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Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses
Environmental Research Section A 85, 226}231 (2001) doi:10.1006/enrs.2000.4229, available online at http://www.idealibrary.com on
Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses1 Jorgelina Daruich, Fanny Zirulnik, and MarmH a Sof mH a Gimenez Ca& tedra de Bioquı& mica Molecular, Area Quı& mica Biolo& gica, Facultad de Quı& mica, Bioquı& mica y Farmacia, Universidad Nacional de San Luis, 5700 San Luis, Argentina Received September 14, 2000
complexation in water with ions, e.g., Ca2# and Mg2#, sorption to sediment, suspended particles in water and soil, photodegradation in water, uptake by plants, and biodegradation. Data on the occurrence of glyphosate in biota and environmental abiota as part of regular monitoring programs are very scarce. Measurements of daily human intake of glyphosate via food and drinking water (total-diet studies) are not available (Mensink and Janssen, 1994). Pregnancy is a physiological condition in which deep adaptations are produced in the maternal metabolism. So the development and growth of fetuses are safeguarded by those metabolic changes (Strubbe and Gorissen, 1973). The effects of protein deprivation and pesticide exposure have been studied in pregnant rats, and a decrease of enzymes such as glutathion peroxidase and glucose-6-phosphate dehydrogenase was observed (Prabhakaran, et al., 1993). Other studies suggest that maternal exposure to herbicides during pregnancy induces a variety of functional abnormalities in the brain of offspring dependent both on the pharmacological action of chemicals and on the stage of gestation, even with a single exposure (Fujii, 1997). The aim of this work was to study the activity of some enzymes with a function in the pathways of NADPH generation, isocitrate dehydrogenase (ICD), glucose-6-phosphate dehydrogenase (G6PD), and malate dehydrogenase (MD) in liver, heart, and brain of pregnant rats and their fetuses which were exposed to the herbicide glyphosate.
To prevent health risk from environmental chemicals, particularly for progeny, we have studied the effects of the herbicide glyphosate on several enzymes of pregnant rats. Glyphosate is an organophosphorated nonselective agrochemical widely used in many countries including Argentina and acts after the sprout in a systemic way. We have studied three cytosolic enzymes: isocitrate dehydrogenase-NADP dependent, glucose-6-phosphate dehydrogenase, and malic dehydrogenase in liver, heart, and brain of pregnant Wistar rats. The treatment was administered during the 21 days of pregnancy, with 1 week as an acclimation period. The results suggest that maternal exposure to agrochemicals during pregnancy induces a variety of functional abnormalities in the speciAc activity of the enzymes in the studied organs of the pregnant rats and their fetuses. 2001 Academic Press Key Words: glyphosate; dehydrogenases; pregnancy; fetuses.
INTRODUCTION
Glyphosate is a weak organic acid consisting of a glycine and a phosphonomethyl moiety. Glyphosate is usually formulated as a salt of the deprotonated acid of glyphosate and a cation, e.g., isopropylamine or trimethyl sulfonium. It is a postemergent, systemic, and nonselective herbicide that is used in both agricultural and nonagricultural areas all over the world. Environmental exposure may occur because of deposition due to drift and accidental releases. The most important processes of dissipation that may be involved after application of glyphosate are
MATERIALS AND METHODS 1
This work was supported by a CONICET Grant (PIP 4931) and by a grant from the Universidad Nacional de San Luis, Argentina (Proyecto 8104).
