acetate
for 30 min and Reynolds (1963) lead citrate for 5 min. The ultrathin sections were viewed with a Philips CM10 transmission
electron microscope (Eindhoven, The Netherlands).

Animal body weight

Animal body weight was recorded at the end of each week
(Fig. 2). The body weight of rats in group G1 rats increased signicantly from week 1 to 6 in comparison with 0 week weight.
In G2, the body weight increased in the same manner as the
control. Rats in G3 experienced a decrease in body weight
throughout the experimental period compared with the saline
control. Although rats in group G4 had a uctuating weight,
there was no signicant increase in body weight compared with
the beginning of the experiment.

Statistical Analysis
Statistical analysis was performed on all the experimental parameters using SPSS 11.0 (SPSS Inc.). The MannWhitney rank order
test was used to determine the signicance in comparison to control values. The limit of statistical signicance was set at P 0.05.

Result
Acetylcholinesterase activity of RBC

J. Appl. Toxicol. 2013; 33: 10361043

Figure 2. Body weight in percentage, from 06 weeks of treatment. Note

that Pox prevented the loss of body weight in the streptozotocin-induced
diabetic rats.

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1039

Acetylcholinesterase enzyme activity of RBC was determined as

a percentage of baseline. The enzyme level in the RBC of the

S. M. Nurulain et al.
Table 2. Blood glucose level (mg dl1) at weekly intervals
Groups

Baseline

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Mean

Saline control (G1)

Paraoxon only (G2)
Diabetes only (G3)
Diabetes + Paraoxon (G4)

66  7
71  14
433  50
349  48

59  3
56  5
448  74
328  67

61  5
66  6
359  52
343  61

59  3
54  4
358  45
310  72

67  8
70  6
332  106
362  79

58  6
60  8
261  102
341  87

72  12
71  10
423  93
389  42

63  6
63  7
363  67
346  28

Results are expressed as mean  standard deviation. The means of 6 weeks were compared with the baseline of respective groups.
Glucose level
The measurement of the blood glucose level revealed no significant changes in all four groups throughout the experiment
(Table 2). The average blood glucose level was 63  6 mg dl1
in the control and 63  7 in POX-treated group. The blood glucose levels for the diabetes control group (G3) and diabetic rats
treated with POX (G4) were 363  67 and 346  28 mg dl1,
respectively. The MannWhitney rank order test on the data
showed no signicant differences between G1 vs. G2 and
G3 vs. G4.
Organ weight/body weight ratio
To determine the effect of long-term sub-lethal exposure of POX
on the liver and kidney of non-diabetic and STZ-induced
diabetic rats, the liver and kidney were weighed at the end of
6 weeks and the individual organ weight was divided by

corresponding animal body weight. The ratio of kidney/body

weight and liver/body weight was almost the same in all groups
(Tables 3A and B) and no signicant differences were observed.
Urine analysis
Notable differences amongst the four groups were the presence
of bilirubin and erythrocytes in the urine samples of G2 and G4
respectively (Table 4).
Electron microscopy
Figure 3 shows the electron microscopy of four groups of rats.
No morphological changes were observed between the treated
and control groups. The structure of pancreatic beta and other
endocrine cells was intact, suggesting that insulin and other
hormone production was normal in the POX-treated rats (G2).
The morphology of pancreatic islet cells of the diabetes only
group and those of diabetic rats treated with POX was similar,

Table 3. A. Liver/body weight ratio at the end of 6 weeks

Saline control
Paraoxon treated
Diabetes only
Diabetes + paraoxon

Body weight (g) (Mean  SD)

Liver weight (g) (Mean  SD)

Ratio (Mean  SD)

254.666  24.784
275.166  42.780
205.250  21.313
221.200  32.754

9.252  1.513
9.417  1.843
9.665  0.984
10.042  1.243

0.036  0.004
0.034  0.002
0.047  0.047
0.046  0.005

Kidney weight (g) (Mean  SD)

1.932  0.2543
1.8667  0.2703
2.3225  0.0929
2.5960  0.3617

Ratio (Mean  SD)

0.0074  0.0004
0.0068  0.0004
0.0114  0.0008
0.0119  0.0019

B. Kidney/body weight ratio at the end of 6 weeks.

Body weight (g) (Mean  SD)
Saline control
254.6667  24.7844
Paraoxon treated
275.1667  42.7804
Diabetes only
205.2500  21.3131
Diabetes + paraoxon
221.2000  32.7543

Table 4. Biochemical analysis of urine samples

pH
Protein (mg dl1)
Glucose (mg dl1)
Bilirubin (mg dl1)
Blood cells (Ery ml1)

Saline control

Paraoxon (Pox)

Diabetes

Diabetes + Pox

6.00  0.00
13.33  12.91
40  0.00
0.60  0.31
< 5  0.00

6.00  0.00
17.50  13.69
40  0.00
2.30  1.88
90.00*  89.44

5  0.00
44.00  31.30
1000  0.00
1  0.00
250.00  0.00

5.00  0.00
40.00  26.46
1000  0.00
2.71**  1.60
250.00  0.00

1040

The results are mean  standard deviation.

