Arch Toxicol (2003) 77: 280284 DOI 10.

1007/s00204-003-0439-x

MOLECULAR TOXICOLOGY

C. Latchoumycandane K.C. Chitra 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 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 eect 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 Hams F-12 medium at 35C. 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 eects of TCDD on the epididymal sperm were due to direct eect of TCDD on epididymis.

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

Introduction
The exposure of humans to environmental contaminants that negatively aect 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 eects 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 signicant 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, 2831 July, 2002. C. Latchoumycandane K.C. Chitra 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

281

sperm production and cauda epididymal sperm counts (Gray et al. 1997; Faqi et al. 1997). There is also much concern that exposure to environmental contaminants induces major pathological eects in the epididymis of men and experimental animals (Hess 1998). The epididymis is responsible for the sustenance, protection, transport, maturations and storage of spermatozoa (Cooper 1999). Maturation of sperm within the epididymis requires interaction between the epididymal epithelium, the luminal uid and sperm (Klinefelter and Hess 1998). TCDD has been reported to produce the epididymis-specic decrease in cauda epididymal sperm counts (Mably et al. 1992; Gray et al. 1995). The epididymal (Mably et al. 1992) and ejaculated (Gray et al. 1995) sperm numbers were much greater than the observed decrease in testicular sperm of TCDD-treated rats, suggesting the epididymis-specic eect. Acute high-dose exposure to TCDD results in oxidative stress in multiple tissues and species (Mohammadpour et al. 1988). Induction of oxidative stress upon exposure to TCDD is considered to be an important mechanism for the toxic eects of TCDD (Stohs 1990; Latchoumycandane and Mathur 2002a; Latchoumycandane et al. 2002a, 2002b). TCDD has been reported to induce superoxide anion, lipid peroxidation and DNA damage in hepatic and brain tissues of rat (Hassoun et al. 2001). Inductions of oxidative stress in the mitochondrial and microsomal fraction of testis and in epididymal sperm (Latchoumycandane et al. 2002a, 2002b) and in mouse liver mitochondria (Senft et al. 2002) after exposure to TCDD have been reported. Excessive generation of reactive oxygen species (ROS) has been shown to cause peroxidative damage to the plasma membrane, which leads to impaired sperm function, and superoxide dismutase depletion is thought to be associated with sperm immotility (Kobayashi et al. 1997). However, the eect of TCDD on the epididymis remains unclear and the epididymal production of free radicals and the functions of antioxidant defense system require further studies. 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. 2001; Chitra et al. 2001; Latchoumycandane and Mathur 2002b, 2002c; Latchoumycandane et al. 2002c). In the present study, we have sought to investigate whether induction of oxidative stress in the epididymal sperm by TCDD was direct eect of TCDD on epididymis.

Animals and treatments Albino male rats of Wistar strain (90 days old) were obtained from the Central Animal House, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India. The animals were maintained under a well-regulated light and dark scheduled (12:12 h) with a temperature of 243C. The rats were fed with standard commercial pelleted feed and water ad libitum. TCDD was dissolved in acetone and olive oil (1:19) and administered orally at doses of 0.1, 1.0, or 10 lg/kg body weight per day for 4 days. Corresponding groups of animals were maintained and administered an equal volume of vehicle alone (acetone and olive oil) to serve as controls.

Necropsy and collection of epididymal sperm The animals were fasted overnight, weighed and killed using anesthetic ether on the day following the last dose. The left and right epididymides were removed and cleared from the adhering tissues and then weighed. The left cauda epididymis was weighed and used for sperm counts. The caput, corpus and cauda epididymides of right epididymis and epididymal sperm were used for biochemical assays. Epididymal sperm were collected by cutting the epididymis into small pieces in Hams F-12 medium at 35C (Latchoumycandane et al. 2002b, 2002c; Chitra et al. 2002). The tissue was washed several times in the same medium in order to remove maximum number of sperm from the epididymis.

Epididymal sperm count The epididymal sperm counts were performed by the method as described previously (Latchoumycandane et al. 2002b, 2002c). Briey, an aliquot of 5 ll of epididymal sperm was diluted with 95 ll of diluent (50 g sodium bicarbonate, 10 ml 35% formalin, and 0.25 g trypan blue were added and made up to a nal volume of 1 l with distilled water). A coverslip was secured to the counting chambers of the improved Neubauer-type hemocytometer. Approximately 10 ll of the thoroughly mixed diluted specimen was transferred to each of the counting chambers of the hemocytometer. The hemocytometer was allowed to stand for 5 min in a humid chamber to prevent drying out. The cells sedimented during this time and were then counted with the help of light microscopy at 200 magnication.