Chemicals. All the chemicals were of reagent grade and were purchased from Sigma and Merck 226
0013-9351/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
227
DARUICH, ZIRULNIK, AND GIMENEZ
Laboratories. Glyphosate is commercialized in Argentina under the name of Herbicygon, from M.F.L. S.R.L. The empirical formula is C3H8NO5P. Animals and diets. Animals were provided by the National University of San Luis. The acclimation period was 1 week. Wistar female virgin rats (210}230 g) at the proestrus stage were housed for 1 night with fertile males, and fertilization was assumed by the presence of spermatozoa in the vaginal smear. That day is designated as day 1 of pregnancy. The rats were then transferred to individual cages and kept in a 22}253C controlled environment with a light}dark cycle of 12 h each. The animals were fed with 20 g of a stock laboratory diet, elaborated by Cargill, with the following ingredients: meat 8our, bone and meat 8our, 7sh meal, blood 8our, soybean meal, toasted soybean, soy expeller, sun8ower 8our, cotton 8our, peanut meal, animal fat, corn, wheat, sorghum, oat, barley, wheat bran, rice bran, gluten meal, vitamins A, E, B, D3, K3, and B12, niacin, pantothenic acid, choline, ascorbic acid, bone ash, salt, calcium carbonate, oyster, manganese oxide, zinc oxide, ferrous sulfate, copper oxide, sodium selenite, iodine, and cobalt. Thirty-7ve milliliters of potable water in a water bottle were administered daily and body weight and food intake were measured daily. The rats were separated into three groups (eight rats/group); I, control group which drank tap water; II, group which drank glyphosate solution 0.5% w/v in tap water (dose: 0.2 ml glyphosate/ml water); and III, group which drank glyphosate solution 1% w/v in tap water (dose: 0.4 ml glyphosate/ml water). Another experiment was carried out to determine whether the obtained results could be attributed solely to glyphosate. Six control rats (IV) were treated with low water and food (10 ml and 10 g, respectively); this treatment began the second week because at this time group III decreased the ingestion of water and food. Group IV is called Low in the tables and 7gures. In the 7rst week the rats of all groups ate about 20 g of food and drank about 35 ml of tap water per day. Experimental conditions. At 8:00}9:00 a.m. of day 21 of gestation, the rats were anesthetized with diethyl ether. Each fetus was delivered by rapid hysterectomy, identi7ed, weighed, and then killed immediately by decapitation with scissors. Maternal and fetal livers, hearts, and brains were immediately removed, washed in a cold saline solution, and kept at !203C until needed for analysis. The fetal organs were pooled. Tissue preparation and enzymatic assays. Livers, hearts, and brains (0.5 g/1 ml buffer) were
homogenized in an Ultra Turrax T25 machine with a 0.5 M Tris}HCl buffer, pH 7.4, containing 1 mM dithiotreitol. The homogenates were centrifuged at 100,000 g for 1 h by a Beckman Model L2 65B ultracentrifuge with a TY rotor to yield the cytosolic fraction, and the enzymatic activities were measured in the supernatant. Isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and malic dehydrogenase were determined by the rate of NADPH formation at 340 nm in a Shimadzu spectrophotometer, according to Farrell (1980), Glock and Mc Lean (1953), and Ochoa (1968), respectively. The results were expressed as lmol NADP/min/mg protein. Protein concentration was measured by the Biuret reaction (Layne, 1957). Statistical analyses. Signi7cant differences among means were considered at a level of P(0.05 and identi7ed by one-way ANOVA, Kolmogorov} Smirnov, and Newman}Keul procedures. In all the cases the variances were homogeneous. RESULTS
Effect of glyphosate on the daily consumption of food and water. Pregnant rat food and water ingestion decreased with both doses of glyphosate. Pregnant rat body and liver weights decreased signi7cantly compared to the controls when glyphosate 1% p/v was administered. Fetus body weights showed no signi7cant differences (Table 1). Effect of glyphosate on the enzymatic activity in liver of pregnant rats. The activity of ICD showed a decrease with 0.5% w/v of glyphosate and a signi7cant increase with a dose of 1% w/v of the herbicide (P(0.01), compared to the control group. The speci7c activity of G6PD decreased with 0.5% w/v and increased to the control value with glyphosate 1% w/v (P(0.05), whereas MD showed no signi7cant differences from the control. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 1). Effect of glyphosate on the enzymatic activity in heart of pregnant rats. ICD activity showed a signi7cant increase with 0.5% w/v and 1% of glyphosate (P(0.005) compared to the control. G6PD showed no differences with 0.5% w/v but increased with a dose of 1% w/v of glyphosate (P(0.01), whereas MD showed no signi7cant differences from the control. The enzymatic activities in the low group, with low water and low food, showed
228
GLYPHOSATE AND ENZYMATIC ACTIVITY IN RATS
TABLE 1 Effect of Glyphosate on Food and Water Ingestion and Body and Organ Weights in Pregnant Rats and Their Fetuses
Pregnant rats Food intake (g/day) Water ingestion (ml/day) Mass balance (%) Total body weight gain (g) Organ weights (g) Liver Heart Brain Fetuses Body weight (g)
Control
0.5% p/v
1% p/v
Low
P
19.05$0.99 32.50$1.05 100 92.00$8.9
15.95$0.24 23.70$0.6 87.7 80.70$11.37
14.53$0.3 25.79$1.56 57.38 52.79$3.14
11.54$0.32 22.49$0.4 53.81 49.51$5.04
(0.01 (0.01 (0.05
8.87$0.53 0.83$0.03 1.45$0.15
7.75$0.31 0.80$0.04 1.71$0.05
7.41$0.38 0.74$0.04 1.57$0.08
7.62$0.26 0.65$0.02 1.47$0.2
(0.05 ns ns
3.20$0.36
3.52$0.03
3.13$0.01
3.06$0.09
ns
Note. Values are means$SD for eight rats/group. Signi7cant differences were determined by one-way ANOVA; ns, not signi7cant. The values of food and water intake are the means of 3 weeks of treatment.
