Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant Vitamins E and C

Reproductive Toxicology 22 (2006) 783–790

Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant Vitamins E and C Baha Oral a , Mehmet Guney a,∗ , Hilmi Demirin b , Meltem Ozguner c , Seren G¨ulsen Giray d , Gulnur Take d , Tamer Mungan a , Irfan Altuntas b b

a Department of Obstetrics and Gynecology, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey Department of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey c Department of Histology and Embryology, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey d Department of Histology and Embryology, Faculty of Medicine, Gazi University, Ankara, Turkey

Received 2 May 2006; received in revised form 20 July 2006; accepted 1 August 2006 Available online 18 August 2006

Abstract We aimed to investigate the effect of subchronic administration of dichlorvos (DDVP) on endometrium and to evaluate ameliorating effects of a combination of Vitamins E and C against DDVP toxicity in the rat. Three groups of rats were used in the experiment. The first group was treated with 4 mg/kg DDVP; the second group was treated with 4 mg/kg body weight DDVP plus Vitamins E and C (DDVP + Vit); the third group was given only corn oil (control). DDVP and DDVP + Vit groups were given DDVP by gavage 5 days a week for 4 weeks at a dose level of 4 mg/kg day by using corn oil as the vechicle. Vitamins E and C were injected at doses of 50 mg/kg i.m. and 20 mg/kg body weight i.p. Histopathological and immunohistochemical examinations for caspase-3 and caspase-9 were accomplished in the endometrium. The level of malondialdehyde (MDA) increased significantly in the DDVP group compared with the control group (p < 0.05). MDA significantly decreased in the DDVP + Vit group compared with the DDVP group (p < 0.05). Administration of Vitamins E and C along with DDVP significantly reduced the histopathological changes and the extent of apoptosis. In conclusion, subchronic DDVP administration caused endometrial damage and that treatment with a combination of Vitamins E and C reduced endometrial damage caused by DDVP. © 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Lipid peroxidation; Dichlorvos; Endometrial toxicant; Vitamin C; Vitamin E

1. Introduction Organophosphorus insecticides (OPIs) are among the most useful and diverse classes of insecticides; however, the widespread use of these insecticides has raised attention to their ecological impact [1]. The toxic effects of OPIs include alterations in metabolism, reproduction, development, carcinogenesis and neurotoxicity [2]. In addition, accidental as well as suicidal poisoning cases related to these pesticides have increased over the years [3]. Dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP) is one of the most widely used organophosphate insecticides in agriculture and public health

∗ Corresponding author at: Modernevler Mahallesi ˙Istanbul, Caddesi Karadayı Apt Kat:1 Daire:2, 32100 Isparta, Turkey. Tel.: +90 246 2238784; fax: +90 246 2370240. E-mail address: [email protected] (M. Guney).

0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2006.08.003

programs. Dichlorvos is also one of the most used OPIs in the region of Isparta, Turkey (Agricultural Ministry Office of Isparta Province). In general, OPIs are neurotoxic in nature by acting as inhibitors of neuronal cholinesterase (ChE) activity. Some studies reported that OPIs caused lipid peroxidation (LPO). In these studies, LPO has been suggested as one of the molecular mechanisms involved in OPIs-induced toxicity [4–6]. OPIs may induce oxidative stress leading to generation of free radicals and alterations in antioxidants or reactive oxygen species (ROS) scavenging enzymes [4,7]. Therefore, OPIs may enhance lipid peroxidation (LPO) by directly interacting with the cellular membrane and ROS [8–10]. Organophosphorus insecticides are also capable of inducing programmed cell death (apoptosis) by multifunctional pathways. Oxidative stress affects quite a variety of cell functions. When cells are exposed to oxidative stress, they often die by apoptosis or necrosis [11]. Under conditions of higher stress, the cellular

