This study evaluates toxicity of a mixture of three pesticides (atrazine, chlorpyrifos and endosulfan; ACE) added to the rodent diet allowing the mice to ingest the equivalent of the Acceptable Daily Intake (ADI: 0.005, 0.01 and 0.006 mg/kg body weight /day, respectively), in addition to oral administration of vitamin E (α-tocopherol; 100 ul / mouse). During gestation (21d), the mouse dams were received one of the following treatments: (a) diet free of ACE; (b) diet enriched with ACE; (c) diet free of ACE + oral vitamin E; and (d) diet enriched with ACE + oral vitamin E. During lactation, the dams were not subjected to any chemical treatments. After weaning (42d), selected organs and blood samples were collected for analyses. Compared with the control results either in dams or their pups, the ACE mixture induced high elevation in AST, ALT, ALP, urea and MDA and high decline in BuChE, SOD and CAT. The pups were more affected than the dams with respect to alterations in MDA and BuChE activities, while the opposite was achieved with respect to SOD activities. The ameliorative effect of vitamin E, based on estimating the “Amelioration Index; AI” revealed the powerful effect of this vitamin in alleviating the oxidative stress exerted by the tested mixture. The findings may encourage further work on the toxicity of pesticide mixtures, especially on pregnant and lactating animals and the reproductive effects on new born child. Also, the search for powerful antioxidants has to receive special attention.
The extensive use of different chemical groups of pesticides caused contamination across the globe. This in turn, resulted in complications affecting human health and impacts on non-target organisms . About 95% of studies related to pesticides were carried out on individual chemicals; however, studies on the effect of chemical mixtures on human systems are rare . Humans are exposed to a cocktail of chemical compounds of multiple routes of exposure (e.g., ingestion, inhalation, skin contact), but food remains an important source of exposure. The impacts of such exposures on human health are still poorly understood, a matter making risk assessment of chemical mixtures uneasy and inaccurate process. Major difficulties in this respect are attributed to differences in the levels and exposure periods and frequency of their occurrence, as well as the diversity of active substances and adjuvants used in the formulations of these compounds . Certain pesticides in a mixture may interact chemically, mainly because the metabolism of one chemical can affect the metabolism of the other. Subsequently, mixtures of pesticides can interact additively, synergistically or antagonistically . Potentiation was reported for the mixture of atrazine, chlorpyrifos and chlorothalonil [4-6], as well as the mixture of cypermethrin, quinolphos and linuron .
To adequately protect the environment from toxicity of chemical mixtures, we may face many challenges including understanding the nature of interactions within an organism, identifying the most commonly mixtures occurring and causing adverse effects, and developing a regulatory plan to minimize their health and environmental impacts .
The present study is a part of collaborative research project between Egypt and France, thus the selection of pesticides was based on compensation between the mutual interests of both sides. The selected three pesticides belong to three different chemical classes; namely: triazines (atrazine), organophosphates (chlorpyrifos) and organochlorines (endosulfan) in tertiary combination (ACE). These pesticides are still used in several countries for combating pests in agricultural and household purposes . They are characterized by their persistence at low doses in the environment and food, as well as their acute toxicity on animal models. Their residues were found even in countries in which they were banned . The concentrations of endosulfan and chlorpyrifos in various foods were respectively found to be around (0.011–0.037 ppm) and (0.002–0.1 ppm) . Atrazine is still a drinking water contaminant, at very low concentrations, in some counties including France .
Atrazine is a systemic triazine herbicide used mainly to control weeds in some crops such as maize and rapeseed. In the EU it has been banned in 2004 due to its long persistence in groundwater . This herbicide has neurotoxic effects, immunotoxicological potential, especially in prenatal exposures, even at very low doses [13,14]. The herbicide atrazine was officially banned in Egypt since 1996.
Chlorpyrifos is an Organophosphorus (OP) insecticide widely used in agriculture and households. Its oxygen analogue metabolite (chlorpyrifos-oxon) affects the central nervous system by inhibiting acetylcholinesterase .and was reported to cause immunologic abnormalities in humans .and animals .and rectal cancer . Growing evidences demonstrated that chlorpyrifos and other OPs possessed endocrine disruptor (ED) properties at low doses . This insecticide is still used in Egypt for combating several pests in agriculture and public health.