no signi7cant differences from that of the control group (Table 2). Effect of glyphosate on the enzymatic activity in brain of pregnant rats. ICD did not change with 0.5% glyphosate but increased with 1% (P(0.01), whereas G6PD and MD did not change with both doses of glyphosate. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 2). Effect of glyphosate on the enzymatic activity in liver of fetuses. The activity of ICD showed no differences between control and 0.5% w/v of glyphosate, but it showed a signi7cant decrease
when glyphosate 1% w/v was administered (P(0.05). G6PD and MD showed no signi7cant differences with both doses of glyphosate used compared to the control. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 3). Effect of glyphosate on the enzymatic activity in heart of fetuses. ICD activity showed a signi7cant increase with 0.5% w/v dose of glyphosate (P(0.01), but there were no differences with the 1% w/v dose. The same behavior was observed with G6PD activity (P(0.05). MD decreased signi7cantly with both doses of glyphosate (P(0.05). The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 4). Effect of glyphosate on the enzymatic activity in brain of fetuses. ICD increased signi7cantly when
TABLE 2 Effect of Glyphosate on the Enzymatic Activity in Heart of Pregnant Rats
Control 0.5% p/v 1% p/v Low FIG. 1. Effect of glyphosate on the enzymatic activity in liver of pregnant rats. Values are means$SD of eight rats/group. a P:0.01; bP:0.05; cP:0.001, as determined by one-way ANOVA.
ICD
G6PD
MD
53.24$7.73 93.07$7.33b 121.88$5.85c 49.65$8.78
1.85$0.66d 1.403$0.57e 4.43$1.58f 2.09$0.68
2.36$1.10 2.673$0.41 2.65$1.38 2.85$1.28
Note. Values are means$SD of eight rats/group (kmol NADP/min/mg prot.). Signi7cant differences are determined by one-way ANOVA. a,b - a,c-e,f: P(0.001; d,e-d,f: P(0.01.
229
DARUICH, ZIRULNIK, AND GIMENEZ
FIG. 2. Effect of glyphosate on the enzymatic activity in brain of pregnant rats. Values are means $SD of eight rats/group. a P:0.01, as determined by one-way ANOVA.
FIG. 4. Effect of glyphosate on the enzymatic activity in heart of fetuses. Values are means $SD of eight rats/group. aP:0.01; b P:0.05; cP:0.05, as determined by one-way ANOVA.
0.5% (P(0.001) and 1% w/v (P(0.01) doses were administered, G6PD showed an increase with 1% w/v dose (P(0.01). MD showed an increase only with 1% w/v dose (P(0.01). The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 5).
Glyphosate produced a signi7cant decrease of food and water consumption. The same effect was observed in pregnant rats and rabbits treated with dinitroaniline herbicides, maternal toxicity was in-
dicated by abortions and/or deaths in conjunction with anorexia and cachexia (Byrd et al., 1995). Probably, the least water ingestion observed could be associated either with the palatability of the glyphosate solution or with the effect of the herbicide and/or its metabolites on the nervous center of the thirst. A signi7cant weight loss during the exposure period was observed, related to the decreased food consumption. After the organs were weighed the liver showed a signi7cant decrease but the heart and brain showed no changes. After having been exposed to drinking water containing 3.0, 12.0, or 48.0 mM sodium chlorate, both male and female
FIG. 3. Effect of glyphosate on the enzymatic activity in liver of fetuses. Values are means $SD of eight rats/group. aP:0.05, as determined by one-way ANOVA.
FIG. 5. Effect of glyphosate on the enzymatic activity in brain of fetuses. Values are means $SD of eight rats/group. a,c,d P:0.01; bP:0.001; as determined by one-way ANOVA.
