Arch Toxicol (2003) 77: 280–284 DOI 10.

1007/s00204-003-0439-x

MOLECULAR TOXICOLOGY

C. Latchoumycandane Æ K.C. Chitra Æ P.P. Mathur

2,3,7,8-Tetrachlorodibenzo-p -dioxin (TCDD) induces oxidative stress in the epididymis and epididymal sperm of adult rats

Received: 19 September 2002 / Accepted: 15 January 2003 / Published online: 26 February 2003 Ó Springer-Verlag 2003

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent environmental contaminants, which has been shown to induce oxidative stress in testis and epididymal sperm of rats. However, the nature and mechanism of action of TCDD on the epididymis is not clear. The aim of the present study was to investigate whether induction of oxidative stress in epididymal sperm was direct effect of TCDD on epididymis. In the present studies, TCDD (0.1, 1.0 and 10 lg/kg body weight per day) was administered orally to rats for 4 days. Twentyfour hours after the last treatment the animals were killed using anesthetic ether. Both epididymides were dissected out and epididymal sperm were collected by cutting the epididymides into small pieces in Ham’s F-12 medium at 35°C. The epididymal sperm and caput, corpus and cauda epididymides were homogenized and used for biochemical studies. Epididymal sperm counts did not decrease in the rats treated with TCDD. Administration of TCDD increased the production of reactive oxygen species such as hydrogen peroxide while the activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were found to be decreased in the epididymal sperm as well as in cauda epididymides. Lipid peroxidation also increased in the epididymal sperm and in the various regions of the epididymides after exposure to TCDD. The results indicated that TCDD induces oxidative stress in the epididymis and epididymal sperm by decreasing the antioxidant enzymes through induction of reactive oxygen species. Thus, the adverse effects of TCDD on the epididymal sperm were due to direct effect of TCDD on epididymis.

Keywords 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Æ Oxidative stress Æ Sperm Æ Epididymis Æ Lipid peroxidation

Introduction
The exposure of humans to environmental contaminants that negatively affect the male reproductive system is increasing (Sharpe 1993). It is important to note that many organochlorine environmental contaminants accumulate in the fatty tissues and, therefore, continuous exposure of humans to increasing numbers and amounts of these contaminants may have cumulative effects that lead to reproductive disorders. Dioxins are a class of persistent polychlorinated aromatic hydrocarbons and some of the most potent environmental contaminants that induce a wide spectrum of toxic responses in experimental animals, including reproductive, developmental and immunologic toxicities as well as carcinogenicity (Williams and Iatropoulos 2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known dioxin, which is formed as an unwanted by-product in the manufacture of chlorinated hydrocarbons. It is also formed in incineration processes, paper and pulp bleaching, and emissions from steel foundries and motor vehicles (Skene et al. 1989). The lipophilicity and low rate of metabolism of TCDD leads to its accumulation and persistence in adipose tissue (Enan et al. 1998). Among the several mechanisms, aryl hydrocarbon receptor (Ah) mechanisms have been proposed to explain the toxicity of TCDD and its congeners (Safe 1995, 2001). Male rats exposed to TCDD display reduced fertility, delayed puberty and altered reproductive organ weights (Gray et al. 1997). TCDD-exposed male rats displayed decreased numbers of sperm (Gray et al. 1997) and increased numbers of abnormal sperm in the epididymis (Faqi et al. 1997). In utero and lactational exposure to TCDD have been shown to cause a significant reduction in the weight of the testis, epididymis, in daily

This paper was presented at the 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, USA, 28–31 July, 2002. C. Latchoumycandane Æ K.C. Chitra Æ P.P. Mathur (&) School of Life Sciences, Pondicherry University, Pondicherry, 605 014, India E-mail: ppmathur@hotmail.com Tel.: +91-413-2655212 Fax: +91-413-2655211