Endosulfan was already banned in Egypt as early as the 1980’s and in several countries. However, it is one of the most toxic pesticides used intensively in Asia (India and China). It is basically a neurotoxic organochlorine insecticide and acaridae of long persistence in the environment. In laboratory animals, it induced toxic effects to the liver, kidneys, the nervous system, and reproductive organs [20,21]. These effects have been attributed to its oxidative stress against antioxidant enzymes; e.g., superoxide dismutase (SOD), glutathione peroxidase (GPX) and Glutathione-S-transferase (GST) .
It has been long recognized that many pesticides may induce oxidative stress following acute exposure in humans and animals [23,24]. Not only exposure to individual pesticides, but also exposure to pesticide mixtures canpromote oxidative stress, by increasing the concentration of reactive oxygen species (ROS) and products of oxidative damage such as lipid peroxides, and therefore affect the activity of antioxidant enzymes (AOE) , . Antioxidant, such as Vitamin E acts as biological free radical scavenger in the cell membranes , and thus protects cells from the damaging effects of the harmful free radicals .
Safety assessment established from various experimental and epidemiological studies are conducted through legal exposure thresholds such as the Acceptable Daily Intake (ADI). Exposure to pesticides at home is described as a significant risk; however, the most important source for contamination of humans remains food and cocktails of pesticides ingested every day . Women, during pregnancy and lactation, may expose to low doses of pesticides from different environmental sources. In the same protocol of the present study we recently investigated the effect of exposure of mouse dams to atrazine , endosulfan  and chlorpyrifos  during the overall period of gestation and lactation, and the indirect effect of such exposure on the offspring.
To the best of our knowledge, there is no data on ameliorative effect of vitamin E against the tested mixture (ACE), but there are many publications on the single pesticides [29-31]. Therefore, it was found of interest, to study and elucidate toxicity of a mixture composed of the above three mentioned pesticides and to assess the ameliorative effect of vitamin E supplementation during either gestation or lactation period. In our previous study, we investigated the effect of exposure to pesticide mixture during the lactation period only .
The present study was planned to evaluate the toxic effect of exposure to mixture of three pesticides (atrazine, chlorpyrifos and endosulfan; ACE) and the protective role of vitamin E during gestation period on mice.
Standard of selected pesticides (atrazine, 97.4%; chlorpyrifos, 99.2% and endosulfan (α & β = 2+1; 99.9%) were purchased from Fluka, Riedel-de Haën, France. D-L α-tocopherol acetate was purchased from Fluka (Riedel-de Haën, France).
Pesticides were mixed with rodent feed at a dose of 25 (atrazine), 50 (chlorpyrifos) and 30 µg /kg (endosulfan) of food as a ternary mixture (ACE). Pesticides levels in feed ware analysed in Eurofins (Nantes, France). The quantities of selected pesticides were around that we expected (i.e., ~ 25, 50 and 30 µg per kg of food as described in our previous study . Moreover, the average food consumption was estimate . The doses of tested pesticides were equalled to the Acceptable Daily Intake (ADI) for humans (0.005, 0.01 and 0.006 mgkg-1bw/ day, respectively) according to FAO/ WHO .
Mice weights 20±2g (adage 20 week) male and female C57 BL/6 J were obtained from Charles River Laboratories (Domaine des Oncins-BP 109, 69592 L’arbresle, Cedex, France). After 2 weeks of acclimatization, mice were divided into 8 cages (sixteen virgin female mice). Then, one male was placed overnight in each cage. The presence of spermatozoa was checked in the vaginal smear in the following morning. This day was connoted as gestation day 0 (GD 0). Pregnant female’s mice were housed individually at 23 ± 2o C; 40% RH, free fed on diet and water. The mouse dams were feed on pesticides during gestation only (20 days). Postnatal day 0 (PND 0) was used as the day of parturition (zero day of lactation). The litter of offspring were reduced to 6 pups, 3 male/3 females . The experimental work on animals was performed in Toxalim Unit, INRA, Toulouse, France, and in accordance to its institutional ethical committees in an accredited animal house.