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impairment is so high that apoptosis is suppressed, leading to cell death by necrosis, which causes further tissue damage and an intense inflammatory response [11]. Caspases are a family of cysteine proteases that are present in cytosol as inactive proenzymes but they become activated when apoptosis is initiated, playing an essential role at various stages of apoptosis [12]. Excessive ROS and lipid peroxidation generation have been found to be involved in many diseases [13]. It is known that ROS can initiate apoptosis and organ lesions [14,15]. Llopis et al. reported that OPIs caused apoptosis in fish through oxidative stress induced inhibition of caspase-3-like activity [12]. ROS have physiological and pathological roles in the female reproductive tract. Numerous animal and human studies have demostrated the presence of ROS in the female reproductive tract: ovaries, fallopian tubes, and uterus [16]. However, it is not known whether OPIs may lead to damage and apoptosis of endometrium induced by LPO. Nonenzymatic antioxidants such as Vitamins C and E can act to overcome the oxidative stress, being part of the antioxidant system. Many studies have reported that a combination of Vitamins E and C can reduce LPO caused by toxic substances [13,17,18]. Antioxidants play an important role in preventing free radical mediated damages by directly scavenging them. ␣-Tocopherol is one of the most widely distributed, naturally occurring and biologically active antioxidants in the biological system. It protects against lipid peroxidation most efficiently through its chain breaking action. Intracellularly, it is associated with lipid-rich membranes such as mitochondria and microsomes. In contrast to ␣-tocopherol, ascorbic acid is hydrophilic and functions better in an aqueous environment than does ␣tocopherol. Furthermore, it can restore the antioxidant property of oxidized tocopherol, suggesting that a major function of ascorbic acid is to recycle the tocopheroxyl radical [19]. Recently, a role for oxidative stress in the induction of apoptosis has been provided by several studies, and it was observed that various antioxidants, such as Vitamins E and C, can inhibit cell death [20,21]. The present study was undertaken to determine the possible negative effects of DDVP on LPO, immunohistochemistry and histopathology of rat endometrium, and additionally to investigate whether there is any preventive effect of a combination of Vitamins C and E given to rats after DDVP administration.

ing this into consideration, we did not constitute a separate vitamin group alone. Vaginal smears were obtained from all rats to determine whether they had regular cycles before drug administration. DDVP and DDVP + Vit groups were treated by gavage 5 days a week for 4 weeks at a dose level of 4 mg/kg body weight DDVP (0.05 of the acute oral LD50 = 80 mg/kg) in corn oil [22]. Corn oil alone was given in the same way to the control rats. The administered volume of each dose of the vehicle corn oil was 4 mL/kg body weight/day and the doses were adjusted for recorded body weight changes during the study. Vitamin E, as ␣-tocopherol acetate (Evigen; Aksu Farma Istanbul, Turkey), Vitamin C, as sodium L-ascorbate (Redoxon; Roche, Basel, Switzerland), were injected at doses of 50 mg/kg i.m. and 20 mg/kg body weight i.p. respectively 30 min after each dose of DDVP in the DDVP + Vit group [13,17,18]. Equivalent amounts of physiological saline instead of Vitamin C, and olive oil instead of Vitamin E were given to rats of the control and DDVP groups. All of the rats were allowed to feed ad libitum, until midnight after they had received the proposed treatment. Daily phases of the estrous cycle and body weight were recorded throughout the experiment. Rats were sacrificed by decapitation under 60 mg/kg i.m. ketamine hydrochloride (Eczacibasi, Istanbul, Turkey) anesthesia, 24 h after the last oral dose administration, at the end of the fourth week, and soon after vaginal smear observation. Venous blood samples were collected by direct heart puncture under ketamine anesthesia. Plasma was removed and stored at −85 ◦ C pending analysis. The uteri were removed, weighed and fixed in 10% formalin for histopathological examination.

2.2. Estrus cycle The phases of estrous cycle were determined by observing the vaginal smear in the morning (08:00–10:00 h) as described by Zarrow et al. [23]. Sacrifice was scheduled to be carried out irrespective of reproductive cycle stage at the end of the fourth week; however, the DDVP and DDVP + Vit treated animals were found to be in diestrous, at the scheduled time of sacrifice. Control rats were found to be either proestrous or estrous on the scheduled day of sacrifice. After the final dose, control animals were not sacrificed until the onset of the diestrous phase.