The levels of hydrogen peroxide (Pick and Keisari 1981) and lipid peroxidation (Buege and Aust 1976) were estimated in the epididymal sperm and caput. 1997. Necropsy and collection of epididymal sperm The animals were fasted overnight. Chitra et al. 1995). corpus and cauda epididymides was prepared in cold normal saline using a glass– Teflon homogenizer. 2002). Latchoumycandane et al. The epididymis is responsible for the sustenance. 1992) and ejaculated (Gray et al. Department of Veterinary Anatomy and Public Health. Chitra et al. TCDD has been reported to produce the epididymis-specific decrease in cauda epididymal sperm counts (Mably et al. Previous studies from our laboratory have shown that lindane and methoxychlor induce oxidative stress in the testis as well as in epididymis and epididymal sperm of rat (Sujatha et al. Merck (Darmstadt. Corresponding groups of animals were maintained and administered an equal volume of vehicle alone (acetone and olive oil) to serve as controls. USA. 1. India. Animals and treatments Albino male rats of Wistar strain (90 days old) were obtained from the Central Animal House. Germany). The tissue was washed several times in the same medium in order to remove maximum number of sperm from the epididymis. The caput. 2002a). 1997). weighed and killed using anesthetic ether on the day following the last dose. 1992. TCDD was dissolved in acetone and olive oil (1:19) and administered orally at doses of 0. 1997). Acute high-dose exposure to TCDD results in oxidative stress in multiple tissues and species (Mohammadpour et al. suggesting the epididymis-specific effect. 2002c. 2002b) and in mouse liver mitochondria (Senft et al. 1995) sperm numbers were much greater than the observed decrease in testicular sperm of TCDD-treated rats. A 1% homogenate of caput. 2002) after exposure to TCDD have been reported. glutathione reductase (Carlberg and Mannervik 1975) and glutathione peroxidase (Paglia and Valentine 1967) in the epididymal sperm and various regions of the epididymides were assayed by the methods described previously (Latchoumycandane et al. an aliquot of 5 ll of epididymal sperm was diluted with 95 ll of diluent (50 g sodium bicarbonate. In the present study. 2001. corpus and cauda epididymides of right epididymis and epididymal sperm were used for biochemical assays.0. The hemocytometer was allowed to stand for 5 min in a humid chamber to prevent drying out. However. The left cauda epididymis was weighed and used for sperm counts. The homogenate was centrifuged at 800 g for 20 min at 4°C. There is also much concern that exposure to environmental contaminants induces major pathological effects in the epididymis of men and experimental animals (Hess 1998). protection. and superoxide dismutase depletion is thought to be associated with sperm immotility (Kobayashi et al. Protein content was estimated by the method of Lowry et al. All other chemicals were of analytical grade and obtained from local commercial sources. 2002c). 2002c. The homogenate was centrifuged at 800 g for 20 min at 4°C. Excessive generation of reactive oxygen species (ROS) has been shown to cause peroxidative damage to the plasma membrane. Stephen Safe. Latchoumycandane et al.281 sperm production and cauda epididymal sperm counts (Gray et al.1. Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER). 2002a. Induction of oxidative stress upon exposure to TCDD is considered to be an important mechanism for the toxic effects of TCDD (Stohs 1990. TCDD has been reported to induce superoxide anion. Faqi et al. corpus and cauda epididymides of rats as described previously (Latchoumycandane et al. or 10 lg/kg body weight per day for 4 days. and the supernatant was used for biochemical studies. All the data were analyzed using one-way analysis of variance followed by Duncan’s mul- Material and methods Chemicals TCDD was obtained as a gift from Dr. the sperm suspensions obtained as described above were centrifuged at 800 g for 20 min at 4°C. (1951). The rats were fed with standard commercial pelleted feed and water ad libitum. Briefly. lipid peroxidation and DNA damage in hepatic and brain tissues of rat (Hassoun et al. Inductions of oxidative stress in the mitochondrial and microsomal fraction of testis and in epididymal sperm (Latchoumycandane et al. 10 ml 35% formalin.25 g trypan blue were added and made up to a final volume of 1 l with distilled water). 2001). 2002b. transport. the effect of TCDD on the epididymis remains unclear and the epididymal production of free radicals and the functions of antioxidant defense system require further studies. Biochemical studies For biochemical assays. The animals were maintained under a well-regulated light and dark scheduled (12:12 h) with a temperature of 24±3°C. 2002c). A & M University. we have sought to investigate whether induction of oxidative stress in the epididymal sperm by TCDD was direct effect of TCDD on epididymis. and the supernatant was used for biochemical estimations. 2002a). A coverslip was secured to the counting chambers of the improved Neubauer-type hemocytometer. Thiobarbituric acid and malondialdehyde were obtained from E. . Pondicherry. 1988). Epididymal sperm count The epididymal sperm counts were performed by the method as described previously (Latchoumycandane et al. The pellet was resuspended in 2 ml normal saline and homogenized with the help of glass–Teflon homogenizer for a few seconds in the cold. Texas. The activities of superoxide dismutase (Marklund and Marklund 1974). maturations and storage of spermatozoa (Cooper 1999). Approximately 10 ll of the thoroughly mixed diluted specimen was transferred to each of the counting chambers of the hemocytometer. 2002a. The epididymal (Mably et al. Latchoumycandane and Mathur 2002a. 2002b). Maturation of sperm within the epididymis requires interaction between the epididymal epithelium. the luminal fluid and sperm (Klinefelter and Hess 1998). Gray et al. The cells sedimented during this time and were then counted with the help of light microscopy at ·200 magnification. and 0. The left and right epididymides were removed and cleared from the adhering tissues and then weighed. which leads to impaired sperm function. Epididymal sperm were collected by cutting the epididymis into small pieces in Ham’s F-12 medium at 35°C (Latchoumycandane et al. Latchoumycandane and Mathur 2002b. 2002b. 2001.