The mouse dames in the present study were subjected to the following treatments: Group I feed on diet free of ACE (control), Group II feed on diet enriched with ACE, Group III feed on diet free of ACE + oral vitamin E (α-to-copherol) at 100 µl per mouse, Group IV feed on diet enriched with ACE + oral vitamin E at 100 µl per mouse.
Vitamin E was given twice a week. Pups were divided into subgroups (males or females) each of 20 animals. At the end of weaning (ca. 42 d), blood samples were taken from the facial artery of each animal (dams and pups), serum was separated. Then, the animals were sacrificed; organs (heart, spleen, liver, kidneys, testes or ovaries) were removed and weighted. Organs were cute to into two pieces, one kept in 10% formalin and used for histopathological investigation. Other pieces of liver were kept in nitrogen (-80o C) and used for biochemical studies.
All biochemical parameters were determined based on the methods descript in kits pamphlet. All kits if biomarkers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), butyryl cholinesterase (BuChE), urea, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) were obtained from Biodiagnostic Co., Dokki, Giza, Egypt. MDA and SOD activities were determined in liver tissues, while the other biochemical parameters were measured in sera.
Oranges (Liver, kidneys, ovaries and testes) were subjected to routine histopathological process and stained by haematoxylin and eosin (H&E). Four sections of each organ (two slides and two sections per slide) for each organ. Tissue injury in the examined organs was scored in different ratings according to .
The resultswereexpressed as means ± SE. All the data were statistically analyzed by means of the Statistical Package for Social Sciences (SPSS – Version 17.0 for windows). The results were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparison between different treatment groups. Statistical significance was set at P≤0.05.
Table 1 presents results of body, absolute and relative organs weights of mouse dams treated with the pesticide mixture during gestation period and the same for their pups just after weaning. There were insignificant differences between control results and those of vitamin E ones, with respect to weights of body and the examined organs, either for dams or their pups. The mixture treatment of dam’s body weight (27.53 g) recorded insignificant difference than control (29.70 g), while those of female (9.86 g) and male (10.84 g) pups recorded significant (P≤ 0.05) and high significant (P≤ 0.01) decreases, respectively compared to their corresponding control values. Co-administration of vitamin E in the male pups’ treatment improved body weight decrease to a pronounced degree.
The weights of liver recorded 0.68 g and 0.75 g for control female and male pups, respectively; values which were significantly higher (P≤ 0.05 - P≤ 0.01) than those obtained for the mixture treatments (Table 1). Co-administration of vitamin E didn’t improve such liver weight decrease. Except the treatment of the mixture to male pups, the kidney’s weights of other treatments didn’t show any significant differences than the corresponding control results. The excepted treatment recorded 0.14 g which were significantly lower (P≤ 0.05) than the control result (0.18 g). Except the treatment of the mixture to female pups, the heart’s weights of other treatments didn’t show any significant differences than the corresponding control results. The excepted treatment recorded 0.07 g which were significantly lower (P≤ 0.05) than the control result (0.09 g). In all experimental treatments there were no significant differences in spleen weights compared with the values recorded for control treatments. The weights of ovaries in control treatments were found 0.35 and 0.02 g, respectively for dams and female pups. In the mixture treatments, such values were significantly decreased. Vitamin E couldn’t alleviate such decrease in the female pups’ treatment. The testes weights of male pups exposed to the mixture or the mixture + vitamin E were significantly lower than the control value(0.08 g) (Table 1).
The relative weights (Table 1) of liver, kidney, ovary and testis of all treatments showed results of non-significant differences when compared with the results of their corresponding controls. The relative weight of heart in control dams recorded 0.87%. The mixture treatment (1.06%) and the mixture + vitamin E treatment (0.71%) were respectively found to be higher and lower than control value at (P≤ 0.05). The relative weight of spleen in the pups exposed to the mixture treatment (0.61%) was significantly higher (P≤ 0.05) than that obtained for the control mouse pups (0.48%) (Table 1).