DISCUSSION
GLYPHOSATE AND ENZYMATIC ACTIVITY IN RATS
Sprague}Dawley rats had signi7cant weight loss during the 90-day exposure period. Also, in these same groups, females had mild but signi7cant decreases in the following relative organ weights; adrenals, thymus, and spleen. In males, the heart, kidneys and liver were mildly decreased (Mc Cauley et al., 1995). On the other hand, according to OMS data (1996), long-term studies of toxicity in rats treated with doses of glyphosate of 30 g/kg of food showed a significant decrease in the body weight of the rats and their pups. A reduction in the weight of the body and organs, such as kidney, liver, and thymus, in rats F344/N and mice B6C3F1 fed with glyphosate was observed. The effect of glyphosate on enzymatic activity in the liver of pregnant rats showed alterations for ICD and MD. Female rats exposed to the herbicide have lower food ingestion and the enzymatic activity could be affected by the alteration in the diet (Asante et al., 1989). On the other hand, diquat-treated hepatocytes maintained in a chemically de7ned medium showed both a time- and a concentration-dependent increase in G6PD enzyme activity. Therefore, the expression of hepatic G6PD would be modulated by free radicals during oxidative stress (Cramer et al., 1995). The activity of ICD, G6PD, and MD showed similar behavior in heart and liver. Both G6PD and ICD are sensitive to lower concentrations of herbicide, but there was a higher enzymatic activity when its concentration was increased, probably as a protection mechanism by increasing the NADPH production. Beuret and colleagues. have observed in our laboratory that glyphosate increases the TBAR’S production under our experimental conditions (C. J. Beuret, personal communication). In brain the enzymatic activity of ICD showed changes similar to those that were observed in heart. The studies of Arisi et al. (1994) with other pesticides showed that the activity of antioxidant enzymes such as G6PD in the brain were not modi7ed by acute lindane treatment. The poor response of the different fetus organs could be a protective mechanism of the placenta. Studies of placental transfer and teratology of pentachlorophenol in rats by Larsen et al. (1975) showed that the concentrations in the placentas and fetuses up to 32 h remained very small, indicating that the amount that passes through the placental barrier is negligible. Our studies show that the enzymatic activity of the dehydrogenases of both pregnant rats and their fetuses are altered by the herbicide glyphosate. The effect of glyphosate, de-
230
pending on its concentration, was speci7c for each enzyme in the different organs. When we studied the effect of low water and low food intake, we found that there were no signi7cant differences from the values of enzymatic activities of the control group. Thus, we can attribute the observed changes in the enzyme activities to the effects of glyphosate. Our study and those of Cox (1995) and Greenpeace (1995) contribute evidence on the effect of glyphosate on the ecological equilibrium. Since it was observed that glyphosate affects the enzyme activities that catalyze NADPH production, the mechanisms by which glyphosate produces such effects should be studied. Agriculturists combine the different commercial herbicides together with glyphosate to obtain the best results. The practice of creating coctails of herbicides could produce a potentiation of their individual properties. As a result, glyphosate could increase its levels of toxicity.
ACKNOWLEDGMENTS The authors are grateful to Lic. Cecilia J. Beuret and Mr. Dominguez for their technical assistance.
REFERENCES Arisi, A. C., Simizu, K., and Kogate, M. (1994). Brain and liver lipid peroxidation levels following acute and short-term lindane administration in the rat. Toxicol. Lett. 74, 61}68. Asante, E. A., Hill, W. G., and Bul7eld, G. (1989). Analysis of lines of mice selected for fat content. 1. Correlated responses in the activities of NADPH generating enzymes. Genet. Res. 54, 155}160. Byrd, R. A., Markham, J. K., and Emmerson, J. L. (1995). Developmental toxicity of dinitroaniline herbicides in rats and rabbits. I: Tri8uoralin. Fund. Appl. Toxicol. 26, 181}190. Cox, C. (1995). Glyphosate, part 2: Human exposure and ecological effects. Herbicide factsheet. J. Pestic. Reform. 15, 14}20. Cramer, C. T., Cooke, S., Ginsberg, L. C., Kletzien, R. F., Stapleton, S. R., and Ulrich, R. G. (1995). Upregulation of glucose-6phosphate dehydrogenase in response to hepatocellular oxidative stress: Studies with diquat. J. Biochem. Toxicol. 10, 293}298. Farrell, H. M. (1980). Puri7cation and properties of NADP-isocitrate dehydrogenase from lactating bovine mammary gland. Arch. Biochem. Biophys. 204, 551}559. Fujii, T. (1997). Transgenerational effects of maternal exposure to chemicals on the functional development of the brain in the offspring. Cancer Causes Control 8, 525}528. Glock, G. E., and Mc Lean, P. (1953). Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase of rat liver. Biochem. J. 55, 400}408.
