Toxicology Letters 153 (2004) 227232

The effects of sub-chronic exposure of Wistar rats to the herbicide Glyphosate-Biocarb

Alo sio Luiz Benedetti a , Cid onia de Lourdes Vituri b , Andra Gonalves Trentin a , Maria Aparecida Custdio Domingues c , Marcio Alvarez-Silva a,
a

Laboratrio de Neurobiologia e Hematologia Celular e MolecularBEGCCBUniversidade Federal de Santa Catarina Campus Universitrio, 88040-900 Florianpolis, SC, Brazil b Departamento de Anlises Cl nicasCCSUniversidade Federal de Santa Catarina, Florianpolis, SC, Brazil c Laboratrio de Hemopatologia, Centro de Hematologia e Hemoterapia de Santa Catarina (HEMOSC), Florianpolis, SC, Brazil Received 14 November 2003; received in revised form 7 April 2004; accepted 7 April 2004 Available online 24 May 2004

Abstract The object of this study was to analyze the hepatic effects of the herbicide Glyphosate-Biocarb (as commercialized in Brazil) in Wistar rats. Animals were treated orally with water or 4.87, 48.7, or 487 mg/kg of glyphosate each 2 days, during 75 days. Sub-chronic treatment of animals starting from the lowest dose of glyphosate induced the leakage of hepatic intracellular enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), suggesting irreversible damage in hepatocytes. We observed the increase of Kupffer cells in hepatic sinusoid of glyphosate-treated animals. This was followed by large deposition of reticulin bers, composed mainly of collagen type III. We may conclude that Glyphosate-Biocarb may induce hepatic histological changes as well as AST and ALT leaking from liver to serum in experimental models. 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Herbicide; Biocarb ; Glyphosate; Rats; ALT; AST; Liver; Kupffer cell; Reticulin

1. Introduction Glyphosate is the active ingredient of Biocarb or Rundup (Brazil), marketed as a non-selective, broad-spectrum, post-emergence herbicide. It is used to control weeds in emerged grasses, broad-leaf weeds, pastures and rice, corn and soy plantations (Smith and Oehme, 1992). The adverse effects of
Corresponding author. Tel.: +55-48-331-6905; fax: +55-48-331-5148. E-mail address: malvarez@ccb.ufsc.br (M. Alvarez-Silva).

glyphosate and others components of its commercial formulations are periodically re-evaluated and toxicity of the pesticide has been described for severe exposure conditions (Williams et al., 2000). It has been suggested that particular toxicity of glyphosate-containing products is related to the surfactant components of the formulation products. For regular usage, several regulatory agencies and scientic institutions worldwide have concluded that there is no indication of any human health concern with glyphosate and Roundup (Williams et al., 2000). However, it has been described that long-term

0378-4274/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2004.04.008

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exposition to glyphosate can cause toxicity in pregnant rats with decient ossication of fetuses (Dallegrave et al., 2003). This teratogenic effect was observed in sub-chronic exposition to Rundup . In other studies, chronic administration using technical glyphosate was conducted on rats. Studies were made at the following periods 19791981 and 19881990 (WHO, 2003). Animals received 3, 10, and 32 mg/kg per day in the rst study and 100, 410, and 1060 mg/kg per day in the second one. In the rst study, a signicant increase in the incidence of interstitial cell tumors was observed in rats, however, the absence of the same effect with the higher doses used in the second one was the basis to exclude glyphosate from the carcinogenic category (WHO, 2003). We report here that long-term treatment of rats with low doses of Glyphosate-Biocarb increased release of hepatic-aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to serum of animals. This was accompanied by changes in hepatic liver tissue, with larger deposition of reticulin bers followed by increase in Kupffer cells, suggesting the increase in connective tissue. These data suggest some degree of hepatic toxicity of this product and raises the necessity of deeper studies about side effects related to its manipulation.

