Dichlorvos-induced testicular toxicity in male rats and the protective role of vitamins C and E

Experimental and Toxicologic Pathology 64 (2012) 821–830

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp

Dichlorvos-induced testicular toxicity in male rats and the protective role of vitamins C and E Enver Kerem Dirican a,∗ , Yusuf Kalender b a b

Memorial Antalya Hospital, Center for Reproductive Medicine, Department of Embryology, Antalya, Turkey Gazi University, Faculty of Arts and Science, Department of Biology, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 11 October 2010 Accepted 7 March 2011 Keywords: Organophosphorus pesticides Dichlorvos Vitamin E Vitamin C Testis Pathology

a b s t r a c t Dichlorvos is an organophosphorus insecticide that is used worldwide for pest control in agriculture and household use. Vitamins C and E are potential antioxidants protecting cells from oxidative stress. Vitamin C + vitamin E, dichlorvos, a combination of vitamin C + vitamin E + dichlorvos, or corn oil (control) were given to rats via oral gavage for 7 weeks. Body and testis weights, sperm parameters, hormone levels, histo- and cytopathological changes in testes were investigated at the end of 24 h and the 4th and 7th weeks comparatively with the control group. Body and testis weights, sperm morphology, FSH, LH, and testosterone levels were decreased significantly at the end of 4th and 7th weeks in the dichlorvosand vitamins + dichlorvos-treated groups. A statistically significant decline in sperm motility and testosterone levels occurred by the end of 7th week in the dichlorvos- and vitamins + dichlorvos-treated groups. Light and electron microscopy revealed necrosis, edema and cellular damage in testicular tissues of the dichlorvos- and vitamins + dichlorvos-treated rats at the end of 4th and 7th weeks. In conclusion, dichlorvos caused subacute and subchronic reproductive toxicity, but vitamins did not confer protection. © 2011 Elsevier GmbH. All rights reserved.

1. Introduction Organophosphorus (OP) compounds represent the most widely used group of pesticides for the control of agricultural pests and disease vectors (Maitra and Mitra, 2008). Due to the wide availability of OP compounds, poisoning is common (Garcia et al., 2003). OP pesticides are toxic to mammals; they inhibit acetylcholinesterase (AChE), leading to the accumulation of acetylcholine and subsequent activation of cholinergic muscarinic and nicotinic receptors (Hazarika et al., 2003). OP pesticides can also inhibit pseudocholinesterase activity (Kalender et al., 2006). People are commonly exposed to OP pesticides through eating fresh and processed vegetables, contacting pesticide-contaminated surfaces, breathing air near pesticide applications (both indoors and outdoors), and drinking pesticide-contaminated water. Approximately 40 organophosphate pesticides are registered with the United States Environmental Protection Agency (EPA). About 70% of insecticides used in the United States are OP pesticides. These compounds are known to cause adverse effects in several mammalian organs, including the liver (Kalender et al., 2005a; Ogutcu et al., 2008), heart (Ogutcu et al., 2006), and kidney (Kalender et al., 2007). In addition, some studies demonstrated that OP pesticides

∗ Corresponding author. Tel.: +90 242 314 66 66; fax: +90 242 314 66 45. E-mail address: [email protected] (E.K. Dirican). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.03.002

could interfere with the male reproduction system (Uzunhisarcikli et al., 2007). Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP), an OP pesticide, is used throughout the world for the protection of stored products and crops and as an insecticide for public health (Choudhary and Gill, 2001). It is effective against aphids, caterpillars, spider mites, thrips, and white flies in greenhouses and outdoor fruit and vegetable crops and bedbugs (Yarsan and Cakir, 2006). Public exposure to dichlorvos may occur via air, water, or food; dichlorvos is readily absorbed through all routes of exposure (Raheja and Gill, 2002). The acute effects of dichlorvos are well documented and are mediated through the inhibition of acetylcholinesterase, an enzyme vital for cholinergic transmission (Sarin and Gill, 1999). Other organs and systems that could be affected by dichlorvos are the respiratory system (Atis¸ et al., 2002), reproductive system (Okamura et al., 2005; Oral et al., 2006), and liver (Ogutcu et al., 2008). Vitamins C and E have been investigated for their effects in studies of pesticide toxicity (Ogutcu et al., 2008; Kalender et al., 2007; Uzunhisarcikli et al., 2007). Vitamin C is water soluble, and vitamin E is lipid soluble (Young and Woodside, 2001). Vitamin E inhibits the formation of free radicals (Kalender et al., 2004, 2005b), perhaps effectively minimizing lipid peroxidation in biological systems (Kalender et al., 2002). In cell membranes and lipoproteins, the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid perox-

822

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

idation (Young and Woodside, 2001; Schneider, 2005). Vitamin C scavenges superoxide, hydrogen peroxide, hydroxyl radicals, aqueous peroxyl radicals, and singlet oxygen (John et al., 2001). Also, ␣-tocopherol-mediated pro-oxidation can be stopped by ascorbic acid (Bowry and Stocker, 1993). Diets high in fruits and vegetables have shown to protect against cardiovascular disease and cancer (Schneider, 2005), but such a protective effect have not been evaluated before on the testicular toxicity of dichlorvos. An environmental agent should disrupt reproductive function in the male at several potential target sites, the most important being the testes, the male gonads, which are the sites of spermatogenesis and androgen production. There are paracrine and autocrine regulations in various components of the testis that are under endocrine influences from the pituitary, responsible for secreting gonadotrophin releasing hormone, and hypothalamus, secreting follicle stimulating hormone (FSH). Leydig cells are the endocrine cells in the testis that produce testosterone from cholesterol via a series of enzymatic pathways and steroidal intermediates under the control of luteinizing hormone (LH) from the pituitary. Testosterone mediates numerous functions throughout the life cycle of the male, including the differentiation and development of the male reproductive tract and the maintenance of spermatogenesis. Toxicant-induced alterations in testosterone production can have a devastating impact on the male reproductive system and its functioning in the adult. Within the testes are the nurse cells that luminal environment is both created and controlled, the Sertoli cells, which are under the influence of FSH from the hypothalamus. In this study, we examined the effects of dichlorvos on body and testis weights, sperm count, motility, and morphology; FSH (follicle stimulating hormone), LH (luteinizing hormone), and testosterone (T) levels; histopathology and fine structure at acute (24 h), subacute (4 weeks), and subchronic (7 weeks) stages; and assessed the protective potential of vitamins C and E in male rats.

inhalation of diethyl ether and then euthanized. Testicular tissue samples were retrieved for the testicular sperm count; analyses of epididymal sperm motility and morphology and light and electron microscopic investigations; and blood samples were retrieved for the measurements of FSH, LH, and testosterone levels. The treatments were administered in the morning (between 09:00 and 10:00 h) of each day, to non-fasted rats. The day of the first exposure to dichlorvos was defined as experimental day 0.