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Greenpeace. (1995). ‘‘Pestizide im grundurasser.’’ Greenpeace Bericht. Larsen, R. V., Born, G. S., Kessler, W. V., Shaw, S. M., and Van Sickel, D. C. (1975). Placental transfer and teratology of pentachlorophenol in rats. Environ. Lett. 10, 121}128. Layne, E. (1957). Spectrophotometric and turbidimetric methods of measuring proteins. III. Biuret method. Methods Enzymol. 3, 450}462. Mc Cauley, P. T., Robinson, M., Daniel, F. B., and Olson, G. R. (1995). The effects of subchronic chlorate exposure in Sprague}Dawley rats. Drug Chem. Toxicol. 2}3, 185}199.
Mensink, H., and Janssen, P. (1994). Glyphosate. In ‘‘Environmental Health Criteria 159.’’ World Health Organization, Geneva. Ochoa, S. (1968). Methods Enzymol. I, 735. Organizacion Mundial de la salud. (1996). Criterios de salud ambiental No. 154. Glifosato. Acta Toxicologica Argentina. 4, 20}22. Prabhakaran, S., Shameem, F., and Devi, K. S. (1993). In8uence of protein de7ciency on hexachlorocyclohexane and malathion toxicity in pregnant rats. Vet. Toxicol. 35, 429}433. Strubbe, J. H., and Gorissen, J. (1973). Meal patterning in the lactating rat. Physiol. Behav. 25, 18}21.
Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses1 Jorgelina Daruich, Fanny Zirulnik, and MarmH a Sof mH a Gimenez Ca& tedra de Bioquı& mica Molecular, Area Quı& mica Biolo& gica, Facultad de Quı& mica, Bioquı& mica y Farmacia, Universidad Nacional de San Luis, 5700 San Luis, Argentina Received September 14, 2000
complexation in water with ions, e.g., Ca2# and Mg2#, sorption to sediment, suspended particles in water and soil, photodegradation in water, uptake by plants, and biodegradation. Data on the occurrence of glyphosate in biota and environmental abiota as part of regular monitoring programs are very scarce. Measurements of daily human intake of glyphosate via food and drinking water (total-diet studies) are not available (Mensink and Janssen, 1994). Pregnancy is a physiological condition in which deep adaptations are produced in the maternal metabolism. So the development and growth of fetuses are safeguarded by those metabolic changes (Strubbe and Gorissen, 1973). The effects of protein deprivation and pesticide exposure have been studied in pregnant rats, and a decrease of enzymes such as glutathion peroxidase and glucose-6-phosphate dehydrogenase was observed (Prabhakaran, et al., 1993). Other studies suggest that maternal exposure to herbicides during pregnancy induces a variety of functional abnormalities in the brain of offspring dependent both on the pharmacological action of chemicals and on the stage of gestation, even with a single exposure (Fujii, 1997). The aim of this work was to study the activity of some enzymes with a function in the pathways of NADPH generation, isocitrate dehydrogenase (ICD), glucose-6-phosphate dehydrogenase (G6PD), and malate dehydrogenase (MD) in liver, heart, and brain of pregnant rats and their fetuses which were exposed to the herbicide glyphosate.
To prevent health risk from environmental chemicals, particularly for progeny, we have studied the effects of the herbicide glyphosate on several enzymes of pregnant rats. Glyphosate is an organophosphorated nonselective agrochemical widely used in many countries including Argentina and acts after the sprout in a systemic way. We have studied three cytosolic enzymes: isocitrate dehydrogenase-NADP dependent, glucose-6-phosphate dehydrogenase, and malic dehydrogenase in liver, heart, and brain of pregnant Wistar rats. The treatment was administered during the 21 days of pregnancy, with 1 week as an acclimation period. The results suggest that maternal exposure to agrochemicals during pregnancy induces a variety of functional abnormalities in the speciAc activity of the enzymes in the studied organs of the pregnant rats and their fetuses. 2001 Academic Press Key Words: glyphosate; dehydrogenases; pregnancy; fetuses.