compared with those in the corresponding groups of control animals. whereas lower doses did not induce the production of hydrogen peroxide (Table 2). 1998). superoxide dismutase.53* 1. The units are expressed as nmol/min per mg protein for SOD. Activities of superoxide dismutase. glutathione reductase and glutathione peroxidase (Table 2) decreased significantly.0 or 10 lg/kg body weight per day for 4 days did not cause any significant change (P>0.23±1.16±3. The production of hydrogen peroxide in the epididymal sperm increased significantly (P<0. Administration of TCDD to adult rats at the dose levels of 1.31 30.1.05. while the levels of lipid peroxidation (Table 2) in the caput and corpus epididymis were found to be Table 1 Effect of 2. 2002a. However. In order to see the direct of TCDD on epididymis we have administered TCDD for 4 days.69 49. catalase.01* 26. and as lmol/mg protein for LPX. In the present studies administration of TCDD increased the production of hydrogen peroxide in the epididymal sperm of adult rats.93 19.1. the cauda epididymis showed significant changes at all the dose levels whereas the caput and corpus epididymides did not show any significant change at the 0. 1.01 2. which has been shown to be appropriate for observation of the direct effect of drugs on epididymis (Latchoumycandane et al.23* 10 14.34±0.72±1.01 47. TCDD is one such environmental contaminant and has been shown to induce reproductive abnormalities in both wildlife and humans by reduction in fertility (El-Sabeawy et al. and data were expressed as means ±SD for four animals per group.26* 26.0 lg/kg dose levels. 1. The activities of antioxidant enzymes such as superoxide dismutase.82* 2. Results Administration of TCDD at the dose levels of 0.96 2. Similar biochemical changes were also noted in the caput.16 23.282 tiple range test.38±3. Discussion There is much concern that exposure to environmental contaminants induces major pathological effects in the epididymis in men and experimental animals (Hess 1998).0 and 10 lg/kg body weight decreased the activities of superoxide dismutase.71±2.05) in the epididymal sperm counts compared with the corresponding counts in groups of control animals (data not shown).03* 41.0 and 10 lg/kg for 4 days increased lipid peroxidation of the epididymal sperm compared with the corresponding values in groups of control animals (Table 1).75±3. Induction of oxidative stress has been reported in the testis and epididymal sperm of rats (Latchoumycandane et al. However.05) in the rats treated with TCDD when compared with the corresponding groups of control animals (Table 1).00±2. glutathione reductase (GRX) and glutathione peroxidase (GPX).51±4. Administration of TCDD only at the dose level of 10 lg/kg body weight per day for 4 days increased the production of hydrogen peroxide in the caput and corpus epididymis of rats. 2002b). catalase.0 and 10 lg/kg for 4 days.06* 28.89±0. compared with that in corresponding groups of control animals. Data represent means±SD for four animals per dose Parameter Control TCDD (lg/kg body weight per day) 0. glutathione reductase and glutathione peroxidase (Table 1) decreased significantly (P<0.11±2.1 SOD CAT GRX GPX H2O2 LPX 21. corpus and cauda epididymides of the TCDD-treated rats.05 was considered as significant against control. GRX.16* 24.40±0.69±2. 1998).23 32.11±0. GPX and H2O2.01 2. 2002c).88±2.30* 2. considered significant against corresponding control . catalase (CAT).30±0. The activities of catalase. It has been reported that an evaluation of sperm in the proximal cauda epididymis 4 days following the onset of toxicant exposure avoids any possible contribution of testicular sperm to the results (Klinefelter and Hess 1998).32±1. we have sought to investigate whether induction of oxidative stress in the epididymal sperm was due to the direct effect of TCDD on epididymis. 1.7. The antioxidant enzyme superoxide dismutase greatly accelerates the dismutation of the *P<0.1.21±3.99±0.8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities of superoxide dismutase (SOD). as lmol/min per mg protein for CAT.18±2. In the present studies.71±0.11* 2.03±4.26* 43.27* increased significantly in the animals exposed to TCDD at the dose level of 10 lg/kg body weight per day for 4 days.0 or 10 lg/kg body weight per day.0 16. and these doses have shown to alter male reproduction of rat when administered intraperitoneally for 1 week during postnatal days 21 to 28 (El-Sabeawy et al. Rats were administered TCDD at doses of 0. However.26* 1. administration of TCDD for 4 days at the dose levels of 0. glutathione reductase and glutathione peroxidase while the levels of lipid peroxidation (Table 2) increased significantly in the cauda epididymis of adult rats compared with those in corresponding groups of control animals. administration of TCDD for 4 days increased the production of hydrogen peroxide in the cauda epididymis of adult rats with respect to that in the corresponding groups of control animals (Table 2).16 20.87* 2. The journey of sperm from the caput to the cauda epididymis takes approximately 4 days in rats.3. 1995).1 and 1. and the levels of hydrogen peroxide (H2O2) generation and lipid peroxidation (LPX) in the epididymal sperm of adult rats. glutathione reductase and glutathione peroxidase were found to be decreased while the levels of lipid peroxidation increased significantly in the epididymal sperm of rats. TCDD has been reported to produce an epididymis-specific decrease in cauda epididymal sperm counts (Gray et al.05) in the epididymal sperm of rats treated with TCDD at the dose levels of 1. catalase. A P-value of less than 0.46±2.89±0.