The detection limit of the test strip is: pH value = 59; protein (albumin): 6500 mg dl1. The negative result is assigned ve in the
above-mentioned average calculation; glucose: 401000 mg dl1. 40 mg dl1 is considered normal; Bilirubin: 0.56 mg dl1. A negative
result is assigned a value of 0.4 mg dl1; blood cells: 5250 ery ml1.
* Signicant difference compared with the saline control.
** Signicant difference compared with the diabetes control.

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J. Appl. Toxicol. 2013; 33: 10361043

Paraoxon and diabetes mellitus

Figure 3. Transmission electron microscopy of pancreatic beta cells.

Note that the structure of the granules and other cytoplasmic organelles
is intact in all the four groups. Magnication: 14 000  .

suggesting that POX did not alter the structure of cells of the
islet of Langerhans, either in normal or diabetic conditions.

Discussion

J. Appl. Toxicol. 2013; 33: 10361043

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1041

There is a wide variety of OPCs ranging from mild to highly toxic.

OPCs are structurally different from each other but have a common
mechanism of action, i.e. inhibition of the neurotransmitter enzyme,
acetylcholine esterase at nerve junctions. Excessive acetylcholine
produces unwanted muscarinic effects (miosis, bradycardia,
glandular hypersecretion and gastrointestinal dysfunction) and
nicotinic effect, which includes muscular twitching and seizure.
The literature review shows that exposure to OPCs can impair
glucose metabolism, through a variety of factors, as highlighted
in the introduction and Table 1AD.
This study showed that contrary to previous reports on other
OPCs, POX, at sub-lethal and sub-chronic exposure, with up to
49% RBC-AChE inhibition (a mild effect according to Balali-Mood
and Balali-Mood, 2008) did not induce diabetes mellitus in
experimental rats. This is the rst report showing that an extremely
toxic OPC, such as POX does not cause diabetes mellitus. Nurulain
et al. (unpublished data) worked on another extremely toxic OPC
with the same protocol and observed similar result. SadeghiHashjin et al. (2008) did not attribute any diabetogenic effect
to azinphos methyl, a class Ib OPC. However, it must be noted
that the treatment with azinfos-methyl was only for 8 days.
The present study was conducted with a sub-lethal dose of
POX for 6 weeks. Many previous reports have linked exposure
to other OPCs such as parathion, a precursor of POX, to the
development of diabetes worldwide (Begum and Rajini, 2011;
Kamath and Rajini, 2007; Rezg et al., 2006; Sadeghi-Hashjin et al.,
2008; Slotkin, 2011). Other investigators have also shown that
malathion, dimethoate and other OPCs can cause diabetes
mellitus in animal and clinical studies (Table 1AD). It is not clear
why POX does not cause diabetes in accordance with other OPCs.