INTRODUCTION
Glyphosate is a weak organic acid consisting of a glycine and a phosphonomethyl moiety. Glyphosate is usually formulated as a salt of the deprotonated acid of glyphosate and a cation, e.g., isopropylamine or trimethyl sulfonium. It is a postemergent, systemic, and nonselective herbicide that is used in both agricultural and nonagricultural areas all over the world. Environmental exposure may occur because of deposition due to drift and accidental releases. The most important processes of dissipation that may be involved after application of glyphosate are
MATERIALS AND METHODS 1
This work was supported by a CONICET Grant (PIP 4931) and by a grant from the Universidad Nacional de San Luis, Argentina (Proyecto 8104).
Chemicals. All the chemicals were of reagent grade and were purchased from Sigma and Merck 226
0013-9351/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
227
DARUICH, ZIRULNIK, AND GIMENEZ
Laboratories. Glyphosate is commercialized in Argentina under the name of Herbicygon, from M.F.L. S.R.L. The empirical formula is C3H8NO5P. Animals and diets. Animals were provided by the National University of San Luis. The acclimation period was 1 week. Wistar female virgin rats (210}230 g) at the proestrus stage were housed for 1 night with fertile males, and fertilization was assumed by the presence of spermatozoa in the vaginal smear. That day is designated as day 1 of pregnancy. The rats were then transferred to individual cages and kept in a 22}253C controlled environment with a light}dark cycle of 12 h each. The animals were fed with 20 g of a stock laboratory diet, elaborated by Cargill, with the following ingredients: meat 8our, bone and meat 8our, 7sh meal, blood 8our, soybean meal, toasted soybean, soy expeller, sun8ower 8our, cotton 8our, peanut meal, animal fat, corn, wheat, sorghum, oat, barley, wheat bran, rice bran, gluten meal, vitamins A, E, B, D3, K3, and B12, niacin, pantothenic acid, choline, ascorbic acid, bone ash, salt, calcium carbonate, oyster, manganese oxide, zinc oxide, ferrous sulfate, copper oxide, sodium selenite, iodine, and cobalt. Thirty-7ve milliliters of potable water in a water bottle were administered daily and body weight and food intake were measured daily. The rats were separated into three groups (eight rats/group); I, control group which drank tap water; II, group which drank glyphosate solution 0.5% w/v in tap water (dose: 0.2 ml glyphosate/ml water); and III, group which drank glyphosate solution 1% w/v in tap water (dose: 0.4 ml glyphosate/ml water). Another experiment was carried out to determine whether the obtained results could be attributed solely to glyphosate. Six control rats (IV) were treated with low water and food (10 ml and 10 g, respectively); this treatment began the second week because at this time group III decreased the ingestion of water and food. Group IV is called Low in the tables and 7gures. In the 7rst week the rats of all groups ate about 20 g of food and drank about 35 ml of tap water per day. Experimental conditions. At 8:00}9:00 a.m. of day 21 of gestation, the rats were anesthetized with diethyl ether. Each fetus was delivered by rapid hysterectomy, identi7ed, weighed, and then killed immediately by decapitation with scissors. Maternal and fetal livers, hearts, and brains were immediately removed, washed in a cold saline solution, and kept at !203C until needed for analysis. The fetal organs were pooled. Tissue preparation and enzymatic assays. Livers, hearts, and brains (0.5 g/1 ml buffer) were
homogenized in an Ultra Turrax T25 machine with a 0.5 M Tris}HCl buffer, pH 7.4, containing 1 mM dithiotreitol. The homogenates were centrifuged at 100,000 g for 1 h by a Beckman Model L2 65B ultracentrifuge with a TY rotor to yield the cytosolic fraction, and the enzymatic activities were measured in the supernatant. Isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and malic dehydrogenase were determined by the rate of NADPH formation at 340 nm in a Shimadzu spectrophotometer, according to Farrell (1980), Glock and Mc Lean (1953), and Ochoa (1968), respectively. The results were expressed as lmol NADP/min/mg protein. Protein concentration was measured by the Biuret reaction (Layne, 1957). Statistical analyses. Signi7cant differences among means were considered at a level of P(0.05 and identi7ed by one-way ANOVA, Kolmogorov} Smirnov, and Newman}Keul procedures. In all the cases the variances were homogeneous. RESULTS