16±3.41±2.08±5.14* 50. The reduction in the activities of superoxide dismutase. The units are expressed as nmol/min per mg protein for SOD.67±0.46* 52. Acknowledgements The authors acknowledge Dr.L.41±3.73±0.62±0.22 39. glutathione reductase (GRX) and glutathione peroxidase (GPX) and the levels of hydrogen peroxide (H2O2) generation and lipid peroxidation (LPX) in the caput.98 2.03 2.23 36.283 Table 2 Effect of 2. India.91* 51.19* 30. USA.21 38.0 32.60* 2. indicating TCDD-induced oxidative stress in the epididymis. acknowledges the Indian Council of Medical Research. Griveau and Lannou (1997) reported that a ROS such as H2O2 appears to be a key agent in the cytotoxic effects in spermatozoa. 1989).16* 33.82 14.06±3.11* 26. Furthermore.81 2.03 15.20 2.71±2.12±3.86±1.P. catalase (CAT). corpus and cauda epididymides of Tissue Parameter Control adult rats. New York for financial assistance (Grant Nos.31 40.63±0.3.62 60.14 2. for a Senior Research Fellowship. which is known to inactivate catalase activity (Kono and Fridovich 1982). as lmol/min per mg protein for CAT.05.40 2.61 13.14±2.11 61. cauda epididymis was more susceptible to TCDD-induced oxidative stress than the caput and corpus epididymides. Clarkson JS. This may lead to disruption in functional integrity of cell organelles and to the onset of sperm damage under TCDD-induced pathological conditions.63±0.62* 2. J Androl 10:214–220 Buege JA.24±2.02±2. B99. Pondicherry University for various facilities.91 59. 1999). Cytoplasm of the spermatozoa is extremely limited in volume and localization so the polyunsaturated fatty acids that are bound in the sperm plasma membrane are very susceptible to ROS attack (Aitken et al.42 2.048R/ICMC).71±2.11 14.17±3. Catalase degrades hydrogen peroxide to water and oxygen and the glutathione peroxidase/reductase system catalyses the degradation of hydrogen peroxide and lipid hydroperoxide by using reduced glutathione.22* 30.20 38.01±2.15 39.21±1.26 14. and the staff of the Bioinformatics Center.25 37.68 15.63±0.57±3.30±0.26* 1.16 2. glutathione reductase and glutathione peroxidase could reflect the adverse effect of TCDD on the antioxidant enzymes in epididymal sperm.49±3.74±5.56±0.26 2.01* 2.65 67. the accumulated H2O2 has been shown to cause inactivation of superoxide dismutase (Sinet and Garber 1981).26±1. 1979).99±0.23 36.11±0.75±0.61* 2.41±4.C. considered significant against corresponding control superoxide anion to hydrogen peroxide (H2O2). References Aitken RJ. Data represent means±SD for four animals per dose TCDD (lg/kg body weight per day) 0.78±0. Aust SD (1976) Lactoperoxidase catalyzed lipid peroxidation of microsomal and artificial membranes. GRX.86±3.08* 56. In conclusion.62±1. and in addition to their direct effect on cellular constituents produce oxidative stress by decreasing the enzymatic defenses.14* 20.71±0.7. for a Junior Scholarship. Texas. The results have suggested that the graded doses of TCDD elicit depletion of the antioxidant defense system in the epididymal sperm and various regions of the epididymis.16±2. The increase in the levels of hydrogen peroxide and lipid peroxidation could reflect the adverse effect of TCDD on the membrane systems of the epididymal sperm and cauda epididymides of rats. Hargreave TB (1989) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.98±0.81±3.10±2.62±0.19 40.11±3.1 Caput epididymis SOD CAT GRX GPX H2O2 LPX SOD CAT GRX GPX H2O2 LPX SOD CAT GRX GPX H2O2 LPX 33. and P.74±3.52±0.11±1.30 64.87±3. catalase.80±4.19* Corpus epididymis Cauda epididymis *P<0.29* 30.14±2.48±0. GPX and H2O2.31±1.16 34.M.93* 2.04±1.68±3.86±2.29 36.61* 60.04±2.82±1.31* 10 26.11±1.22* 34. and as lmol/mg protein for LPX. for the generous gift of TCDD. Biochim Biophys Acta 444:192–201 .87* 17.13±1. The sperm membranes have been reported to undergo permeability changes following enhanced lipid peroxidation and glutathione depletion (Chance et al. acknowledges the Population Council.80* 3.17±0. the adverse effect of TCDD on the epididymal sperm was due to the direct effect of TCDD on the epididymis. New Delhi. Stephen Safe.38±3.91±0.98 62. Under high ROS concentrations pre-damaged spermatozoa are exposed to lipid peroxidation by polyunsaturated fatty acids (Ichikawa et al.36* 3.48±3.68* 3.C acknowledges Lady Tata Memorial Trust.61* 18.45±4. C. Catalase or glutathione peroxidase fails to eliminate H2O2 from the cell. It has been reported that a reduction in the activity of superoxide dismutase causes an increase in the level of superoxide anion.98±0.01* 24.50±2. K.13±0.48±0.24 36.8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities of superoxide dismutase (SOD).19* 30.68±2. Mumbai.16 13.87* 19.26 36.01* 2.047P-9/ICMC and B99.34±0.08 2.31±5.12 2.65 2.01 2.79* 3.71±0.42±5.26 33.46±2.01 2.11±2.08 61.