It is possible that a longer exposure period may predispose the

animals to diabetes, but this has to be thoroughly investigated.
Moreover, there is also the possibility that different OPCs cause
a diabetogenic effect at different periods of exposures. Kuehn
(2008) studied the link between an OPC pesticide, dichlorvos,
and agriculture workers. He postulated that the incidence of diabetes increased with cumulative days of exposure. The unique toxic
prole of each OPC may also be a factor. Some OPCs may cause
diabetes while others do not. Therefore, from the results of this
study, it may be concluded that a generalized hypothesis may
not be appropriate to conclude that all OPCs are diabetogenic,
whether in acute, chronic or sub-chronic exposure.
The body weight of rats in the POX-treated groups was
slightly but not signicantly lower than the control groups. A
decrease in body weight in the OPC-exposed group is a common phenomenon, which has been reported previously (Begum
and Rajini, 2011). The relative decrease in body weight may be
because of a decrease in appetite or gastrointestinal dysfunction
induced by OPCs poisoning.
Although the liver/body weight ratio was slightly lower in the
POX-treated group than the control, it was not signicantly different. The same pattern was observed in monocrotophos-treated
(Begum and Rajini, 2011) and diazinon-treated (Ueyama et al.,
2007) rats. The lower liver/body weight ratio was attributed to
a disturbance in glucose homeostasis and enhanced glucose
output (Joshi and Rajini, 2009; Rahimi and Abdollahi, 2007).
The electron microscopy of the islet cells of POX-treated rats
showed no morphological changes. The pancreatic beta and other
endocrine cells were intact, suggesting that insulin and other
hormone production was normal in the treated rats. However,
Hagar and Fahmy (2002) reported ultrastructural changes in pancreatic islet cells after 2 months of chronic dimethoate treatment.
Hagar and Fahmy (2002) showed that pancreatic beta cells were
the most affected among the damaged islets cells compared with
the control. They showed that most of the beta cells displayed a
marked reduction in the number of cytoplasmic granules, with
multiple vacuoles. Gokcimen et al. (2007) observed that the
diazinon effect is dose dependent and 1015% of the LD50
dose caused fat necrosis, cellular and glandular degeneration.
Pournourmohammadi et al. (2007) investigated the effects of
malathion on insulin secretion from rat pancreatic islets. Their light
microscopic examination in semithin sections revealed that malathion causes patchy degenerative changes in pancreatic islets in
a dose-dependent manner. In terms of toxicity, dimethoate, diazinon and malathion belong to the mildly toxic group of OPCs and
have been reported to cause diabetes mellitus (Table 1A, B). Their
toxic effect on pancreatic islet cells is thus obvious. However, the
point to note is that they produced the effect in a dose-dependent
manner. In this study, the 100 nmol dose was the maximum tolerable dose for the 6-week treatment. Mortality was noted in the rst
week of treatment when 150 nmol was used as the tentative next
higher dose of POX. However, neither hyperglycemia nor glycosuria was observed. The absence of morphological alterations in
pancreatic beta cells is convincing evidence that POX does not
cause diabetes mellitus at sub-lethal chronic exposure.
Urinalysis in the POX-treated group (G2) revealed a normal
glucose level compared with the control (G1). However, a small
but not signicant increase in protein and bilirubin level in urine
was noted in the POX-treated group compared with the control.
The presence of protein and bilirubin indicates a dysfunction of
the kidney and liver, respectively. Liver and kidney disorders can
be caused by OPC (Hayes et al., 1978).

S. M. Nurulain et al.
The second part of the study examined the effect of sub-lethal
sub-chronic exposure of POX in STZ-induced diabetic rats. The
RBC-AChE measurement in diabetes control rats showed a 15%
inhibition in acetylcholinesterase activity, which is of course a mild
and insignicant inhibition, but indicated that diabetes is associated with impaired AChE activity in RBC. An abnormal AChE level
may occur as a resultof various physiological and pathological
conditions including diabetes mellitus (Jokanovic and Maksimovic,
1997). A signicantly decreased level of cholinesterase has also
been reported in clinical diabetes (Rizvi and Zaid, 2001; Suhail
and Rizvi 1989, 1990) and in animal models (Sanchez-Chavez and
Salceda, 2000, 2001). However, the extent of inhibition (2040%)
in all of these reports comes under mild level of poisoning.
Many factors have been speculated for the decreased level of
AChE in diabetes mellitus. For instance, in the absence of an
inhibitor, impaired synthesis may lead to a decreased synthesis
of AChE (Szutowicz et al., 1994). The altered molecular form
and isoforms of cholinesterase (Sanchez-Chavez and Salceda,
2000, 2001), altered level of pre-existing enzymes (Milatovic
and Dettbarn, 1996), the modied dynamic properties of the
erythrocyte membrane (Testa et al., 1988; Watala, 1993) or an
alteration in the cholinergic system because of diabetes
(Wahaba and Soliman, 1988) may also be possible factors for
RBC-AChE inhibition in diabetes.
POX (100 nmol per rat) inhibited the same level of RBC-AChE
inhibition (49% of baseline) in diabetic rats compared with the
control. This shows that although diabetes is associated with a
low level of RBC-AChE, it does not lead to excessive reduction
after the administration of 100 nmol of POX. Moreover, the addition of POX does not exacerbate diabetes mellitus in STZ-treated
rats. This is in contrast to previous reports. According to Begum
and Rajini (2011), monochrotofos at a sub-lethal dose, signicantly increased the hyperglycemic outcome in STZ-induced
diabetic rats, more than 56% above control rats. Ueyama et al.
(2007) reported that diazinon-oxon, a metabolite of diazinon,
caused hyperglycemia in STZ-induced diabetic rats. In the present study, no such effects were observed, suggesting that POX
has a different toxic prole regarding glucose metabolism when
compared with other OPCs.
In conclusion, POX neither induces diabetes mellitus nor
increases the glycemic level of diabetes rats indicating that
not all OPCs are diabetogenic.

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