2. Material and methods

Body and testis weights of the control and treated rats were measured at the end of 24 h and the 4th and 7th weeks with an automatic balance (AND GX-600, Japan). Rats were anesthetized with diethyl ether after measurement of body weights, and testes were removed and weighed immediately.

2.1. Animals Seventy-two sexually mature male Wistar rats (weighing approximately 310–340 g, 90 days old) were obtained from the Refik Saydam Central Hygiene Institute, Ankara, Turkey. The animals were housed in plastic cages and were fed a standard laboratory diet and water ad libitum. Rats were exposed to a 12 h light/dark cycle at a temperature of 20 ± 2 ◦ C. Animals were quarantined for 10 days before the start of experiments. All rats were handled in accordance with the standard guide for the care and use of laboratory animals. 2.2. Chemicals Dichlorvos (purity 98%) was obtained from the Central Research Institute of Agricultural Defense, Ankara, Turkey. Vitamin E (dl␣-tocopherol acetate) was supplied from Merck (Germany), and vitamin C (l-ascorbic acid) was supplied from Carlo Erba (Milano, Italy).

2.3.1. Control group Corn oil at a dose of 1 ml/kg body weight (bw) was given via oral gavage to rats once per diem. 2.3.2. Vitamin C + vitamin E-treated group (vitamins-treated group) Vitamin C (200 mg/kg bw) was administered via oral gavage to rats once per diem. After the administration of vitamin C, vitamin E (200 mg/kg bw per day) was administered via oral gavage to rats once per diem. Vitamins C and E were dissolved in water (1 ml/kg bw) and corn oil (1 ml/kg bw), respectively. 2.3.3. Dichlorvos-treated group Dichlorvos was prepared at a dose of 1.6 mg/kg bw (1/50 LD50 oral dose) each day in corn oil and given via oral gavage to rats once per diem. 2.3.4. Vitamins + dichlorvos-treated group Vitamin C (200 mg/kg bw, once per diem in water) and vitamin E (200 mg/kg bw, once per diem in corn oil) were administered via oral gavage 30 min before the administration of dichlorvos (1.6 mg/kg bw, once per diem in corn oil) via oral gavage. 2.4. Measurement of body and organ weights

2.5. Testicular sperm count One testis of each rat was placed in 1 ml phosphate buffered saline immediately after dissection. The tunica albuginea was cut by surgical blades and removed, and the remaining seminiferous tubules were mechanically minced using surgical blades in the 1 ml phosphate buffered saline. The testicular cell suspension was pipetted several times and vortexed to make a homogenous cell suspension. One drop of the suspension was placed on a counting chamber (Makler Counting Chamber, Sefi Medical Instruments, Israel), and the concentration of testicular sperm was evaluated as millions of sperm cells per ml of suspension under 200× magnification using a phase contrast microscope (Olympus CX 31, Tokyo, Japan). 2.6. Sperm motility analysis

2.3. Animal treatment schedule Rats were randomly divided into two main groups: a control (n = 18) and experimental groups (n = 54). Rats in the experimental group were further divided into three subgroups: a vitamin C + vitamin E-treated group (vitamins-treated group, n = 18), a dichlorvos-treated group (n = 18), and a vitamins + dichlorvostreated group (n = 18). At the end of 24 h and the 4th and 7th weeks, six rats from each of the four groups were anesthetized by

Epididymal sperm were collected as quickly as possible after dissection. The cauda epididymis was cut by surgical blades into small pieces in a 1 ml solution of phosphate buffered saline at 37 ◦ C. The solution was pipetted several times and vortexed to homogenize the sperm suspension, and one drop of the suspension was placed on a slide, covered by a 24 × 24 mm coverslip, and evaluated under 200× magnification using a phase contrast microscope (Olympus CX 31, Tokyo, Japan). Sperm motility was categorized

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

into “motile” or “immotile”. Results were recorded as percentage of sperm motility.

2.7. Epididymal sperm morphology After the evaluation of the epididymal sperm motility, one drop of the above suspension was prepared for an analysis of sperm morphology. One drop of each suspension was smeared on glass slides and stained by the Spermac stain (Stain Enterprises Inc., Wellington). Two thousand sperm cells were evaluated on each slide and results were recorded as percentage of abnormal sperm for each slide. Abnormal heads and tails were viewed and photographed on a light microscope (Olympus BX 51, Tokyo, Japan) with an attached camera (Olympus C-5050, Olympus Optical Co. Ltd., Japan) and were evaluated using the criteria of Okamura et al. (2005), Nahas et al. (1989) and Mori et al. (1991).

2.8. Hormone assays At the end of 24 h and the 4th and 7th weeks, blood samples were obtained from the hearts of the rats and placed into sterile tubes. Blood samples were centrifuged at 3500 rpm for 20 min, and serum was separated. LH and FSH levels were measured by an automated immunofluorescent assay on a Brahms Kryptor immunoassay analyzer using commercial kits (Brahms LH Kryptor 820.050, Brahms FSH Kryptor 818.050). T levels were measured by a chemiluminescence immunoassay on an Access immunoassay analyzer (Beckman Coulter) using commercial kits (Access testosterone 33560).

2.9. Light microscopic studies For histological light microscopic examinations, the testicular tissues were dissected, and the tissue samples were fixed in Bouin’s solution for 14–18 h, processed in a series of graded ethanol solutions, and embedded in paraffin. Paraffin sections were cut with a microtome to a 5 ␮m thickness and stained with hematoxylin and eosin for light microscopic examination. The sections were viewed and photographed on a light microscope (Olympus BX51, Tokyo, Japan) with an attached camera (Olympus C-5050, Olympus Optical Co. Ltd., Japan).