2.3. Biochemical analysis Plasma LPO was evaluated by the fluorometric method based on the reaction between MDA and thiobarbutiric acid (TBA). Briefly, 500 ␮L of plasma was added to a 750 ␮L 440 mM H3 PO4 and 250 ␮L 42 mM TBA solution to give 1.5 mL final volume. The mixture was incubated for 1 h at 100 ◦ C, then an aliquot of 500 ␮L was added to 500 ␮L of methanol: 1 M NaOH (91:9, v:v) mixture. After vortexing, at 4000 rpm for 5 min 30 ␮L of supernatant was injected to the HPLC (Thermo Finnigan) system. Fluorescence of the butanol extract was measured at an excitation wavelength of 539 nm and emission wavelength of 533 nm. 1,1,3,3-Tetraethoxypropane (Sigma) was used as the standard solution, and the values were presented as ␮mol/L. An autoanalyzer, Abbott Aeroset (Abbott Park, III., USA), was used to determine the serum activites of ChE.

2.4. Histopathological examination 2. Materials and methods 2.1. Animals and treatment Twenty-one sexually mature female Wistar-albino rats, showing at least three consecutive regular estrous cycles, weighing 160 ± 10 g and 16 weeks of age were used. The rats were obtained from Laboratory Animal Production Unit of Suleyman Demirel University and kept in our laboratory for at least 3 weeks before the experiments. All animal protocols were reviewed and approved by the Animal Care Laboratory of S¨uleyman Demirel University Faculty of Medicine Institutional Review Board. The 21 rats were randomly divided into three experimental groups each consisting of seven rats, as follows: control group, DDVP-treated group (DDVP), and DDVP plus Vitamin E plus Vitamin C treated group (DDVP + Vit). In previous studies, no statistically significant biochemical and ultrastructural changes were reported when control group compared with vitamin groups alone. Tak-

Following sacrifice, the endometrium was dissected and fixed in 10% formaldehyde solution at room temperature. The tissue samples were dehydrated and embedded in paraffin using standard histologic procedures. Serial cross sections of 5 ␮m were prepared from the endometrium. The sections were mounted and stained with hematoxylin-eosin. At least 10 histologic sections from the endometrium were assessed at a magnification of 20× and 40× objectives and photographed using an Olympus BX50 (Japan) photomicroscope. Two independent observers who were blind to the treatment regimen performed the histopathological evaluations separately.

2.5. Immunohistochemical methods Tissue samples from each groups were fixed in neutral formalin for 72 h and processed for paraffin embedding. Sections of 4–5 ␮m thickness were processed for on polylysine-coated microscope slides. For immunohistochemical

B. Oral et al. / Reproductive Toxicology 22 (2006) 783–790 examination, slides were stored in a microwave oven (LabVision, Cat AP-9005500, USA) in 0.01 M Tris–HCl buffer for heat induced antigen retrieval through microwave irradiation so as to increase the sensitivity of the immunohistochemical detection. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide (Lot AHP40114, Cot TA-125-HP, LabVision, USA). Epitopes were stabilized by application of serum blocking solution (Lot AUB40204, Cot TA125-UB, LabVision, USA) and then split into groups by slides. First group was incubated with Caspase-3 (rabbit polyclonal antibody Ab-4, 1mg/ml, Lot 1197P209, Cat RB-1197-PO, NewMarker, USA), second group was incubated with Caspase-9 (rabbit polyclonal antibody Ab-4, 1 mg/ml, Lot 1205P306, Cat RB-1205-PO, NewMarker, USA) diluted in UltraAb Diluent (Lot AUD40706, Cot TA-125-UD, LabVision, USA) 60 min. at room temparature. After that, the biotinated secondary antibody (Goat antirabbit, Lot RBN40218, Cot TR125-BN, LabVision, USA), was applied. Then, streptavidin peroxidase (Lot SHR40211, Cot TS-125-HR, Lab Vision, USA) was applied to the slides, and AEC (Lot AH41013, Cot TA-125-HA, Lab Vision, USA) was used as chromogen. Afterwards, the slides were counterstained with Mayer’s hematoxylin and examined using an Olympus BH2 photo-light microscope. Negative control was made in the primary antibody application stage. Two independent observers who were blind to the treatment regimen performed the immunolabelling scores evaluations separately. The labelling intensity was graded semiquantitatively and the HSCORE was calculated using the following equation: HSCORE = Pi (i + 1), where i is the intensity of labelling with a value of 1, 2 or 3 (weak, moderate or strong, respectively) and Pi is the percentage of labelled epithelial and stromal cells for each intensity, varying from 0% to 100%. Statistical differences were calculated using the Mann–Whitney U test. Results are expressed as the mean ± SE. A p value <0.05 was considered significant. Color prints were made using 100 ASA film (Fuji; Tokyo, Japan).