trolled humidity and on a 12 h-light/dark cycle (lights on from 09:00 to 21:00 h), with free access to standard laboratory rat chow and water. All breeding phases and all experiments were in agreement with the Ethics and Animal Experimentation Committee of our Institution. We performed two independent experiments and the number of animals per group was not similar, in one experiment we used 38 rats while in another independent group we used 20 animals. Rats were organized in four groups. The control experimental group (n = 16) received distilled water while experimental groups (n = 14 for each group) received 4.87, 48.7, or 487 mg/kg of Glyphosate-Biocarb diluted in water. The dose regimen of treated groups was based on no observed adverse effect level (NOAEL) for developmental toxicity in rats which was 1000 mg/kg glyphosate (Williams et al., 2000). Our values were within the limits of NOAEL and equivalent to 1/1000, 1/100, and 1/10 of LD50 in rats as described elsewhere (Larini, 1999). Glyphosate-Biocarb was administered orally by gavage in a volume of 0.5 ml/kg each 2 days, during 75 days. At the end of this period, blood samples and liver tissue were collected. 2.3. Measurement of liver enzymes Blood samples were collected from all animals by percutaneous cardiac puncture immediately before euthanasia. Blood samples were transferred to test tubes and allowed to stand for 30 min to clot before being centrifuged at 300 g for 10 min. Sera were obtained by centrifugation and stored at 4 C, for subsequent analysis of alanine aminotransferase and aspartate aminotransferase enzyme levels. AST and ALT levels were determined by UV kinetics methodology using AST and ALT substrate, respectively in commercial kit (Biotcnica, SP). Tests were performed in automatic system Express Plus (Ciba Corning). Enzyme activity was expressed in International Units per liter (IU/L). All analyses were checked for accuracy by concurrent analyses of AMES control sera for AST and ALT. 2.4. Liver biopsies Liver samples were obtained from all animals by surgical processing after euthanasia. It was

2. Materials and methods 2.1. Chemicals The glyphosate formulation (Glyphosate-Biocarb , So Jos dos Pinhais, Brazil) consisting of 360 g/L glyphosate (N-phosphonomethylglycine) and 18% (w/v) polyoxyethyleneamine (the surfactant) was used. The solutions of Glyphosate-Biocarb formulation were prepared by the addition of appropriate volumes of distilled water. 2.2. Animals and treatment Adult male Wistar rats (90-day-old, 280310 g) bred in the animal facilities of our own Department were used. All animals were housed in polyethylene (65 cm 25 cm 15 cm) home cages, with sawdust-covered oors. Animals were maintained in a colony room at 22 2 C under conditions of con-

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removed a standard sample of 0.5 cm always from the same hepatic lobe. Samples were taken from all the animals and xed with 10% formaldehyde in phosphate-buffered saline for 24 h. Then, they were washed with tap water, dehydrated in alcohol and embedded in parafn. Sections of 2 m were mounted in glass slides covered with saline. Tissue was evaluated after haematoxylineosin staining. The biopsies were assessed for the deposition of reticulin bers by Gomory trichromic staining (de Boer et al., 2000; Fakoya, 2002). 2.5. Statistical analysis Parametric data, expressed as mean S.E.M., were analyzed by one-way variance NewmanKeuls for ALT and AST tests. Differences were considered to be statistically signicant when P < 0.05.

serum ALT and AST, indicating the hepatic toxicity induced by glyphosate. 3.2. Liver biopsies To test the presence pathological lesions, we performed histological observations of liver lobule. Despite the apparent normal aspect of hepatocytes we observed the local increase in the number of Kupffer cells in hepatic sinusoid in all animals of glyphosate-treated group with 487 mg/kg as indicated by the large increase in arrows that point to Kupffer cells (Fig. 1). Kupffer cells did not increase in animals treated with lower doses of glyphosate. This may be due to local proliferation of these cells since no apparent inltrate of perivascular Kupffer cells or lymphocytes were observed in portal tract. No evidence of inammatory lesions was observed. Interestingly we observed the increase in connective tissue deposition in all glyphosate-treated animals with 487 mg/kg (Fig. 2). This was not observed in animals treated with lower doses of glyphosate. These data suggest that in glyphosate-treated animals hepatic modulation may occur where collagen or other extracellular matrix proteins were induced. To test this hypothesis we prepared reticulin silver staining (Fig. 3). This method has been helpful to identify collagen bers (Wang et al., 1999). We observed that deposition of reticulin bers increased in 487 mg/kg glyphosate-treated animals, while cellularity apparently remained unaltered. We also observed that reticulin bers were larger in glyphosate-treated animals than in controls. Such modications were not detected with lower doses of glyphosate than 487 mg/kg, so it is not possible to establish quantitative dose response for this effect in our studies.
Table 2 Serum activity of AST in Wistar rats

3. Results 3.1. Effects of glyphosate on serum transaminase activities in rats Hepatic toxicity was monitored by quantitative analysis of the ALT and AST activities, which were used as the biochemical markers of liver injury. As shown in Table 1, serum ALT levels in control rats were 28.5 1.81 U/L. In glyphosate-treated rats serum ALT increased about two-fold at the end of sub-chronic treatment. For serum AST we observed that control groups were 84.3 4.67 (Table 2). In glyphosate-treated rats, serum AST elevated to about one and half-fold at the end of treatment. Although low this difference was signicant due to automatic system used to measure
Table 1 Serum activity of ALT in Wistar rats Group Control 4.87 mg/kg Glyphosate-Biocarb 48.7 mg/kg Glyphosate-Biocarb 487 mg/kg Glyphosate-Biocarb n 16 14 14 13 X S.E.M. U/L 28.5 45.5 47.9 55 1.81 6.7 6.1 6.8