823

2.10. Electron microscopy For electron microscopic examinations of testicular tissues, primary fixation was performed in 3% glutaraldehyde (Agar Sci. Ltd., Essex, England) in sodium phosphate buffer (200 mM, pH 7.4) (Merck, Alfred Paluka Co., Turkey) for 3 h at 4 ◦ C. Materials were washed with the same buffer and postfixed in 1% osmium tetroxide (Agar Sci. Ltd., Essex, England) and in sodium phosphate buffer pH 7.4 for 1 h at 4 ◦ C. Tissue samples were washed with the same buffer for 3 h at 4 ◦ C, than dehydrated in graded ethanol series (Agar Sci. Ltd., Essex, England) and were embedded in Araldite (Agar Sci. Ltd., Essex, England). Thin sections were cut with a Reichert OM U3 (Leica Co., Austria) ultramicrotome. Samples were stained with 2% uranyl acetate and lead citrate. The sections were viewed and photographed on a Jeol 100 CX II transmission electron microscope (TEM) (Jeol Ltd., Japan) at 80 kV. 2.11. Statistical analysis Data were analyzed using SPSS 11.0 for Windows. Statistical significance was calculated using a one-way analysis of variance (ANOVA) followed by Tukey’s procedure for multiple comparisons. P < 0.05 was considered statistically significant. 3. Results In this study, none of the parameters investigated were altered significantly after 24 h of treatment in control and experimental groups. 3.1. Evaluation of body and organ weights No animals died during the experimental period. However, food intake of the dichlorvos- and vitamins + dichlorvos-treated rats was reduced. Body weight, absolute testis weight, and relative testis weight did not significantly differ during the experiment between the vitamins-treated group and the control group. Body weight, absolute testis weight, and relative testis weight had significantly decreased in the dichlorvos- and vitamins + dichlorvos-treated groups, compared to the control group (P < 0.05), by the end of the 4th and 7th weeks. No statistically significant differences in body weight, absolute testis weight, and relative testis weight were observed between the vitamins + dichlorvostreated group and the dichlorvos-treated group at the end of 24 h, 4th and 7th weeks (Table 1).

Table 1 Body weight, testis weight, and relative testis weight of control and experimental rats. Groups

Body weight (g) Initial (g)

Absolute testis weight (g) Final (g)

Control Vitamins Dichlorvos Vitamins + dichlorvos

326 326.98 327.56 325.92

± ± ± ±

2.72 2.7 5.42 6.32

326.55 327.9 326.61 327.45

± ± ± ±

3.04 2.86 2.87 5.22

0.22 0.27 −0.28 0.45

Control Vitamins Dichlorvos Vitamins + dichlorvos

326.78 328.18 326.95 326.13

± ± ± ±

1.72 2.17 7.01 7.44

347.57 352.82 303.18 304.68

± ± ± ±

4.59 5 4.74 7.67

6.36 7.5 −7.28 −6.58

Control Vitamins Dichlorvos Vitamins + dichlorvos

327.3 330.15 326.08 326.72

± ± ± ±

3.28 1.97 6.23 6.86

363.03 366.7 281.98 284.15

± ± ± ±

3.91 4.76 5.53 7.89

10.91 11.06 −13.44 −13.04

a

Relative testis weight (g/100 g body weight)

% Change 24 h ± 0.1 ± 0.14 ± 0.91 ± 0.74 4th week ± 0.85 ± 0.86 ± 0.67a,b ± 0.31a,b 7th week ± 0.14 ± 0.8 ± 0.59a,b ± 1.2a,b

2.99 2.98 2.96 3.04

± ± ± ±

0.02 0.02 0.03 0.13

0.9137 0.9117 0.9083 0.93

± ± ± ±

0.008 0.004 0.011 0.036

3.04 3.08 2.4 2.42

± ± ± ±

0.02 0.03 0.59a,b 0.08a,b

0.8733 0.875 0.7967 0.795

± ± ± ±

0.005 0.005 0.01a,b 0.01a,b

3.15 3.19 2.19 2.18

± ± ± ±

0.02 0.02 0.08a,b 0.06a,b

0.8685 0.8733 0.7767 0.77

± ± ± ±

0.004 0.005 0.019a,b 0.012a,b

Comparison of control and other groups. Comparison of vitamins C and E-treated group with dichlorvos- and vitamins C and E + dichlorvos-treated groups. Values are means ± SD for six rats in each group. Significance at P < 0.05. b

824

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Fig. 1. Effects of vitamins, dichlorvos, and vitamins + dichlorvos treatments on testicular sperm count in rats. Values are means ± SD of six animals in each group.

3.2. Evaluation of testicular sperm count The control group was compared with all other groups at the end of 24 h and the 4th and 7th weeks. In addition, the dichlorvostreated group was compared to the vitamins + dichlorvos-treated group. Changes in testicular sperm count are shown in Fig. 1. No statistically significant changes in testicular sperm count were observed between the experimental groups and the control group at the end of 24 h, 4th and 7th weeks (Fig. 1). No statistically significant differences in testicular sperm count were observed between the vitamins + dichlorvos-treated group and the dichlorvos group at the end of 24 h, 4th and 7th weeks (Fig. 1). 3.3. Evaluation of sperm motility The control group was compared with all other groups at the end of 24 h and the 4th and 7th weeks. In addition, the dichlorvostreated group was compared to the vitamins + dichlorvos-treated group. Changes in total sperm motility are shown in Fig. 2. No statistically significant differences in total sperm motility were observed in the dichlorvos- and vitamins + dichlorvos-treated groups compared to the control group at the end of 24 h and the 4th week. A significant decrease in total epididymal sperm motility was detected at the end of the 7th week in the dichlorvos- and vitamins + dihlorvos-treated groups compared with the control group

Fig. 2. Effects of vitamins, dichlorvos, and vitamins + dichlorvos treatments on epididymal sperm motility in rats. a Comparison of control and other groups (P < 0.05). b Comparison of vitamins-treated group with dichlorvos- and dichlorvos + vitaminstreated groups (P < 0.05). Values are means ± SD of six animals in each group.

Fig. 3. Effects of vitamins, DDVP, and vitamins + DDVP treatments on epididymal sperm morphology in rats. a Comparison of control and other groups (P < 0.05). b Comparison of vitamins-treated group with DDVP- and DDVP + vitamins-treated groups (P < 0.05). Values are means ± SD of six animals in each group.

(P < 0.05). No changes in total epididymal sperm motility between the vitamins + dichlorvos-treated group and the dichlorvos-treated group were observed at the end of 24 h, 4th and 7th weeks (Fig. 2).