Some biochemical parameters including liver, kidney and ant oxidative stress enzymes were estimated in mouse dams and their offspring following exposure of mothers during gestation period to the mixture of pesticides (Table 2). The serum activity of AST in control mice was found 69.66, 70.33 and 70.00 U/L, respectively in dams, female pups and male pups. The vitamin E treatments showed values of insignificant differences than those of controls. The treatment of the mixture caused high significant elevation (P≤ 0.05), accounted to 92.00, 92.66 and 93.66 U/L, respectively for dams, female pups and male pups. Co-administration of vitamin E with the pesticide mixture achieved some improvement in the enzyme activity but still significantly (P≤ 0.05) higher than control values. A significant elevation (P≤ 0.05) in serum activity of ALT was obtained for the mixture treatments (47.00, 45.33 and 46.00 U/L), compared with the control values (31.66, 32.66 and 33.33 U/L), respectively for dams, female pups and male pups. Co-administration of vitamin E with the pesticide mixture normalized the enzyme activity to a great extent. The mixture treatment caused very high elevation (P≤ 0.01) in ALP activity (142.00, 106.66 and 107.00 U/L) compared with the results of control treatments (75.33, 56.33 and 56.00 U/L), respectively for dams, female pups and male pups. Co-administration of vitamin E with the mixture achieved some improvement in the enzyme activity but still significantly (P≤ 0.05) higher than control values. Concentrations of urea in the mixture treatments were relatively little, however they were significantly higher (P≤ 0.05) than the concentrations in control treatments. Co-administration of vitamin E with the pesticide mixture normalized the enzyme activity to a great extent (Table 2). The serum activity of butyryl cholinesterase (BuChE) in control mice was found 3419.00, 1548.00 and 1503.33 U/L, respectively in dams, female pups and male pups (Table 2).
The vitamin E treatments showed values of insignificant differences than those of controls. The treatment of the mixture caused high significant decrease (P≤ 0.01), accounted to 3026.00, 1241.33 and 1281.66 U/L, respectively for dams, female pups and male pups. Co-administration of vitamin E with the pesticide mixture caused elevation of the enzyme activity but with values significantly lower (P≤ 0.05), than control values. A high significant elevation (P≤ 0.01) in the activity of lipid peroxidation (LPO), in terms of malondialdehyde (MDA) was obtained for the mixture treatments (100.10, 95.60 and 84.33 nmol/g tissue), compared with the control values (82.00, 51.33 and 52.33 nmol/g tissue), respectively for dams, female pups and male pups. Co-administration of vitamin E with the pesticide mixture resulted in normalizing the MDA value of the dam’s treatment (85.33 nmol/g tissue). While that for female and male pups (71.03 and 65.33 nmol/g tissue, respectively) were still significantly higher (P≤ 0.05) than control values. The activity of superoxide dismutase (SOD) in control mice was found 210.00, 190.66 and 190.00 U/g tissues, respectively in dams, female pups and male pups. These values were significantly decreased (P≤ 0.01) in the mice treated with the pesticide mixture and recorded 168.00, 165.33 and 166.66 U/g tissues, respectively. Co-administration of vitamin E with the pesticide mixture improved SOD activity but with values still significantly lower (P≤ 0.05) than control values. The activity of serum catalase (CAT) in control mice was found 401.66, 381.66 and 385.33 µmol/min/ml, respectively in dams, female pups and male pups. These values were significantly decreased (P≤ 0.01) in the mice treated with the pesticide mixture and recorded 256.33, 239.66 and 243.00 µmol/min/ml, respectively. Co-administration of vitamin E with the pesticide mixture improved CAT activity but with values still significantly lower (P≤ 0.05) than control values (Table 2)
Body, absolute and relative organs weights of mouse dams and their pups treated with the mixture, with and without supplementation of vit E, during gestation periods.
Activities and/or concentration levels of biochemical parameters in serum / tissue of mouse dams and their pups following exposure during gestation to a mixture of pesticides, with and without administration of vitamin E.