Biochemical studies For biochemical assays, the sperm suspensions obtained as described above were centrifuged at 800 g for 20 min at 4C. The pellet was resuspended in 2 ml normal saline and homogenized with the help of glassTeon homogenizer for a few seconds in the cold. The homogenate was centrifuged at 800 g for 20 min at 4C, and the supernatant was used for biochemical studies. A 1% homogenate of caput, corpus and cauda epididymides was prepared in cold normal saline using a glass Teon homogenizer. The homogenate was centrifuged at 800 g for 20 min at 4C, and the supernatant was used for biochemical estimations. Protein content was estimated by the method of Lowry et al. (1951). The levels of hydrogen peroxide (Pick and Keisari 1981) and lipid peroxidation (Buege and Aust 1976) were estimated in the epididymal sperm and caput, corpus and cauda epididymides of rats as described previously (Latchoumycandane et al. 2002a). The activities of superoxide dismutase (Marklund and Marklund 1974), 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. 2002a). All the data were analyzed using one-way analysis of variance followed by Duncans mul-

Material and methods

Chemicals TCDD was obtained as a gift from Dr. Stephen Safe, Department of Veterinary Anatomy and Public Health, A & M University, Texas, USA. Thiobarbituric acid and malondialdehyde were obtained from E. Merck (Darmstadt, Germany). All other chemicals were of analytical grade and obtained from local commercial sources.

282 tiple range test, and data were expressed as means SD for four animals per group. A P-value of less than 0.05 was considered as signicant against control.

Results
Administration of TCDD at the dose levels of 0.1, 1.0 or 10 lg/kg body weight per day for 4 days did not cause any signicant change (P>0.05) in the epididymal sperm counts compared with the corresponding counts in groups of control animals (data not shown). The production of hydrogen peroxide in the epididymal sperm increased signicantly (P<0.05) in the rats treated with TCDD when compared with the corresponding groups of control animals (Table 1). The activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase (Table 1) decreased signicantly (P<0.05) in the epididymal sperm of rats treated with TCDD at the dose levels of 1.0 and 10 lg/kg for 4 days, compared with those in the corresponding groups of control animals. Administration of TCDD to adult rats at the dose levels of 1.0 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). 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, whereas lower doses did not induce the production of hydrogen peroxide (Table 2). However, 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). Activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase (Table 2) decreased signicantly, while the levels of lipid peroxidation (Table 2) in the caput and corpus epididymis were found to be
Table 1 Eect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRX) and glutathione peroxidase (GPX), and the levels of hydrogen peroxide (H2O2) generation and lipid peroxidation (LPX) in the epididymal sperm of adult rats. The units are expressed as nmol/min per mg protein for SOD, GRX, GPX and H2O2, as lmol/min per mg protein for CAT, and as lmol/mg protein for LPX. Data represent meansSD for four animals per dose Parameter Control TCDD (lg/kg body weight per day) 0.1 SOD CAT GRX GPX H2O2 LPX 21.721.96 2.400.23 32.182.69 49.383.93 19.712.01 2.340.16 20.692.01 2.300.31 30.753.01 47.514.16 23.462.82* 2.710.26* 1.0 16.321.87* 2.110.26* 26.002.26* 43.213.01* 26.882.30* 2.890.23* 10 14.231.53* 1.890.16* 24.163.03* 41.034.06* 28.112.11* 2.990.27*

increased signicantly in the animals exposed to TCDD at the dose level of 10 lg/kg body weight per day for 4 days, compared with that in corresponding groups of control animals. However, administration of TCDD for 4 days at the dose levels of 0.1, 1.0 and 10 lg/kg body weight decreased the activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase while the levels of lipid peroxidation (Table 2) increased signicantly in the cauda epididymis of adult rats compared with those in corresponding groups of control animals.

Discussion
There is much concern that exposure to environmental contaminants induces major pathological eects in the epididymis in men and experimental animals (Hess 1998). 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. 1998). TCDD has been reported to produce an epididymis-specic decrease in cauda epididymal sperm counts (Gray et al. 1995). Induction of oxidative stress has been reported in the testis and epididymal sperm of rats (Latchoumycandane et al. 2002a, 2002b). In the present studies, we have sought to investigate whether induction of oxidative stress in the epididymal sperm was due to the direct eect of TCDD on epididymis. Rats were administered TCDD at doses of 0.1, 1.0 or 10 lg/kg body weight per day, 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. 1998). In order to see the direct of TCDD on epididymis we have administered TCDD for 4 days, which has been shown to be appropriate for observation of the direct eect of drugs on epididymis (Latchoumycandane et al. 2002c). The journey of sperm from the caput to the cauda epididymis takes approximately 4 days in rats. 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). In the present studies administration of TCDD increased the production of hydrogen peroxide in the epididymal sperm of adult rats. The activities of catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase were found to be decreased while the levels of lipid peroxidation increased signicantly in the epididymal sperm of rats. Similar biochemical changes were also noted in the caput, corpus and cauda epididymides of the TCDD-treated rats. However, the cauda epididymis showed signicant changes at all the dose levels whereas the caput and corpus epididymides did not show any signicant change at the 0.1 and 1.0 lg/kg dose levels. The antioxidant enzyme superoxide dismutase greatly accelerates the dismutation of the