Effect of glyphosate on the daily consumption of food and water. Pregnant rat food and water ingestion decreased with both doses of glyphosate. Pregnant rat body and liver weights decreased signi7cantly compared to the controls when glyphosate 1% p/v was administered. Fetus body weights showed no signi7cant differences (Table 1). Effect of glyphosate on the enzymatic activity in liver of pregnant rats. The activity of ICD showed a decrease with 0.5% w/v of glyphosate and a signi7cant increase with a dose of 1% w/v of the herbicide (P(0.01), compared to the control group. The speci7c activity of G6PD decreased with 0.5% w/v and increased to the control value with glyphosate 1% w/v (P(0.05), whereas MD showed no signi7cant differences from the control. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 1). Effect of glyphosate on the enzymatic activity in heart of pregnant rats. ICD activity showed a signi7cant increase with 0.5% w/v and 1% of glyphosate (P(0.005) compared to the control. G6PD showed no differences with 0.5% w/v but increased with a dose of 1% w/v of glyphosate (P(0.01), whereas MD showed no signi7cant differences from the control. The enzymatic activities in the low group, with low water and low food, showed
228
GLYPHOSATE AND ENZYMATIC ACTIVITY IN RATS
TABLE 1 Effect of Glyphosate on Food and Water Ingestion and Body and Organ Weights in Pregnant Rats and Their Fetuses
Pregnant rats Food intake (g/day) Water ingestion (ml/day) Mass balance (%) Total body weight gain (g) Organ weights (g) Liver Heart Brain Fetuses Body weight (g)
Control
0.5% p/v
1% p/v
Low
P
19.05$0.99 32.50$1.05 100 92.00$8.9
15.95$0.24 23.70$0.6 87.7 80.70$11.37
14.53$0.3 25.79$1.56 57.38 52.79$3.14
11.54$0.32 22.49$0.4 53.81 49.51$5.04
(0.01 (0.01 (0.05
8.87$0.53 0.83$0.03 1.45$0.15
7.75$0.31 0.80$0.04 1.71$0.05
7.41$0.38 0.74$0.04 1.57$0.08
7.62$0.26 0.65$0.02 1.47$0.2
(0.05 ns ns
3.20$0.36
3.52$0.03
3.13$0.01
3.06$0.09
ns
Note. Values are means$SD for eight rats/group. Signi7cant differences were determined by one-way ANOVA; ns, not signi7cant. The values of food and water intake are the means of 3 weeks of treatment.
no signi7cant differences from that of the control group (Table 2). Effect of glyphosate on the enzymatic activity in brain of pregnant rats. ICD did not change with 0.5% glyphosate but increased with 1% (P(0.01), whereas G6PD and MD did not change with both doses of glyphosate. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 2). Effect of glyphosate on the enzymatic activity in liver of fetuses. The activity of ICD showed no differences between control and 0.5% w/v of glyphosate, but it showed a signi7cant decrease
when glyphosate 1% w/v was administered (P(0.05). G6PD and MD showed no signi7cant differences with both doses of glyphosate used compared to the control. The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 3). Effect of glyphosate on the enzymatic activity in heart of fetuses. ICD activity showed a signi7cant increase with 0.5% w/v dose of glyphosate (P(0.01), but there were no differences with the 1% w/v dose. The same behavior was observed with G6PD activity (P(0.05). MD decreased signi7cantly with both doses of glyphosate (P(0.05). The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 4). Effect of glyphosate on the enzymatic activity in brain of fetuses. ICD increased signi7cantly when
TABLE 2 Effect of Glyphosate on the Enzymatic Activity in Heart of Pregnant Rats
Control 0.5% p/v 1% p/v Low FIG. 1. Effect of glyphosate on the enzymatic activity in liver of pregnant rats. Values are means$SD of eight rats/group. a P:0.01; bP:0.05; cP:0.001, as determined by one-way ANOVA.
ICD
G6PD
MD
53.24$7.73 93.07$7.33b 121.88$5.85c 49.65$8.78
1.85$0.66d 1.403$0.57e 4.43$1.58f 2.09$0.68
2.36$1.10 2.673$0.41 2.65$1.38 2.85$1.28
Note. Values are means$SD of eight rats/group (kmol NADP/min/mg prot.). Signi7cant differences are determined by one-way ANOVA. a,b - a,c-e,f: P(0.001; d,e-d,f: P(0.01.
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FIG. 2. Effect of glyphosate on the enzymatic activity in brain of pregnant rats. Values are means $SD of eight rats/group. a P:0.01, as determined by one-way ANOVA.
FIG. 4. Effect of glyphosate on the enzymatic activity in heart of fetuses. Values are means $SD of eight rats/group. aP:0.01; b P:0.05; cP:0.05, as determined by one-way ANOVA.