3.4. Evaluation of epididymal sperm morphology The control group was compared with the experimental groups at the end of 24 h and the 4th and 7th weeks. In addition, the dichlorvos-treated group was compared to the vitamins + dichlorvos-treated group. Changes in epididymal sperm morphology are shown in Fig. 3. No statistically significant changes were observed between the vitamins-treated group and the control group at the end of 24 h, 4th and 7th weeks. A significant increase in the levels of abnormal sperm morphology was detected at the end of the 4th and 7th weeks in the dichlorvos- and vitamins + dichlorvos-treated groups compared to the control group (P < 0.05) (Fig. 3). No changes in the levels of abnormal sperm morphology between the vitamins + dichlorvos-treated group and the dichlorvos-treated group were observed at the end of 24 h, 4th and 7th weeks (Fig. 3). Fig. 4 shows some normal and abnormal rat sperm cells.

Fig. 4. Normal (N) and abnormal (A) spermatozoa from the epididymis (×1000).

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Fig. 5. Effects of vitamins, dichlorvos, and vitamins-dichlorvos treatments on FSH levels in rats. a Comparison of control and other groups (P < 0.05). b Comparison of vitamins-treated group with dichlorvos- and dichlorvos + vitamins-treated groups (P < 0.05). Values are means ± SD of six animals in each group.

3.5. Hormone analyses 3.5.1. FSH levels The control group was compared with all other groups at the end of 24 h and the 4th and 7th weeks. In addition, the dichlorvostreated group was compared to the vitamins + dichlorvos-treated group. Changes in FSH levels are shown in Fig. 5. No statistically significant changes were observed between the vitamins-treated group and the control group at the end of 24 h, 4th and 7th weeks. A significant decrease in FSH levels in the dichlorvos- and vitamins + dichlorvos-treated groups compared to the control group (P < 0.05) was observed at the end of the 4th and 7th weeks (Fig. 5). FSH levels did not change significantly between the vitamins + dichlorvos-treated group and the dichlorvos-treated group at the end of 24 h, 4th and 7th weeks (Fig. 5). 3.5.2. LH levels LH levels in the control group were compared with those in the experimental groups at the end of 24 h and the 4th and 7th weeks after vitamins, dichlorvos, and vitamins + dichlorvos were given to rats. In addition, levels in the dichlorvos-treated group were compared to those in the vitamins + dichlorvos-treated group. Changes in LH levels are shown in Fig. 6. No statistically signif-

825

Fig. 7. Effects of vitamins, dichlorvos, and vitamins + dichlorvos treatments on testosterone levels in rats. a Comparison of control and other groups (P < 0.05). b Comparison of vitamins-treated group with dichlorvos- and dichlorvos + vitaminstreated groups (P < 0.05). c Comparison of dichlorvos + vitamins-treated group with dichlorvos-treated group (P < 0.05). Values are means ± SD of six animals in each group.

icant changes in LH levels between the vitamins-treated group and the control group were observed at the end of 24 h, 4th and 7th weeks. A significant decrease in LH levels in the dichlorvosand vitamins + dichlorvos-treated groups compared to the control group (P < 0.05) was observed at the end of the 4th and 7th weeks (Fig. 6). No changes in LH levels between the vitamins + dichlorvostreated group and the dichlorvos-treated group were observed at the end of 24 h, 4th and 7th weeks (Fig. 6). 3.5.3. Testosterone levels Testosterone levels in the control group were compared to those in the experimental groups at the end of 24 h and the 4th and 7th weeks after vitamins, dichlorvos, and vitamins + dichlorvos were given to rats. In addition, the dichlorvos-treated group was compared to the vitamins + dichlorvos-treated group. Changes in testosterone levels are shown in Fig. 7. No statistically significant changes were observed between the vitamins-treated group and the control group at the end of 24 h, 4th and 7th weeks (Fig. 7). A significant decrease in testosterone levels in the dichlorvosand vitamins + dichlorvos-treated groups, compared to the control group (P < 0.05), was observed at the end of the 4th and 7th weeks (Fig. 7). A significant recovery in testosterone levels was observed in the vitamins + dichlorvos-treated group compared to the dichlorvos-treated group at the end of the 4th and 7th weeks (Fig. 7). 3.6. Histological changes in the testis

Fig. 6. Effects of vitamins, dichlorvos, and vitamins + dichlorvos treatments on LH levels in rats. a Comparison of control and other groups (P < 0.05). b Comparison of vitamins-treated group with dichlorvos- and dichlorvos + vitamins-treated groups (P < 0.05). Values are means ± SD of six animals in each group.

Fig. 8 shows the histology of seminiferous tubules and interstitial tissues. Spermatogenic cells and Sertoli cells in the seminiferous tubules of rats in the vitamins-treated and control groups were structurally normal. Leydig cells and blood vessels were present in the interstitial connective tissue between the seminiferous tubules (Fig. 8A). No pathological alterations were detected in seminiferous tubules and interstitial tissues in the dichlorvos- and vitamins + dichlorvos-treated groups at the end of 24 h (Fig. 8B and C). After 4 weeks of dichlorvos and vitamins + dichlorvos exposure, structural abnormalities were detected in the seminiferous epithelium in some of the seminiferous tubules; and edema in interstitial tissues was also observed (Fig. 8D and E). After 7 weeks of dichlorvos exposure, necrosis was observed in some seminiferous tubules and edema in the interstitial tissue of testes (Fig. 8F). After vitamins + dichlorvos were given to rats for 7

826

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Fig. 8. (A) Testicular section of control rats. No pathological changes in testicular section after 24 h of dichlorvos (B) and vitamins + dichlorvos (C) exposure. Structural disruption in seminiferous epithelium () and edema in interstitial tissue (

) after 4 weeks of dichlorvos (D) and vitamins + dichlorvos (E) treatment to rats. (F) Necrosis

) in interstitial tissue after 7 weeks of dichlorvos treatment to rats. (G) Necrotic areas () in seminiferous tubules after 7 weeks () in seminiferous tubules and edema ( of vitamins + dichlorvos treatment to rats. (A) ×200, (B) ×200, (C) ×200, (D) ×200, (E) ×200, (F) ×300, (G) ×400.

weeks, necrotic areas in some seminiferous tubules were observed (Fig. 8G). 3.7. Ultrastructural alterations in testicular tissues Fine structure of testicular cells is shown in Fig. 9. No pathological changes were observed in the spermatogenic cells of rats in the dichlorvos- and vitamins + dichlorvos treated groups at the

end of 24 h. After 4 weeks of dichlorvos and vitamins + dichlorvos exposure, morphological abnormalities in spermatozoa and mitochondrial swellings and vacuolizations were detected in the cytoplasm of Sertoli cells (Fig. 9B and C). After 7 weeks of dichlorvos and vitamins + dichlorvos treatment to rats, dense swellings and vacuolizations in the mitochondria of Sertoli cells, increase in lysosomal structures and severe morphological abnormalities in spermatozoa were observed (Fig. 9D and E).