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6.0%. However the net body weight gain of the animals treated with DDVP was markedly less and was 1.8% only, as compared to the controls. DDVP + Vit treated animals resulted in a net body weight gain of 1.9%. Severe fasciculations were observed after every DDVP administration in rats. Fatigue symptoms were less severe in DDVP + Vit group compared with the DDVP group. The fasciculations continued for approximately 1–1.5 h, after which symptoms of fatigue were observed. There were no deaths in any of the groups. 3.2. Estrus cycle Corn oil-treated control rats showed normal estrus cycles and normal duration of each phase of the estrous cycle over the 4week treatment. Treatment with DDVP and DDVP + Vit resulted in a significant decrease in the number of estrus cycles with a decrease in the duration of each phase (other than diestrus), and concomitant increase in the duration of diestrus when compared with control rats. 3.3. Biochemical findings The results are shown in Figs. 2 and 3. The level of MDA increased significantly in the DDVP group compared with the control group (p < 0.05). MDA significantly decreased in the

2.6. Statistical analysis For statistical analysis, the non-parametric Kruskal–Wallis test and the Mann–Whitney U-test were used to compare the values of the groups. Results are expressed as the mean ± S.D. The p-value <0.05 was considered as significant.

3. Results 3.1. Uterine and body weight There were no significant changes in mean weights of the uterus between control, DDVP, and DDVP + Vit groups. The percent increase in body weight of control, DDVP, and DDVP + Vit groups were 6.0, 1.8, and 1.9%, respectively, when compared with that of their initial body weights (Fig. 1). During the course of present investigations, it was observed that the body weights of the untreated control animals increased progressively throughout the study and recorded a net body weight gains of

Fig. 1. Initial and final body weights of rats in control group, DDVP group and DDVP + Vit group. Values are means ± S.D. for seven rats in each group.

Fig. 2. Plasma levels of MDA in control group, DDVP + Vit group and DDVP group. * p < 0.05 DDVP vs. control, # p < 0.05 DDVP + Vit vs. DDVP, ** p < 0.05 DDVP + Vit vs. control. Values are means ± S.D. for seven rats in each group.

Fig. 3. Activities of ChE in control group, DDVP + Vit group and DDVP group. * p < 0.05 DDVP vs. control, # p < 0.05 DDVP + Vit vs. DDVP, ** p < 0.05 DDVP + Vit vs. control. Values are means ± S.D. for seven rats in each group.

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DDVP + Vit group compared with the DDVP group (p < 0.05). There were also statistical differences in the levels of MDA between DDVP + Vit group and the control group (p < 0.05). ChE activities were decreased in the DDVP group compared with the control group (p < 0.05) and the DDVP + Vit group (p < 0.05). When DDVP was given concurrently with Vitamins E and C, the decrease in ChE activity compared with control was significantly less (p < 0.05) than for DDVP alone. 3.4. Histopathological observations In control uteri, the endometrium was of normal histological appearance which consisted of columnar epithelium and a lamina propria. The lamina propria was intact with rich glandular component and highly cellular stroma. The mitotic figures were visible in both surface epithelial cells and glandular epithelial cells. The myometrium and uterine serosa were of normal histologic appearance (Fig. 4A). In the DDVP group, there were significant changes in the endometrium when compared with the control group. The epithelial lining of the endometrium was irregular and there was disorganization of the glandular epithelium. Shrinkage of epithelial cells in the endometrial glands and pyknotic nucleus in many of the epithelial cells were also observed (Fig. 4B). In the DDVP + Vit group, the epithelial lining of endometrium was regular although there was significant histological changes in the glandular epithelium when compared with the control group. Comparing the DDVP and the DDVP + Vit groups, shrinkage of the epithelial cells and pyknotic nuclei in the epithelial cells were similar and irregularity of the glandular epithelium was seen (Fig. 4C). 3.5. Immunohistochemical observations 3.5.1. Caspase-3 reactivity In the control group, a negative apical cytoplasmic reactivity was seen in uterine epithelium. In endothelial cells of stromal capillaries there was weak peripheral staining. No caspase-3 immunoreactivity was detected in glandular cells and stromal cells (Fig. 5A). In the DDVP applied group, a negative to very weak cytoplasmic reactivity was detected. In stromal cells and endothelial cells of stroma there was moderate to strong immunostaining (Fig. 5B). In the DDVP + Vit treated group, epithelial staining was seen and stromal reactivity was observed to be decreased in this group compared with the DDVP group. Weak to moderate staining was seen in the stromal component (Fig. 5C). Immunolabelling intensities of caspase-3 in epithelial and stromal cells of the endometrium increased significantly in the DDVP and DDVP + Vit groups compared with the control group (p < 0.01). Immunolabelling intensities of caspase3 in epithelial and stromal cells of the endometrium significantly decreased in the DDVP + Vit group compared with the DDVP group (p < 0.01). There were statistically significant differences in immunolabelling intensities of caspase-3 between DDVP + Vit group and the control group (p < 0.01). The results are shown in Table 1.