Microscopic examination of sections from liver, kidneys, ovaries and testes prepared from the animals subjected to different treatments in the present study was performed. Figure 1 illustrates the histopathologic structure of the studied organs prepared from the normal (control) groups. The latter was considered as a base for comparing the histopathologic alterations in the pesticide mixture groups, with and without vitamin E. Sections of control organs (Fig. 1) were characterized by normal histopathologic structure, observed on hepatic lobules, central veins and appearance of hepatocytes. Renal paranchyma and tubules showed normal histopathological structure of kidneys. Graffian follicle and corpus luteum were characterized in normal ovary. Sections of testes were characterized by normal seminiferous tubules, and ovaries showed normal corpus luteum (Fig. 1a–i).
Based on Brunt et al. (1999) such alterations were adopted and scored in terms of degree of cell damage, as follows: (0) = no change; (1) = mild change (e.g., <25% of cells showing damage); (2) = moderate change (e.g., 25–50% cell damage); and (3) = severe change (e.g., >50% cell damage).
Liver: moderate hepatic vacuolar degeneration of hepatocytes (dams, male and female pups); severe cytoplasmic vacuolization of hepatocytes (dams and male pups), but to less extend in the female pups. Moderate and severe congestion, respectively in dams and female pups accompanied with mild Kupffer cells activation (dams). Pyknosis of nuclei was scored as mild in female pups, but moderate in dams and male pups. Co-administration of vitamin E diminished all the above cellular damages in female pups and minimized the others in male pups and dams.
Kidneys: moderate and mild vacuolation of glomerular tuft, respectively in dams and female pups epithelial lining renal tubules and glomerular tuft (dams, male and female pups); moderate focal renal hemorrhage (dams); moderate thickening of parietal layer of Bowman’s capsule (dams) and mild congestion of intertubular blood vessels (female pups); severe vacuolation of epithelial lining renal tubules in dams and male pups. Moderate focal renal haemorrhage was seen in dam’s sections. Co-administration of vitamin E diminished or minimized all the above cellular damages to a great extent.
Ovary: mild and moderate -atretic follicles in dams and female pups, respectively and severe degeneration of corpus luteum (dams). Also, there were moderate hyperplasia of interstitial cells and follicles of different stages of development in dams. Co-administration of vitamin E retained normal histopathological picture to the examined ovaries.
Testis: severe degeneration of spermatogoneal cells lining seminiferous tubules were seen in male pups exposed the pesticide mixture. Also, moderate necrosis of germ cells, intertubular oedema and atrophy of seminiferous tubules were manifested. Co-administration of vitamin E diminished or minimized all the above cellular damages in testes to a great extent.
Large quantities of pesticides are used globally to control pests in agricultural crops and households. Humans may expose at all stages of their life to pesticides even when the babies are in the mother’s womb. Also, children are not adequately protected from the adverse effects of pesticides . Epidemiological studies revealed an association between spontaneous abortions and fetal death after maternal exposure to pesticides . Indeed, little is known about impacts of low doses of pesticide mixtures on human health . On the other hand, evaluation of the impact of exposure to low doses of pesticide mixtures on humans is still uncertain. As a consequence, impacts of pesticide combinations have to be examined carefully, especially at very low doses .
The purpose of this study was to investigate toxicity of a mixture composed of three pesticides (atrazine,chlorpyrifos, endosulfan, ACE) to mouse dams during gestation period and the effect on their pups. Also, to evaluate the ameliorative effect of vitamin E supplementation. To the best of our knowledge, there are no studies on the tested mixture with respect to hepato-renal dysfunction and oxidative stress that may be caused to pregnant animals and protective effects of antioxidants. Only, in vitro studies the concerned mixture was evaluated on liver cell defense systems using human and mice cultured hepatocytes and liver cells . However, studies were previously conducted on the single compounds with special concern to their adverse effects during pregnancy and lactation and the ameliorative effects of various antioxidants. For instance, in the same protocol of the present study we previously investigated toxicity of atrazine ; endosulfan ; chlorpyrifos and the protective effect of vitamin E (α-tocopherol) on mouse dams and their pups.