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Fig. 9. (A) Electron micrograph of control rats, mitochondria (M), spermatozoa (Sp) and Sertoli cell cytoplasm (Sc). (B) Abnormal spermatozoa (

827

), mitochondrial swelling

) and mitochondrial swelling (M) after 4 weeks (M) and vacuolizations (V) after 4 weeks with dichlorvos treatment to rats, Sp: spermatozoa. (C) Abnormal spermatozoa ( of vitamins + dichlorvos treatment to rats. (D) Mitochondrial swelling (M) and increase in lysosomes (L) of Sertoli cells after 7 weeks of dichlorvos treatment to rats, Sp: spermatozoa. (E) Mitochondrial swelling (M) of Sertoli cells and abnormal spermatozoa (⇒) after 7 weeks of vitamins + dichlorvos treatment to rats. G: Golgi complex. (A) ×7500, (B) ×8500, (C) ×7500, (D) ×12,500, (E) ×10,000.

4. Discussion Experimental and human epidemiological data indicate that the OP compounds, including dichlorvos, being lipid soluble, can be quickly absorbed from the skin, gastrointestinal tract, and especially pulmonary route. As these chemicals can easily pass through not only the blood-brain barrier but also the placental barrier, they can cause disorders of the fetal organs when they enter the maternal reproductive system during pregnancy (Desi and Nagymajtenyi, 1999). The acute oral LD50 of dichlorvos is 80 mg/kg for male rats (Okamura et al., 2005). Dichlorvos is not only toxic to mammals but also negatively affects Pisces, Aves, and other non-target invertebrates (Ural MS¸ and Köprücü, 2006). The spermatogenetic cycle is highly organized throughout the testis in vertebrates. In the rat, the total duration of spermatogenesis is 50 days (Rosiepen et al., 1995). If a toxicant affects the immature spermatogonia, the effect can be detectable as a change in mature sperm after 7 weeks of exposure. Effects on more mature germ cells would be detected sooner. In the present study, subchronic toxicity was evaluated after 7 weeks of exposure due to

the complete duration of the cycle of seminiferous epithelium in the rat. Oral et al. (2006) applied 4 mg/kg dichlorvos to rats orally and observed apoptosis and endometrial damage in the female reproductive tract. Ogutcu et al. (2008) applied 1.6 mg/kg dichlorvos to male rats and detected hepatotoxic effects. In the present study, even though dichlorvos was given at 1/50 LD50 oral dose, we observed pathological changes in rat testes and decreases in FSH, LH, and T levels; however, no rats died during the experimental period. Many studies imply that OP insecticides cause reduction of body and organ weights in experimental animals (Ogutcu et al., 2006; Kalender et al., 2007; Uzunhisarcikli et al., 2007). In the present study, reduced body, absolute testis, and relative testis weights were observed 4 and 7 weeks after dichlorvos and vitamins + dichlorvos treatment. One of the causes of this reduction is due to the suppression of food intake. Food consumption was lower in rats in the dichlorvos- and vitamins + dichlorvostreated groups than in rats in the control group in our study. According to the present study, dichlorvos reduced the desire

828

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

for food of rats. Other possible cause of this reduction is due to the toxic effect or hormonal imbalance in any step in the hypothalamo–hypophysial–testicular axis, possibly induced by DDVP exposure. Vitamins C and E did not show a protective effect on body weight, absolute testis weight, or relative testis weight. Pesticides cause various histopathological and cytopathological changes in the male reproductive system (Maitra and Mitra, 2008; Uzunhisarcikli et al., 2007; Contreras and Bustos-Obregon, 1999; Joshi et al., 2003; Jaiswal et al., 2005; Zhang et al., 2008; Sarabia et al., 2009). In the present study, dichlorvos caused pathological changes in the seminiferous tubules after 4 and 7 weeks of exposure. Especially after 7 weeks, it caused apparent necrosis and edema in the seminiferous tubules and interstitial tissue. In addition, the same pathological findings were observed in the vitamins + dichlorvos-treated rats also. Mitochondria are the key organelles representing cellular damage (Kalender et al., 2005a) and pesticide derived mitochondrial pathologies are well known (Contreras and Bustos-Obregon, 1999; Joshi et al., 2003). Our study showed a clear incline towards dichlorvos induced cellular damage after subacute and subchronic dichlorvos exposure indicated by mitochondrial vacuolizations and swellings and increased lysosomal structures; and these alterations did not show any sign of recovery by the addition of vitamins in diet. Dichlorvos exposure, regardless of vitamin supplementation, also resulted in ultrastructural abnormalities in spermatozoa. In toxic interactions, both direct and indirect mechanisms may be involved. Certain vitamins and various nutrients can likewise influence toxic outcomes. Considerable experimental and clinical evidence supports the importance of mitochondrial oxidative damage as a critical event in toxic oxidative stress-related diseases and situations, induced by toxicants. Antioxidants such as vitamin E directed to mitochondria have been shown to protect cells against toxic oxidative stress (Young and Woodside, 2001). Though the critical mitochondrial events responsible for oxidative stress-mediated cell injury and death have yet to be defined in detail, oxidative damage to mitochondrial lipids, nucleic acids and proteins appear to be important events in these toxic processes. Studies support the use of dietary vitamin E supplementation as a potential therapeutic strategy to fight against oxidative stress (Kalender et al., 2004, 2005b, 2002; Schneider, 2005; John et al., 2001; Bowry and Stocker, 1993). On the other hand, cholesterol is an important cellular membrane component and is a precursor for steroids hormones like testosterone and other biologically active lipids that function in cell–cell signaling. Also derived from precursors of cholesterol biosynthesis are the fat soluble vitamins, which have diverse functions including protection against oxidative damage to cells by vitamin E (Young and Woodside, 2001). As an electron donor, vitamin C acts as a cofactor for various enzymes involved in collagen hydroxylation, biosynthesis of carnitine and nor epinephrine, tyrosine metabolism and amidation of peptide hormones. Vitamin C is also a powerful water-soluble antioxidant and, at physiological concentrations, probably does not produce reactive intermediates. It protects low-density lipoproteins from oxidation and reduces harmful oxidants (Young and Woodside, 2001). On the contrary, our light and electron microscopic findings represent a typical damage to both spermatogenic and interstitial cells, which is not recovered by vitamins C and E. As these vitamins are the main factors in cellular struggle against reactive oxygen species and lipid peroxidation (Young and Woodside, 2001; Kalender et al., 2004, 2005b, 2002; Schneider, 2005; John et al., 2001; Bowry and Stocker, 1993), and vitamin treatment did not have any protective role on dichlorvos exposure in our study, we speculate that the histopathological alterations induced by dichlorvos in the testicular tissues are probably not caused by oxidative stress.