Fig. 4. (A) Control group. Histologic section of endometrium showing mitotic figures in columnar surface epithelium and glandular epithelium which is representative of normal histology. (B) DDVP group. Histologic section of endometrium showing severe damage including pyknotic nucleus in some cells of surface epithelium and disorganization and focal atrophy of glandular epithelium. (C) DDVP + Vit group. Histologic section of endometrium showing a moderate decrease in the surface and glandular epithelial damage when compared with the DDVP group (H&E, 400×).

3.5.2. Caspase-9 immunoreactivity In the control group, a very weak apical cytoplasmic reactivity was seen in the uterine epithelium. No caspase-9 staining was detected in glandular epithelium, stromal components,

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Table 1 Immunolabelling intensities of caspase-3 and caspase-9 in epithelial and stromal cells of the endometrium of control, DDVP + Vit, and DDVP groups Caspase-3

Caspase-9

Control group Epithelial cells Stromal cells

55.4 ± 1.03 66.0 ± 1.1b

56.8 ± 1.46a 33.2 ± 0.8c

DDVP + Vit group Epithelial cells Stromal cells

214.0 ± 2.41a 160.6 ± 1.35b

56.6 ± 1.04 34.4 ± 0.49d

DDVP group Epithelial cells Stromal cells

245.7 ± 1.57a 262.8 ± 2.46b

55.8 ± 1.05 61.2 ± 2.29c,d

HSCORE values are expressed as means ± S.E.M. a p < 0.01 control vs. DDVP + Vit and DDVP groups. b p < 0.01 control vs. DDVP + Vit and DDVP groups. c p < 0.01 control vs. DDVP groups. d p < 0.01 DDVP + Vit vs. DDVP groups.

group, DDVP + Vit group and the control group (p > 0.05). Immunolabeling intensities of caspase-9 in stromal cells of the endometrium were increased significantly in the DDVP and DDVP + Vit groups compared with the control group (p < 0.01). There were no statistical differences in the label intensities of the DDVP + Vit group compared with the control group (p > 0.05). The results are shown in Table 1. 4. Discussion

Fig. 5. Immunolabelling of caspase-3 activity. Weak staining in endothelial cells of stromal capilleries in the uterine epithelium of control group, but negative staining in glandular and stromal cells for caspase-3 were detected (A). Moderate to strong immunostaining were detected in stromal cells and endothelial cells of stroma of DDVP group (B). Weak to moderate immunostaining was detected in stromal components of DDVP + Vit treated group (C).

capillary endothelial cells and stromal cells (Fig. 6A). In the DDVP applied group, a weak cytoplasmic reactivity was seen in the uterine epithelium; however a moderate staining was detected in stromal components (Fig. 6B). In the DDVP + Vit group, a negative to weak cytoplasmic staining was seen in the uterine epithelium compared with the DDVP group. A negative caspase-9 reactivity was observed in the stromal components but a weak to moderate staining was observed in capillary endothelial cells (Fig. 6C). There were no statistically significant differences in immunolabeling intensities of caspase-9 in epithelial cells of the endometrium between the DDVP