0.5% (P(0.001) and 1% w/v (P(0.01) doses were administered, G6PD showed an increase with 1% w/v dose (P(0.01). MD showed an increase only with 1% w/v dose (P(0.01). The enzymatic activities in the low group, with low water and low food, showed no signi7cant differences from that of the control group (Fig. 5).
Glyphosate produced a signi7cant decrease of food and water consumption. The same effect was observed in pregnant rats and rabbits treated with dinitroaniline herbicides, maternal toxicity was in-
dicated by abortions and/or deaths in conjunction with anorexia and cachexia (Byrd et al., 1995). Probably, the least water ingestion observed could be associated either with the palatability of the glyphosate solution or with the effect of the herbicide and/or its metabolites on the nervous center of the thirst. A signi7cant weight loss during the exposure period was observed, related to the decreased food consumption. After the organs were weighed the liver showed a signi7cant decrease but the heart and brain showed no changes. After having been exposed to drinking water containing 3.0, 12.0, or 48.0 mM sodium chlorate, both male and female
FIG. 3. Effect of glyphosate on the enzymatic activity in liver of fetuses. Values are means $SD of eight rats/group. aP:0.05, as determined by one-way ANOVA.
FIG. 5. Effect of glyphosate on the enzymatic activity in brain of fetuses. Values are means $SD of eight rats/group. a,c,d P:0.01; bP:0.001; as determined by one-way ANOVA.
DISCUSSION
GLYPHOSATE AND ENZYMATIC ACTIVITY IN RATS
Sprague}Dawley rats had signi7cant weight loss during the 90-day exposure period. Also, in these same groups, females had mild but signi7cant decreases in the following relative organ weights; adrenals, thymus, and spleen. In males, the heart, kidneys and liver were mildly decreased (Mc Cauley et al., 1995). On the other hand, according to OMS data (1996), long-term studies of toxicity in rats treated with doses of glyphosate of 30 g/kg of food showed a significant decrease in the body weight of the rats and their pups. A reduction in the weight of the body and organs, such as kidney, liver, and thymus, in rats F344/N and mice B6C3F1 fed with glyphosate was observed. The effect of glyphosate on enzymatic activity in the liver of pregnant rats showed alterations for ICD and MD. Female rats exposed to the herbicide have lower food ingestion and the enzymatic activity could be affected by the alteration in the diet (Asante et al., 1989). On the other hand, diquat-treated hepatocytes maintained in a chemically de7ned medium showed both a time- and a concentration-dependent increase in G6PD enzyme activity. Therefore, the expression of hepatic G6PD would be modulated by free radicals during oxidative stress (Cramer et al., 1995). The activity of ICD, G6PD, and MD showed similar behavior in heart and liver. Both G6PD and ICD are sensitive to lower concentrations of herbicide, but there was a higher enzymatic activity when its concentration was increased, probably as a protection mechanism by increasing the NADPH production. Beuret and colleagues. have observed in our laboratory that glyphosate increases the TBAR’S production under our experimental conditions (C. J. Beuret, personal communication). In brain the enzymatic activity of ICD showed changes similar to those that were observed in heart. The studies of Arisi et al. (1994) with other pesticides showed that the activity of antioxidant enzymes such as G6PD in the brain were not modi7ed by acute lindane treatment. The poor response of the different fetus organs could be a protective mechanism of the placenta. Studies of placental transfer and teratology of pentachlorophenol in rats by Larsen et al. (1975) showed that the concentrations in the placentas and fetuses up to 32 h remained very small, indicating that the amount that passes through the placental barrier is negligible. Our studies show that the enzymatic activity of the dehydrogenases of both pregnant rats and their fetuses are altered by the herbicide glyphosate. The effect of glyphosate, de-
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pending on its concentration, was speci7c for each enzyme in the different organs. When we studied the effect of low water and low food intake, we found that there were no signi7cant differences from the values of enzymatic activities of the control group. Thus, we can attribute the observed changes in the enzyme activities to the effects of glyphosate. Our study and those of Cox (1995) and Greenpeace (1995) contribute evidence on the effect of glyphosate on the ecological equilibrium. Since it was observed that glyphosate affects the enzyme activities that catalyze NADPH production, the mechanisms by which glyphosate produces such effects should be studied. Agriculturists combine the different commercial herbicides together with glyphosate to obtain the best results. The practice of creating coctails of herbicides could produce a potentiation of their individual properties. As a result, glyphosate could increase its levels of toxicity.
ACKNOWLEDGMENTS The authors are grateful to Lic. Cecilia J. Beuret and Mr. Dominguez for their technical assistance.
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