Oeda T. Toxicology 176:67–75 Latchoumycandane C. Mathur PP (2002b) Effect of methoxychlor on the antioxidant system of mitochondrial and microsome-rich fractions of rat testis. Shertzer HG (2002) Dioxin increases reactive oxygen production in mouse liver mitochondria. Raven Press. Mathur PP (2001) Effect of lindane on antioxidant enzymes in epididymis and epididymal sperm of adult rats. Kelce WR (1997) A dose-response analysis of the reproductive effects of a single gestational dose of 2. Sies H.8-tetrachlorodibenzo-p-dioxin on the antioxidant system in mitochondrial and microsomal fractions of rat testis. Chitra KC. Miyazaki T. Hum Reprod 6:987–991 Kono Y.7. A review. Stohs JJ (1988) 2. Randall RJ (1951) Protein measurement with the Folin phenol reagent. Genter MB. Bebert DW.3. Toxicol Appl Pharmacol 114:97–107 Marklund S. Mathur PP (2002c) The effect of methoxychlor on the epididymal antioxidant system of adult rats.7. pp 959–1000 .3.3. Moore RW.8-tetrachlorodibenzo-p-dioxin (TCDD). J Biol Chem 193:265–275 Mably TA. Abushaban A. Chitra KC.8-tetrachlorodibenzo-p-dioxin. Enan E (1998) Treatment of rats during pubertal development with 2. J Biol Chem 257:5751–5754 Latchoumycandane C. Ostby JS. Hum Toxicol 8:177–203 Stohs SJ (1990) Oxidative stress induced by 2. J Reprod Fertil 53:247–59 Ichikawa T. Teratology 56:403 Gray LE Jr.8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced lipid peroxidation in genetically responsive and non-responsive mice. Int J Androl 20:61–69 Hassoun EA.284 Carlberg I.8-tetrachlorodibenzo-p-dioxin (TCDD) in offspring of rats exposed during pregnancy and lactation.3.7.3. J Appl Toxicol 22:345–351 Latchoumycandane C. Lasley B (1998) Mechanisms of gender-specific TCDD-induced toxicity in guinea pig adipose tissue. Reprod Toxicol 16:161–172 Lowry OH. Marcel Decker. Mathur PP (2002a) The effect of 2. Asian J Androl 3:205–208 Chitra KC. Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Nozawa S (1991) Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Arch Toxicol 76:113– 118 Latchoumycandane C. Lasley B. Cell Immunol 59:301–318 Safe S (1995) Modulation of gene expression and endocrine response pathways by 2. Mathur PP (2002b) Induction of oxidative stress in the rat epididymal sperm after exposure to 2. lipid peroxidation and DNA damage in the hepatic and brain tissues of rats after subchronic exposure to mixtures of TCDD and its congeners. San Diego. Ohmori H. Fridovich I (1982) Superoxide radical inhibits catalase.3. Hutchinson RJ.7. Rosebrough NJ. Matsumura F. Wang S. Mathur PP (2002) Induction of oxidative stress in the epididymal sperm of rat after exposure to nonylphenol. Arch Toxicol 76:545–551 Cooper TG (1999) Epididymis. Merker HJ.7. Pharmacol Ther 67:247–281 Safe S (2001) Molecular biology of the Ah receptor and its role in carcinogenesis. Academic Press. New York. Iatropoulos (2001) Principles of testing for carcinogenic activity. Physiol Rev 59:527–534 Chitra KC.8-tetrachlorodibenzo-p-dioxin and related compounds. Mathur PP (2002a) Effect of vitamin E on reactive oxygen species mediated 2. Arch Toxicol 76:692– 698 Latchoumycandane C. Asian J