Recent findings indicate that toxic manifestations induced by OPIs may be associated with an enhanced production of ROS [4,7]. Among ROS superoxide anions, hydroxyl radicals and hydrogen peroxide enhance the oxidative process and induce lipid peroxidative damage of cell membranes. Some studies have reported that OPIs such as dichlorvos, phosphomidon, trichlorfon, fenthion, phosalone, diazinon, and methidathion induce oxidative stress, as shown by enhanced MDA production [6,13,24–26]. MDA is a marker of membrane LPO resulting from the interaction of reactive oxygen species and the cellular membrane [8]. The increase in MDA formation may be due to the OPI’s themselves inducing LPO or possibly due to an increase in ROS induced by OPI’s. Also, histopathological and immunohistochemical changes were accompanied by LPO in rat plasma. These results support the hypothesis whereby LPO has been suggested as one of the molecular mechanisms involved in OPI-induced endometrial damage. The cell has several ways of alleviating the effects of oxidative stress, either by repairing the damage (damaged nucleotides and LPO by-products) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and nonenzymatic antioxidants. Nonenzymatic antioxidants such as Vitamins E and C can act to overcome the oxidative stress, being a part of total antioxidant system. Vitamin E is the most important lipophilic antioxidant and resides mainly in the cell membranes, thus helping to maintain membrane stability [27]. Vitamin C is the most important hydrophilic free-radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and

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Fig. 6. Immunolabelling of caspase-9 activity. Very weak apical cytoplasmic reactivity in surface epithelial cells was detected, whereas negative immunostaining was detected in glandular epithelium and in endothelial cells and stromal cells of control group (A). Moderate immunostaining was detected in stromal components, whereas weak cytoplasmic reactivity was detected in surface epithelial cells of DDVP group (B). Negative to weak cytoplasmic staining in surface epithelial cells was detected and negative caspase-9 reactivity was observed in stromal components of DDVP + Vit treated group (C).

protecting biomembranes from peroxidative damage [28]. In addition to its antioxidant effects, Vitamin C is involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane. Thus, Vitamins E and C can have interactive effects [29]. Ramanathan et al. showed that administration of Vitamins C and E decreased the rate of DNA fragmentation in arsenic exposed rats [21]. The antioxidants Vitamins E and C maintain the Bcl-2 protein in its functional form by their membrane stabilizing action and thus inhibit the release of cytochrome C from

mitochondria. Vitamins E and C can decrease the level of TNF␣ in human monocytes and increase the function of Bcl-2 in human endothelial cells. Modulation of the activities of TNF-␣ and Bcl-2 by Vitamins E and C further decreases the activation of caspase-3 and subsequently DNA fragmentation. In our previous studies, OPIs such as chlorpyrifos-ethyl, methidathion, and fenthion caused a significant increase in LPO and, in addition, treatment with a combination of Vitamins C and E after the administration of OPIs led to a significant decrease in LPO [5,7,25]. It is known that the subchronic and chronic toxicity of OPIs may cause histopathological changes in different tissues [30]. Yavuz et al. and Sutcu et al. have reported that subchronic toxicity of OPIs induced histopathological changes in liver, vascular wall, and heart of the experimental rat [31,17,18]; however it is not known whether the subchronic and or chronic toxicity of OPIs cause histopathological and immunohistochemical changes in endometrium. Nanda and Kaliwal showed that subchronic toxicity of OPIs can lead to significant decreases in the weights of the uterus when compared with those of hemicastrated oil-treated control animals [32]. In the present study these histopathological changes were significant in the DDVP group as glandular epithelium showed signs of degeneration. Therefore, the combination of Vitamins E and C improved DDVP induced histologic effects. It did not completely bring the histopathological changes to normal. In the DDVP + Vit group, a combination of Vitamins E and C should presumably lead to direct protective effect on endometrium; however a combination of Vitamins E and C only partially prevented DDVP-induced endometrial damage. Since the recovery is incomplete, it is possible that some reactive species other than LPO may play a role. Saleh et al. reported that paraxon induces apoptosis in vitro through mitochondrial dysfunction causing caspase-3 activation [33]. Masoud et al. reported that the cytotoxicity of malathion at noncholinergic doses is mediated through caspase-dependent apoptosis [34]. However, few experiments have been done in vivo concerning the apoptosis induced by DDVP. Caspases have cysteine residues in the catalytic domain that are prone to oxidation or thiol alkylation, and therefore, are regulated by the oxidative stress and intracellular redox state. Many studies have demonstrated that oxidant-induced inhibition of caspase-3-like activity is sufficient to switch apoptosis to necrosis [35]. Llopis et al. results suggest that fish individual differences in the homeostasis of the hepatic glutathione redox status can determine the degree of dichlorvos-induced oxidative stress and caspase-3-like inactivation, resulting in hepatic cell death by necrosis [12]. In their study antioxidant (N-acetylcysteine) treatment diminished caspase-3-like inhibition, allowing higher fish tolerance to dichlorvos. In the present study, we studied semiquantitatively the tissue localization of the apoptosis-caspase-3 and caspase-9 by using immunohistochemistry and investigated their significance and involvement in the induction of endometrial apoptosis and ameliorating effects of Vitamins E and C. These antioxidant vitamins directly inhibit free radical mediated apoptosis by directly eliminating them. Apart from the free radical scavenging property, antioxidants are known to regulate the expression of number of genes and signal regulatory pathways and thereby may prevent the incidence of cell death