Histopathological sections for organs (liver, kidney, ovary and testis) prepared from normal mice for comparison with mixture-treated mice, with and without vitamin E, during gestation and lactation periods (H&E stain 400×). (a) Liver of control mouse dam showing the normal histological structure of hepatic lobule from central vein (CV) and normal hepatocytes (H) (H&E stain 400×). (b) Liver of control male offspring showing the normal histological structure of hepatic lobule from central vein (CV)and normal hepatocytes (H) (H&E stain 400×). (c) Liver of control female offspring showing the normal histological structure of hepatic obule from central vein (CV) and normal hepatocytes (H) (H&E stain 400×). (d) Kidney of control mouse dam showing the normal histological structure of renal parenchyma. Note normal glomerulus (G) and normal renal tubules (T) (H&E 400×). (e) Kidney of control male offspring showing the normal histological structure of renal parenchyma. Note normal glomerulus (G) and normal renal tubules (T) (H&E 400×). (f) Kidney of control female offspring showing the normal histological structure of renal parenchyma. Note normal glomerulus (G) and normal renal tubules (T) (H&E 400×). (g) Ovary of control mouse dam showing normal graffian follicle (GF) and normal corpus luteum (CL). (H&E stain 400×). (h) Ovary of female offspring showing normal graffian follicle (GF) (H&E stain 400×). (i) Testis of control male offspring showing normal seminiferous tubules (ST) (H&E 400×).
In toxicological studies, evaluation of organ toxicity is an important criterion. Generally, increase or decrease of organs weights than normal may be considered as a sign of toxicity. The results of the present study revealed that ACEtreated rats during gestation have shown a high significant decrease in the body weight of male pups compared with the females, while the dam’s body weights were not affected significantly (Table 1). According to , such decrease in body weight may be attributed to excessive break down of tissue proteins. On the other hand, the significant increase in relative liver weight in pups may be attributed to induction of hepatomegaly which was designated as an important reason for losing of body weight . Fortunately, the ACE treatments didn’t cause significant changes in the relative weights of kidney, ovary and testis of the experimental mice (Table 1). Supplementation of vitamin E in conjunction with ACE treatments showed body and organs weight values insignificantly differed than the corresponding control ones. These results coincide with the effect of vitamin E on atrazine , endosulfan  and chlorpyrifos  when tested individually at ADI values against pregnant and lactating mice.
The release of intracellular enzymes (e.g., AST, ALT & ALP) in the circulation following exposure to pesticides is one of the most sensitive indicators of hepatocyte injury. The elevated activity of these enzymes, as observed here, is an indicative of cellular leakage and loss of functional integrity of the liver cell membranes . Severe liver injury occurs when released extra amount of ALT and ALP reach three and two times of upper limits of normal levels (ULN), respectively . This gave an indication of severe liver injury due to ACE treatments according to the values obtained for ALT and ALP (Table 2). Kidney is one of the target organs attacked by acute and chronic exposure to pesticides . Elevation of urea concentrations in serum of treated rats (Tables 2) may be an indicative of kidney dysfunction as a result of oxidative damage .
Cholinesterase (ChE), or pseudo cholinesterase, is synthesized mainly in hepatocytes and secreted into the blood stream. In liver dysfunction, its activity is declined due to reduced synthesis in contrast to other serum enzymes of liver function whose activities increase as a result of increased release from their cellular sources following damage of cell membrane . In this respect, changes in ChE activity reflect the changes in hepatocellular functions and have been regarded as sensitive indicators of the diminished synthetic capacity of the hepatic parenchyma . In the present study, the decline of BuChE seemed to occur in a manner resembling that previously reported [29-31] for each individual compound of the tested mixture (ACE) in the present study. Following sub-chronic and chronic exposure to OP pesticides, it was found a correlation between AChE inhibition and lipid peroxidation levels in erythrocytes .