Androl 3:135–138 Williams GM. Lannou DL (1997) Reactive oxygen species and human spermatozoa: physiology and pathophysiology. Chahoud I (1997) Effect on male fertility and testis concentrations of low dose of 2. Ligensa A. Int J Androl 22:37–42 Klinefelter GR. Overstreet J. vol 2. Mathur PP (2001) Effect of lindane on testicular antioxidant system and steroidogenic enzymes in adult rats. Chitra KC. Hess RA (1998) Toxicology of the male excurrent ducts and accessory sex glands. Monosson E. In: Knobil E. Peterson RE (1992) In utero and lactational exposure of male rats to 2. Keisari Y (1981) Superoxide anion and H2O2 production by chemically elicited peritoneal macrophages-induction by multiple nonphagocytic stimuli. Latchoumycandane C.3. Ostby JS. Marklund G (1974) Involvement of superoxide anion radical in autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Birnbaum LS (1995) Exposure to TCDD during development permanently alters reproductive function in male Long Evans rats and hamsters: reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Garber P (1981) Inactivation of human Cu–Zn superoxide dismutase during exposure to O2) and H2O2. Miller M. pp 553–591 Kobayashi T. Overstreet J. Stohs SJ (2001) Production of superoxide anion. Free Radic Biol Med 9:79–90 Sujatha R. Mannervik B (1975) Purification and characterization of the flavoenzyme glutathione reductase from rat liver. Latchoumycandane C. Neil JD (eds) Encyclopedia of reproduction. Toxicology 171:127–135 Latchoumycandane C. Farr AL. Li F. Dalton TP. Schill WB (1999) Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa. Greenberg M (1989) Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans: the risk to human health. Reprod Toxicol 12:357–369 Faqi AS.8tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat offspring.8-tetrachlorodibenzo-p-dioxin alters both signaling kinase activities and epidermal growth factor receptor binding in the testis and the motility and acrosomal reaction of sperm.7. epididymis and fertility.3. Sujatha R. Natori M. Dewhurst IC. Toxicol Appl Pharmacol 146:11–20 Griveau JF. Arch Biochem Biophys 212:411–416 Skene SA. Toxicol Appl Pharmacol 150:427–442 Enan E. Arch Environ Contam Toxicol 17:645–650 Paglia DE. In: Hayes AW (ed) Principles and methods of toxicology.7. In: Korach KS (ed) Reproductive and developmental toxicology. Eur J Biochem 47:469–474 Mohammadpour H. New York. Dalsenter PR. Valentine WN (1967) Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Toxicol Appl Pharmacol 131:108–118 Gray LE. Murray WJ. pp 1–17 El-Sabeawy F. J Lab Clin Med 70:158–169 Pick E.8-tetrachlorodibenzop-dioxin.7.3.8-tetrachlorodibenzop-dioxin toxicity in rat testis. Mathur PP (2002c) Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor. Kelce WR. Toxicol Lett 120:1–7 Senft AP.3. Latchoumycandane C. J Biol Chem 250:5474–5480 Chance B.7. J Appl Toxicol 21:211–219 Hess RA (1998) Effects of environmental toxicants on the efferent ducts. Toxicol Appl Pharmacol 178:15–21 Sharpe RM (1993) Declining sperm counts in men – is there an endocrine cause? J Endocrinol 136:357–360 Sinet PM. El-Sabeawy F.7. Chitra KC.