Group Control 4.87 mg/kg Glyphosate-Biocarb 48.7 mg/kg Glyphosate-Biocarb 487 mg/kg Glyphosate-Biocarb

n 15 14 14 13

X S.E.M. U/L 84.3 101.8 112.9 124.8 4.67 6.77 10.7 8.56

Data are reported as mean S.E.M. There was no signicant difference among glyphosate-treated groups, however, they were different from control ( P < 0.05; P < 0.01, NewmanKeuls). Sera with hemolysis were discharged in these assays.

Data are reported as mean S.E.M. There was no signicant difference among glyphosate-treated groups, however, they were different from control ( P < 0.05; P < 0.01, NewmanKeuls). Sera with hemolysis were discharged in these assays.

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Fig. 1. (A) Histological section of liver lobule in control animal or (B) 487 mg/kg glyphosate-treated rat. Arrows indicate Kupffer cells. Sections were HE stained. Magnication 400.

Fig. 2. (A) Histological section of portal tract in control animal or (B) 487 mg/kg glyphosate-treated rat. Arrows indicate deposition of connective tissue. Sections were HE stained. Magnication 100.

Fig. 3. (A) Histological section of liver lobule in control animal or (B) 487 mg/kg glyphosate-treated rat. Arrows indicate enhanced amount of reticulin. Sections were stained by Gomory Silver technique. Magnication 400.

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4. Discussion Our results demonstrated that Glyphosate-Biocarb affects liver metabolism. The leakage of certain intracellular enzymes suggested damage in hepatocytes. In this case, serum ALT and AST activities frequently served as an index of liver injury (de Boer et al., 2000; El et al., 2002; Kuester et al., 2002). In our studies we tested low doses of glyphosate in long-term administration, maintaining a time interval of 48 h between glyphosate treatment. We could detect the leakage of enzymes to serum at the end of the treatment even with the lowest dose studied, i.e. 4.87 mg/kg. With the administration of 487 mg/kg we observed higher leakage of AST and ALT to serum, suggesting qualitative dose response. This modication was followed by changes in cellularity of tissue, with increase in connective tissue and collagen deposition. This may result from some hepatocyte damage with minute substitution by connective tissue with certain brous expansion of some portal areas, without short brous septa (stage 1) without inammatory evidence (grade 0) as observed in mild chronic hepatitis (Ishak et al., 1995). Since AST/ALT leaking to serum is very sensitive marker of hepatocyte injury, increase in serum enzymes may occur due to minor liver injury in response to drugs or chemicals with low histological changes (Williams and Iatropoulos, 2002). To achieve apparent toxic effects is necessary 1.000 mg/kg (Williams et al., 2000). Acute toxicity of Glyphosate-Biocarb appears to largely exceed experimental protocols we used in our experimental design. The residual levels of glyphosate in soil or water are much lower, being at nanomolar quantities present almost constantly. Cellular injury may occur at millimolar amounts of glyphosate for prolonged periods of time due to accumulative effects. Very low levels of glyphosate could be partially compensated by the great difference in time exposure. It has been shown that glyphosate, in Roundup formulation, can produce toxic effects, such as diarrhea (Adam et al., 1997), immunosuppression (Blakley, 1997), teratogenicity (Dallegrave et al., 2003, Daruich et al., 2001), modication in cell cycle activity (Marc et al., 2002). Although these effects were observed at higher doses, they strongly support our studies indicating that cellular injury may occur with lower doses. Our results therefore question whether human health could be affected by glyphosate.