In fact, the toxicity of biologically active substances (e.g., pesticides) is associated with the formation of reactive oxygen species (ROS). These ROS are responsible of inducing oxidative stress in the tissues and chronic permanent damage . The harmful effects of ROS are balanced by the antioxidant action of no enzymatic and enzymatic antioxidants which are molecules containing an unshared electron. It is well documented that many pesticides may induce oxidative stress following acute exposure in humans  and animals [49,50]. Increased lipid peroxidation (LPO) in various tissues may be one of the molecular mechanisms involved in pesticides - induced toxicity . LPO is a marker of oxidative damage caused by many substances including pesticides. Malondialdehyde (MDA) is a stable end product of LPO and therefore can be used as an indirect measure of the cumulative LPO. Superoxide dismutase (SOD) provides the first line of defense against oxygen derived free radicals and decreases oxidative stress by dismutation O2- . Elevation of MDA and decline of SOD activities by ACE mixture could be attributed to the oxidative stress effects of the tested mixture. Several substances including vitamin E were used to alleviate toxic hazards of pesticides-induced oxidative stress in experimental animals. Antioxidants, such as vitamin E are an important biological free radical scavenger in the cell membranes , and it protects cells from the damaging effects of free radicals . Interestingly, the vitamin showed its ability to ameliorate the oxidative stress induced by the ACE mixture (Table 2).
The hepatic and renal dysfunction results (Table 2) corroborated the histopathological lesions observed in the present study (Table 3). Also, the results revealed the protective effect of vitamin E against ACE-induced histopathological impairments in liver, kidney, ovary and testis of the experimental mice. These results coincided with that previously reported by  on chlorpyrifos.
Pregnant women are considered as a special risk group due to possibility of increased risk to acute lymphocytic leukemia to childhood when these women use pesticides during pregnancy . The fetus is more vulnerable to the toxic effects of environmental exposures than are children or adults . Highly lipophilic compounds, such as organic pesticides, have the ability to cross the placenta during pregnancy and reach the fetus .
The literature offers information about toxic effects of the individual pesticides (atrazine, chlorpyrifos and en-dosulfan) on female mice during gestation and lactation periods and their offspring, as well as the ameliorative effect of vitamin E co-administration [29-31]. To the best of our knowledge, such studies on the tested mixture (ACE) here may not be preceded before. Only, green tea polyphenols (as an example for natural polyphenols) or butylated hydroxytoulene (as an example for artificial polyphenols) were found to attenuate toxicity of a mixture (chloropyrifos, fenitrothion and lambada cyhalothrin) against male rats .
It may be difficult to generalize the toxic effect of a pesticide mixture with respect to effects of its individual components. For examples, the mixture of atrazine, chlorpyrifos and endosulfan (ACE) was reported to affect the cell defense system in mice liver in vitro tests. Such effect mimicked that of the most potent pesticide . Also, the same mixture was reported to induce significantly higher cytotoxic effects on human hepatocytes as compared with its individual pesticides .
On the other hand, a mixture of three pesticides (Chloropyrifos, fenitrothion and lambada cyhalothrin) administered to male rats induced significant inhibition in plasma cholinesterase (ChE), damage in liver confirmed with elevation of plasma ALT, AST, as well as elevation in oxidative stress (OS) marker malodialdehyde (MDA) . Also, a mixture of carbendazim (CBZ) fungicide and chlorpyrifos (CPF) insecticide was reported to cause significant decrease in the body weight gain with concomitant increases in the relative kidney and spleen weights of female rats, as well as significant increase in the levels of AST, ALT, urea and creatinine, and significant decreases in both antioxidant enzymes activities and nonenzymatic antioxidant level .
The physiological and biochemical alterations induced by ACE mixture in the present investigation were coincided, to some extent, with the results of , how ever with different pesticide mixture. Also, the pattern of biochemical alterations induced by the ACE mixture in the present investigation (Table 2) resembled that previously obtained with the individual pesticides [29-31]. By other words, aminotransferases (AST & ALT), ALP and MDA were elevated, while BuChE and SOD were declined, both for the individual pesticides or the ACE mixture, and nearly in equal values. Supporting our findings are those previously reported by  that the effect of combined exposure to methamidophos and chlorothalonil on the development of suckling rats was not found to have a greater toxic effect than that resulting from exposure to only one of the two insecticides.