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[20,21]. Administration of Vitamins E and C along with DDVP significantly reduced the extent of apoptosis Matkovics et al. have shown that the ChE inhibiting action of OP insecticides is better compensated by vitamin [36]. In our earlier study, methidathion caused a significant decrease in the ChE activity [5,13,17,18]. Furthermore, the use of a combination of Vitamins E and C after the administration of methidathion caused a significant increase in ChE activity compared with methidathion alone. In the present study, the subchronic DDVPtreated group showed a significant decrease in ChE activities whereas the use of the vitamin combination caused a partial restoration of the ChE activity compared with DDVP alone, concurrent with the previous studies. These data also showed that a combination of Vitamins E and C might partially restore the activity of ChE in subchronic DDVP-treated rats. The net body weight gains of the animals treated with DDVP were markedly reduced compared with the normal controls. These findings are in line with the previous work, where it was shown that there was a dose-dependent decrease in body weight gain in animals treated with leptophos, isoprocarp, chlorpyrifos, and metyl parathion [37]. This finding is important because nutritional deficiencies have been shown to alter reproductive function [38]. In our study there has not been a nutritional deficiency that would be responsible for the observed reproductive effect. From these results, it can be concluded that subchronic administration of DDVP results in the induction in LPO, suggesting that reactive oxygen species may be involved in the toxic effects of DDVP leading to endometrial damage and apoptosis. Treatment with a combination of Vitamins E and C after the administration of DDVP diminished endometrial damage and apoptosis induced by DDVP has pointed the way towards a possible therapeutic implementation of these drugs to women exposed to DDVP. References [1] Abdollahi M, Mostafalou S, Pournourmohammadi S, Shadnia S. Oxidative stress and cholinesterase inhibition in saliva and plasma of rats following subchronic exposure in malathion. Comp Biochem Physiol C: Toxicol Pharmacol 2004;137(1):29–34. [2] Guilermo RN, Teresita LA, Alberto RO, Cristina FM. Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels. Life Sci 2005;78(9):1015–20. [3] Vittozzi L, Fabrizi L, Di Consiglio E, Testai E. Mechanistic aspects of organophosphorothionate toxicity in fish and humans. Environ Int 2001;26(3):125–9. [4] Bagchi D, Bagchi M, Hassoun EA, Stohs SJ. In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 1995;104:129–40. [5] Altuntas I, Delibas N, Sutcu R. The effects of organophosphate insecticide methidathion on lipid peroxidation and anti-oxidant enzymes in rat erythrocytes: role of Vitamins E and C. Hum Exp Toxicol 2002;21: 681–5. [6] Yamano T, Morita S. Hepatotoxicity of trichlorfon and dichlorvos in isolated rat hepatocytes. Toxicology 1992;76:69–77. [7] Gultekin F, Delibas N, Yasar S, Kilinc I. In vivo changes in antioxidant systems and protective role of melatonin and a combination of Vitamin C and Vitamin E on oxidative damage in erythrocytes induced by chlorpyrifosethyl in rats. Arch Toxicol 2001;75:88–96.

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