Previous studies report that OP insecticides decrease sperm count and motility and increase levels of abnormal sperm morphology (Uzunhisarcikli et al., 2007; Farag et al., 2000; Burruel et al., 2000). Impairments reported in these parameters show a dose dependent manner. However, some recent studies represent conflicting results. In particular, the sperm concentration seems not to be affected by OP exposure (Okamura et al., 2005; Kamijima et al., 2004). In the present study, sperm count was also unchanged during the experimental period. Evaluation of sperm concentration after 24 h, 4th and 7th weeks showed no statistically significant differences between the control group and the dichlorvos- and dichlorvos + vitamins-treated groups. Xu et al. (2004) reported that daily sperm production decreased when phoxim, an OP insecticide, with fenvalerate, a pyrethroid insecticide, were administrated to male rats. In our study, dichlorvos exposure did not affect sperm production in the testes of rats. In addition, vitamin treatments did not increase testicular sperm production independently. Significant increases in the production of dead or abnormal sperm in experimental animals after exposure to certain pesticides have been reported (Uzunhisarcikli et al., 2007; Okamura et al., 2005; Burruel et al., 2000; Latchoumycandane et al., 2002). Exposure to the pesticide ethylene dibromide (ED) has been reported to cause a significant reduction in sperm motility and viability, suggesting that ED exposure may affect accessory sex glands (Schrader et al., 1988). In the present study, a decrease in epididymal sperm motility was observed at the end of the 7th week. Vitamin treatments had no positive effect on the epididymal sperm motility. A reduction in sperm motility in the pesticide-exposed rats may be explained by more than one mechanism. It is known that pesticides may reduce mitochondrial enzyme activity of spermatozoa (Contreras and Bustos-Obregon, 1999), which will result in a reduction of sperm motility. On the other hand, fructose synthesis and secretion by the accessory glands is dependent upon the secretion of testosterone by Leydig cells (Mann, 1964), thus, the observed reduction in sperm motility may be attributed to the reduction of serum testosterone levels. The disruption of microtubule structure in spermatozoa may also cause a reduction in sperm motility, or an increase in the proportion of abnormal sperm might result in a reduction in sperm motility (Uzunhisarcikli et al., 2007). In our study, dichlorvos treatment caused an increase in the count of abnormal spermatozoa and a decrease in testosterone levels. Therefore, the reduction in sperm motility might be related to the decrease of testosterone levels and the increase in abnormal sperm forms (Uzunhisarcikli et al., 2007). A disruption of spermatogenesis and an increase in counts of abnormal sperm are important indicators of genetic damage in the mammalian species exposed to pesticides (Burruel et al., 2000). Pesticides may damage testicular DNA (Sarabia et al., 2009; Topham, 1980). In the present study, an increase in counts of abnormal sperm occurred after 4 and 7 weeks of dichlorvos treatment. Dichlorvos might have increased the rate of abnormal sperm by affecting spermatogenesis/spermiogenesis. Vitamins C and E did not show a protective effect on sperm morphology. Significant alterations in the levels of FSH, LH and testosterone have been reported after exposure to certain pesticides. Exposure to the pesticide quinalphos has been reported to cause a significant reduction in levels of FSH and LH (Ray et al., 1992). In our study, the levels of FSH and LH in the dichlorvos- and vitamins + dichlorvostreated groups were significantly lower than those in the control group at the end of the 4th and 7th weeks. Dichlorvos exposure suppressed the secretion of FSH and LH. In addition, vitamins C and E treatment did not show a protective effect on levels of FSH and LH. The antagonistic property of dichlorvos on androgen receptors might explain these results. Any antagonist of androgen receptors may alter glycosylation of gonadotrophins and result in suppression of the levels of FSH and LH (Naz, 1999).

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Testosterone is the key hormone for regulating spermatogenesis, together with gonadotrophins. Its secretion from the Leydig cells is dependent upon the secretion of LH from the pituitary gland. Various OP compounds have been studied for their effects on the levels of testosterone. Significant reduction in plasma concentrations of testosterone, along with reduction in levels of testicular testosterone, has been reported after quinalphos treatment (Ray et al., 1992). In contrast to Okamura et al. (2005), our study, at the end of the 4th and 7th weeks, showed a decrease in the level of plasma testosterone. Administration of vitamins E and C resulted in a statistically significant increase in levels of plasma testosterone in the dichlorvos + vitamins group relative to the dichlorvos group, but the recovery was insufficient to elevate the concentrations of testosterone to levels seen in the control and vitamins groups. The recovery in the concentration of testosterone after administration of vitamin E and C suggests that dichlorvos impairs the function of Leydig cells by deteriorating their plasma membranes via oxidative damage and that vitamins show their protective roles on the plasma membranes of Leydig cells. Lipid-soluble vitamin E is present in biological membranes (Senthil Kumar et al., 2004). It protects the cell against lipid peroxidation, most efficiently through its chain-breaking anti-oxidant action (Serbecic and Beutelspacher, 2005). In this reaction, vitamin E is converted to a weak free radical (␣-tocopherol radical) (Zaken et al., 2001). Ascorbic acid is hydrophilic and functions better in an aqueous environment. Moreover, it can restore the anti-oxidant properties of oxidized tocopherol, suggesting that a major function of ascorbic acid is to recycle the tocopheroxyl radical (Serbecic and Beutelspacher, 2005). Uzunhisarcikli et al. (2007) have reported that administration of vitamins C and E resulted in an improvement in the sperm count, sperm motility, and histopathology. In our study, sperm motility, abnormal sperm morphology, and levels of FSH, LH, and testosterone assessed after vitamins + dichlorvos treatment were statistically different from those of the control group. We did not see any differences in these parameters except in the levels of testosterone in the vitamins + dichlorvos-treated group compared to the dichlorvos-treated group. In other words, antioxidant treatment did not result in any recovery of these parameters except for levels of testosterone. Therefore, dichlorvos probably has systemic toxic effects on the entire organism, and the deteriorating effects of this compound on reproductive parameters may not be due to the changes in levels of lipid peroxidation and production of free radicals. Still, it is difficult to generalize our results to men. The number of sperm cells in a human ejaculate is usually only two to fourfold more than the number of sperm cells at which fertility is significantly reduced, but in rats epididymal sperm counts can be reduced as much as 90% without a significant loss of fertility (Mori et al., 1991). Men have a much smaller relative size of the testis, and lower rate of daily sperm production per gram testis, and lower percentages of morphologically normal sperm cells in semen than any of the animal models studied (Mori et al., 1991). The observation that humans have experienced increased incidences of developmental, reproductive, and carcinogenic effects and the formulation of a working hypothesis that these adverse effects may be caused by environmental chemicals acting to disrupt the endocrine system that regulates these processes are supported by observations of similar effects in wildlife species. In other words, a common theme runs through both human and wildlife reports (Mori et al., 1991). Whether the young and the adults are capable of regulating minor changes to the reproductive milieu is uncertain. But diet is a major source of pesticide exposure for children. According to a 2008 US report, detectable concentrations of the organophosphate malathion were found in 28% of frozen blueberry samples, 25% of strawberry samples, and 19% of celery samples.