Based on  it was possible to assess the effect of ACE mixture on liver and kidney biomarkers, as well as on antioxidant enzymes (AOEs), in a “quantitative manner” by calculating the percentage of change in ACE-treated groups relative to control untreated groups. On the other hand, estimation of the “Amelioration Index; AI” by comparing the results of AOEs (e.g., MDA, SOD, CAT) in ACE mixture + Vit. E groups with the results of control groups, to assess the ameliorative effect of the vitamin. As AI was approaching “1”, the amelioration reaches high degree of normalization to the control value.
Table 4 presents percent of change in the different biochemical parameters in dams and their pups due to exposure to ACE mixture. The change in MDA activity accounted to 34.27%, 88.19% and 61.34%, respectively in dams, female and male pups. By other word, the estimated values reflect how much deviation than normal was occurred in MDA activity due to exposure to the mixture. Changes in MDA activity in the mice dams were less than in the pups, but female pups were more affected than the male ones. Changes in SOD, CAT and BuChE activities could be easily depicted from the results given in Table 4.
Histopathologic changes based on scoring severity of injury in different organs from mouse dams and their pups following exposure to pesticide mixture with and without vitamin E during gestation.
Assessment of oxidative stress of the pesticide mixture and the ameliorative effect of vitamin E based on measured biochemical parameters from gestation experimental results.
The efficiency of vitamin E to alleviate the oxidative stress of ACE mixture is expressed in terms of “Amelioration Index, AI” (Table 4). The AI of vit. E for MDA equaled 1.04, 1.38 and 1.25, respectively for dams, female and male pups. The AI of vit.E for SOD was (0.86, 0.91 & 0.90), (0.91, 0.95 & 0.93) for CAT and (0.99, 0.95 & 0.98) for BuChE, respectively for dams, female and male pups. Values of AI exceeding 1.0 may refer to either better improvement or negligible experimental errors. The present results are supported by our previously published investigations on the single compounds of the tested ACE mixture [29-31], and revealed ability of vitamin E to ameliorate toxic effects caused by this mixture.
This study indicates that the mixture (ACE) containing atrazine, chlorpyrifos and endosulfan, each at a dose equivalent to the respective ADI value, was able to induce hepato-renal dysfunction and oxidative stress in female mice treated during gestation period. Similar effects were manifested in the pups which were not directly treated with the ACE mixture. The pups were more affected than the dams with respect to alterations in MDA and BuChE activities, while the opposite was achieved with respect to SOD activities. Alteration in catalase activity for dams was nearly equaled to that for pups. No obvious gender-related effects. Co-administration of vitamin E in conjunction with the ACE mixture was resulted in pronounced ameliorative effects towards all the tested animals. The overall findings may reveal the ability of the ACE mixture to exert its toxic effects through placenta; however, such adverse effects could be alleviated, to a great extent, by vitamin E supplementation. The study may support the need to further investigating the adverse effects of exposure to low doses of commonly used pesticides, especially during pregnancy and breast-feeding as well as effects on newborn child.
the aim of this study reveals the toxic effects of the mixture of pesticides at the very low dosages, and the transfer of their toxicities to the mouse offspring through placenta and breast-feeding. Fortunately, co-administration of vitamin E alleviated the toxic effects of the tested pesticides to a great extent. The findings of the present investigation may support the need to further investigating the adverse effects of exposure to low doses of commonly used pesticides, especially during pregnancy as well as effects on newborn child.
The authors thank the late Professor Sameeh A. Mansour, National Research Centre, Dokki, Giza for the kind supervision. Moreover, thank the National Institute of Agricultural Research (INRA, France) and Academy of Scientific Research & Technology (ASRT, Egypt) for supporting this study within the REF BHC IMHOTEP 2011 Project No. 25382 YG. We also thank Dr. Noha N. Yassen, Pathology Department, National Research Centre, Dokki, Giza for reading the histopathological slides.
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