Liver is the main tissue in detoxication and metabolism of chemicals. This may impair its regular function due to xenobiotic modication in detoxication processes. Hepatic lesions may occur soon or during lifetime. This may result in severe injury to liver (Ghany and Hoofnagle, 2001). Although no marked differences were noted in hepatocytes morphology or liver loss of architecture, Kupffer cells may control somewhat growth factor production, extracellular matrix deposition and local microcirculation (Decker, 1998; Kmie, 2001; Williams and Iatropoulos, 2002). We should consider that deposition of collagen may result from toxic effects to liver. In our studies we observed the deposition of reticulin bers. These bers are mainly composed of collagen type III (Fakoya, 2002). We observed changes in the number and the thickness of reticulin bers in experimental groups treated with 487 mg/kg. Large deposition of collagen bers may cause modications in the diffusion of solutes, as proteins, between hepatocytes and plasma. This may impair hepatocyte function. In inammation, cytokines such as TNF-, TNF- e IL-1 can stimulate activation of Ito cells to broblast-like phenotype, followed by deposition of type I and type III collagen in hepatic tissue. Cytokines released by Kupffer cells in response to toxic chemicals can induce the activation of Ito cells without inammatory lesions even in the absence of perceptible loss of hepatic cellular architecture (Pratt and Kaplan, 2001). This can explain why in our studies we did not observe striking morphological changes in hepatocytes or in cellular architecture. Although the leaking of enzymes from liver was not followed by stunning modications in cellular architecture of liver, this may indicate that to detect some kind of liver injury by glyphosate, ALT and AST may be he best choice. Glyphosate formulations contain surfactants, which promote wetting of plant surface and rapid herbicide penetration (Mitchell et al., 1987). In previous reports, the toxicity of Roundup was ascribed to the surfactant component. We showed that glyphosate and its formulation products may act in synergy on the liver metabolism and/or injury. Anyway, the health injury may result from more toxic effects than expected due to accumulative effects. Despite the commercial formulation, glyphosate poses a potential risk for human health. To better understand this fact, additional studies should be carried out to determine the mechanisms

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A.L. Benedetti et al. / Toxicology Letters 153 (2004) 227232 Ghany, M., Hoofnagle, J.H., 2001. Liver and biliary tract disease, approach to the patient with liver disease. In: Braunwald, E., Fauci, A., Kasper, D., Hauser, S., Longo, D.L., Jameson, J.L. (Eds.), Harrisons Principles of Internal Medicine. McGraw-Hill Professional, pp. 17071710. Ishak, K., Baptista, A., Bianchi, L., Callea, F., De, G., Gudat, F., Denk, H., Desmet, V., Korb, G., MacSween, R.N., 1995. Histological grading and staging of chronic hepatitis. J. Hepatol. 22, 696699. Kmie, Z., 2001. Cooperation of liver cells in health and disease. Adv. Anat. Embryol. Cell Biol. 161, 111151. Kuester, R.K., Waalkes, M.P., Goering, P.L., Fisher, B.L., McCuskey, R.S., Sipes, I.G., 2002. Differential hepatotoxicity induced by cadmium in Fischer 344 and SpragueDawley rats. Toxicol. Sci. 65, 151159. Larini, L., 1999. In: Manole (Ed.), Toxicologia dos Praguicidas. pp. 178179. Marc, J., Mulner, L., Boulben, S., Hureau, D., Durand, G., Belle, R., 2002. Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation. Chem. Res. Toxicol. 15, 326331. Mitchell, D.G., Chapman, P.M., Long, T.J., 1987. Acute toxicity of Roundup and Rodeo herbicides to rainbow trout, chinook, and coho salmon. Bull. Environ. Contam. Toxicol. 39, 10281035. Pratt, D.S., Kaplan, M.M., 2001. Liver and biliary tract disease, evaluation of liver function. In: Braunwald, E., Fauci, A., Kasper, D., Hauser, S., Longo, D.L., Jameson, J.L. (Eds.), Harrisons Principles of Internal Medicine. McGraw-Hill Professional, pp. 17111714. Smith, E.A., Oehme, F.W., 1992. The biological activity of glyphosate to plants and animals: a literature review. Vet. Hum. Toxicol. 34, 531543. Wang, R.N., Paraskevas, S., Rosenberg, L., 1999. Characterization of integrin expression in Islets isolated from Hamster, Canine, Porcine, and Human Pancreas. J. Histochem. Cytochem. 47, 499506. WHO, 2003. Glyphosate, Environmental Health Criteria, vol. 159, pp. 1177. Williams, G.M., Iatropoulos, M.J., 2002. Alteration of liver cell function and proliferation: differentiation between adaptation and toxicity. Toxicol. Pathol. 30, 4153. Williams, G.M., Kroes, R., Munro, I.C., 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul. Toxicol. Pharmacol. 31, 117165.

of effects of the commercial formulation on the liver metabolism, function and Kupffer cell proliferation, since humans are also potentially exposed to commercial formulation of glyphosate.

Acknowledgements This work was supported by grants from Coordenao de Aperfeioamento de Pessoal de N vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient co e Tecnolgico (CNPq) and Fundao de Ci encia e Tecnologia (FUNCITEC).

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