829

The United States Environmental Protection Agency first considered a ban on DDVP in 1981. Since then it has been close to being banned on several occasions, but continues to be available. Major concerns are over acute and chronic toxicity. There is no conclusive evidence of carcinogenicity to date and the current data are not available for the mixtures of chemicals that may be able to affect reproductive function in the male. Clearly, more research to fill data gaps and to remove the uncertainty in these unknowns is needed. A recent 2010 study found that each 10-fold increase in urinary concentration of organophosphate metabolites, mainly DDVP, was associated with a 55–72% increase in the odds of ADHD in children, which is a neurobehavioral developmental disorder (Bouchard et al., 2010). However, with few exceptions (e.g., DDT), a causal relationship between exposure to a specific environmental agent and an adverse effect on human reproductive health has not been established yet. A variety of extraneous and internal factors can induce testicular toxicity, leading to poor sperm quality and male factor infertility. Unfortunately, several of these influences like oxidative stress, disrupt the reproductive milieu, and have been underestimated. There is an urgent need to characterize all the factors involved and to develop reliable animal models of testicular toxicity. In conclusion, dichlorvos did not show acute, but it did cause subacute and subchronic reproductive toxicity in male rats in the present study. We did not observe a protective role for vitamins C and E on dichlorvos toxicity. Since in vivo methods are important tools to study the male reproductive system, the complete in vivo assessment of testicular toxicity involves multigenerational studies, now required by most regulatory agencies. These multigenerational studies have a complex design, because testicular function and spermatogenesis are very complicated processes. Future studies examining the breeding success and fertility status of dichlorvos treated rats are essential to interpret our findings properly and to clarify the toxic effects of dichlorvos and protective roles of vitamins C and E. Because of the possible dangers pesticides pose to humans, the U.S. EPA, and the European Union guidelines, limits how much residue can stay on food. But the 2010 ADHD study shows it is possible even tiny, allowable amounts of pesticide may affect brain chemistry. It seems prudent, therefore, to reduce OP pesticide exposure by reducing their use in agriculture, in order to protect individuals from reproductive side effects. Nevertheless, food safety assessment must take into account the potential for toxic interactions of OP compounds, particularly in young populations and further studies on the antioxidant effects of vitamins C and E should be targeted to humans and certain occupational groups at high risk of oxidant damage. Moreover, consumers should limit their OP pesticide exposure by choosing organic products, including frozen organic products, checking the labels on any older pest control or gardening products in the household to make sure that they do not contain OP pesticides, and checking the label on pet care products, avoid flea collars that list OP compounds as active ingredients.

References Atis¸ S, C¸ömeleko˘glu Ü, C¸os¸kun B, Özge A, Ersöz G, Talas D. Electrophysiological and histopathological evaluation of respiratory tract, diaphram, and phrenic nerve after dichlorvos inhalation in rats. Inhal Toxicol 2002;14: 199–215. Bouchard MF, Bellinger DC, Wright RO, Weisskopf MG. Attentiondeficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. Pediatrics 2010;125:1270–7. Bowry VW, Stocker R. Tocopherol-mediated peroxidation. The pro-oxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J Am Chem Soc 1993;115:6029–44.

830

E.K. Dirican, Y. Kalender / Experimental and Toxicologic Pathology 64 (2012) 821–830

Burruel VR, Raabe OG, Overstreet JW, Wilson BW, Wiley LM. Paternal effects from methamidophos administration in mice. Toxicol Appl Pharmacol 2000;165:148–57. Choudhary S, Gill KD. Protective effect of nimodipine on dichlorvos-induced delayed neurotoxicity in rat brain. Biochem Pharmacol 2001;62:1265–72. Contreras HR, Bustos-Obregon E. Morphological alterations in mouse testis by a single dose of malathion. J Exp Zool 1999;284:355–9. Desi I, Nagymajtenyi L. Electrophysiological biomarkers of an organophosphorus pesticide, dichlorvos. Toxicol Lett 1999;1:55–64. Farag AT, Ewediah MH, El-Okazy AM. Reproductive toxicology of acephate in male mice. Reprod Toxicol 2000;14:457–62. Garcia SJ, Abu-Qare AW, Meeker-O’Connell WA, Borton AJ, Abou-Donia MB. Methyl parathion: a review of health effects. J Toxicol Environ Health B Crit Rev 2003;6:185–210. Hazarika A, Sarkar SN, Hajare S, Kataria M, Malik JK. Influence of malathion pretreatment on the toxicity of anilofos in male rats: a biochemical interaction study. Toxicology 2003;185:1–8. Jaiswal A, Parihar VK, Kumar MS, Manjula SD, Krishnanand BR, Shanbhag R, et al. 5-Aminosalicylic acid reverses endosulfan-induced testicular toxicity in male rats. Mutat Res 2005;585:50–9. John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E dimethoate and malathion induced oxidative stress in rat erythrocytes. J Nutr Biochem 2001;12:500–4. Joshi SC, Mathur R, Gajraj A, Sharma T. Influence of methyl parathion on reproductive parameters in male rats. Environ Toxicol Pharmacol 2003;14:91–8. Kalender S, Kalender Y, Ates¸ A, Yel M, Olcay E, Candan S. Protective role of antioxidant vitamin E and catechin on idarubicin-induced cardiotoxicity in rats. Braz J Med Biol Res 2002;35:1379–87. Kalender S, Kalender Y, Ogutcu A, Uzunhisarcıklı M, Durak D, Acıkgöz F. Endosulfaninduced cardiotoxicity and free radical metabolism in rats: the protective effect of vitamin E. Toxicology 2004;202:227–35. Kalender S, Ogutcu A, Uzunhisarcikli M, Ac¸ikgoz F, Durak D, Ulusoy Y, et al. Diazinoninduced hepatotoxicity and protective effect of vitamin E on some biochemical indices and ultrastructural changes. Toxicology 2005a;211:197–206. Kalender Y, Yel M, Kalender S. Doxorubicin hepatotoxicity and hepatic free radical metabolism in rats. The effects of vitamin E and catechin. Toxicology 2005b;209:39–45. Kalender Y, Uzunhisarcikli M, Ogutcu A, Acikgoz F, Kalender S. Effects of diazinon on pseudocholinesterase activity and haematological indices in rats: the protective role of vitamin E. Environ Toxicol Pharmacol 2006;22:46–51. Kalender S, Kalender Y, Durak D, Ogutcu A, Uzunhisarcikli M, Cevrimli BS, et al. Methyl parathion induced nephrotoxicity in male rats and protective role of vitamins C and E. Pestic Biochem Phys 2007;88:213–8. Kamijima M, Hibi H, Gotoh M, Taki K, Saito I, Wang H, et al. A survey of semen indices in insecticide sprayers. J Occup Health 2004;46:109–18. Latchoumycandane C, Chitra KC, Mathur PP. The effect of methoxychlor on the epididymal antioxidant system of adult rats. Reprod Toxicol 2002;16:161–72. Maitra SK, Mitra A. Testicular functions and serum titers of LH and testosterone in methyl parathion-fed roseringed parakeets. Ecotoxicol Environ Saf 2008;71:236–44. Mann T. Fructose, polylos, and organic acids. The biochemistry of semen and the male reproductive tract. New York: John Wiley; 1964. p. 237–64. Mori K, Kaido M, Fujishiro K, Inoue N, Koide O, Hori H, et al. Dose dependent effects of inhaled ethylene oxide on spermatogenesis in rats. Br J Ind Med 1991;48:270–4. Nahas S, Hondt HA, Abdou HA. Chromosome aberrations in spematogonia and sperm abnormalities in Curacon-trated mice. Mutat Res 1989;222:409–14. Naz R. Endocrine disruptors, effects on male and female reproductive systems. Florida: CRC Press; 1999.

Ogutcu A, Uzunhisarcikli M, Kalender S, Durak D, Bayrakdar F, Kalender Y. The effects of organophosphate insecticide diazinon on malondialdehyde levels and myocardial cells in rat heart tissue and protective role of vitamin E. Pestic Biochem Phys 2006;86:93–8. Ogutcu A, Suludere Z, Kalender Y. Dichlorvos-induced hepatotoxicity in rats and the protective effects of vitamins C and E. Environ Toxicol Pharmacol 2008;26:355–61. Okamura A, Kamijima M, Shibata E, Ohtani K, Takagi K, Ueyama J, et al. A comprehensive evaluation of the testicular toxicity of dichlorvos in Wistar rats. Toxicology 2005;213:129–37. Oral B, Guney M, Demirin H, Ozguner M, Giray SG, Take G, et al. Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant vitamins E and C. Reprod Toxicol 2006;22:783–90. Raheja G, Gill KD. Calcium homeostasis and dichlorvos induced neurotoxicity in rat brain. Mol Cell Biochem 2002;232:13–8. Ray A, Chatterjee S, Ghosh S, Bhattacharya K, Pakrashi A, Deb C. Quinalphos induced suppression of spermatogenesis, plasma gonadotropins, testicular testosterone production and secretion in adult rats. Environ Res 1992;57: 181–9. Rosiepen G, Chapin RE, Weinbauer GF. The duration of the cycle of the seminiferous epithelium is altered by administration of 2,5-hexanedione in the adult Sprague–Dawley rat. J Androl 1995;16:127–35. Sarabia L, Maurer I, Bustos-Obregon E. Melatonin prevents damage elicited by the organophosphorous pesticide diazinon on mouse sperm DNA. Ecotoxicol Environ Saf 2009;72:663–8. Sarin S, Gill KD. Dichlorvos induced alterations in glucose homeostasis: possible implications on the state of neuronal function in rats. Mol Cell Biochem 1999;199:87–92. Schneider C. Chemistry and biology of vitamin E. Mol Nutr Food Chem 2005;49:7–30. Schrader SM, Turne TW, Ratcliffe JM. The effects of ethylene bromide on semen quality: a comparison of short term and chronic exposure. Reprod Toxicol 1988;2:191–8. Senthil Kumar J, Banudevi S, Sharmila M, Murugesan P, Srinivasan N, Balasubramanian K, et al. Effects of vitamin C and vitamin E on PCB (Aroclor 1254) induced oxidative stress, androgen binding protein and lactate in rat Sertoli cells. Reprod Toxicol 2004;19:201–8. Serbecic N, Beutelspacher SC. Anti-oxidative vitamins prevent lipid peroxidation and apoptosis in corneal endothelial cells. Cell Tissue Res 2005;320: 465–75. Topham JC. The detection of carcinogen induced sperm head abnormalities in mice. Mutat Res 1980;69:149–55. Ural MS¸, Köprücü SS¸. Acute toxicity of dichlorvos on Fingerling European Catfish, Silurus glanis. Bull Environ Contam Toxicol 2006;76:871–6. Uzunhisarcikli M, Kalender Y, Dirican K, Kalender S, Ogutcu A, Buyukkomurcu F. Acute, subacute and subchronic administration of methyl parathion-induced testicular damage in male rats and protective role of vitamins C and E. Pestic Biochem Phys 2007;87:115–22. Xu L, Zhan N, Liu R, Song L, Wang X. Joint action phoxim and fenvalerate on reproduction in male rats. Asian J Androl 2004;6:337–41. Yarsan E, Cakir O. Effects of dichlorvos on lipid peroxidation in mice on subacute and subchronic periods. Pestic Biochem Phys 2006;86:106–9. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol 2001;54:176–86. Zaken V, Kohen R, Ornoy A. Vitamins C and E improve rat embryonic antioxidant defense mechanism in diabetic culture medium. Teratology 2001;64:33–44. Zhang SY, Ueyama J, Ito Y, Yanagiba Y, Okamura A, Kamijima M, et al. Permethrin may induce adult male mouse reproductive toxicity due to cis isomer not trans isomer. Toxicology 2008;248:136–41.