*P<0.05, considered signicant against corresponding control

283 Table 2 Eect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRX) and glutathione peroxidase (GPX) and the levels of hydrogen peroxide (H2O2) generation and lipid peroxidation (LPX) in the caput, corpus and cauda epididymides of Tissue Parameter Control adult rats. The units are expressed as nmol/min per mg protein for SOD, GRX, GPX and H2O2, as lmol/min per mg protein for CAT, and as lmol/mg protein for LPX. Data represent meansSD for four animals per dose TCDD (lg/kg body weight per day) 0.1 Caput epididymis SOD CAT GRX GPX H2O2 LPX SOD CAT GRX GPX H2O2 LPX SOD CAT GRX GPX H2O2 LPX 33.873.14 2.630.19 40.493.30 64.085.16 13.861.42 2.560.29 36.413.08 2.750.21 38.502.62 60.804.82 14.041.20 2.780.31 40.412.98 2.990.22 39.682.65 67.315.61 13.111.26 2.630.16 34.162.81 2.520.15 39.462.98 62.745.26 14.131.03 2.620.25 37.813.16 2.670.23 36.163.11 61.454.03 15.042.01 2.910.26 36.573.01* 2.730.16* 33.022.61* 60.123.87* 17.261.62* 2.980.26* 1.0 32.683.01 2.480.20 38.743.08 61.425.11 14.821.40 2.710.24 36.173.12 2.630.23 36.012.91 59.712.68 15.621.65 2.980.26 33.414.01* 2.710.19* 30.102.08* 56.113.14* 20.311.36* 3.170.31* 10 26.712.93* 2.300.22* 34.142.91* 51.863.61* 18.211.80* 3.130.29* 30.483.61* 2.620.22* 30.862.46* 52.142.87* 19.111.79* 3.110.19* 30.063.60* 2.340.11* 26.112.14* 50.383.01* 24.242.68* 3.480.19*

Corpus epididymis

Cauda epididymis

*P<0.05, considered signicant against corresponding control

superoxide anion to hydrogen peroxide (H2O2). 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. The reduction in the activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase could reect the adverse eect of TCDD on the antioxidant enzymes in epididymal sperm. It has been reported that a reduction in the activity of superoxide dismutase causes an increase in the level of superoxide anion, which is known to inactivate catalase activity (Kono and Fridovich 1982). Catalase or glutathione peroxidase fails to eliminate H2O2 from the cell; the accumulated H2O2 has been shown to cause inactivation of superoxide dismutase (Sinet and Garber 1981). Griveau and Lannou (1997) reported that a ROS such as H2O2 appears to be a key agent in the cytotoxic eects in spermatozoa, and in addition to their direct eect on cellular constituents produce oxidative stress by decreasing the enzymatic defenses. 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. 1989). Under high ROS concentrations pre-damaged spermatozoa are exposed to lipid peroxidation by polyunsaturated fatty acids (Ichikawa et al. 1999). The sperm membranes have been reported to undergo permeability changes following enhanced lipid peroxidation and glutathione depletion (Chance et al. 1979). The increase in the levels of hydrogen peroxide and lipid peroxidation could reect the adverse eect of

TCDD on the membrane systems of the epididymal sperm and cauda epididymides of rats. 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, indicating TCDD-induced oxidative stress in the epididymis. This may lead to disruption in functional integrity of cell organelles and to the onset of sperm damage under TCDD-induced pathological conditions. Furthermore, cauda epididymis was more susceptible to TCDD-induced oxidative stress than the caput and corpus epididymides. In conclusion, the adverse eect of TCDD on the epididymal sperm was due to the direct eect of TCDD on the epididymis.
Acknowledgements The authors acknowledge Dr. Stephen Safe, Texas, USA, for the generous gift of TCDD, and the sta of the Bioinformatics Center, Pondicherry University for various facilities. C.L. acknowledges the Indian Council of Medical Research, New Delhi, for a Senior Research Fellowship; K.C.C acknowledges Lady Tata Memorial Trust, Mumbai, India, for a Junior Scholarship, and P.P.M. acknowledges the Population Council, New York for nancial assistance (Grant Nos. B99.047P-9/ICMC and B99.048R/ICMC).

References
Aitken RJ, Clarkson JS, Hargreave TB (1989) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 10:214220 Buege JA, Aust SD (1976) Lactoperoxidase catalyzed lipid peroxidation of microsomal and articial membranes. Biochim Biophys Acta 444:192201

284 Carlberg I, Mannervik B (1975) Purication and characterization of the avoenzyme glutathione reductase from rat liver. J Biol Chem 250:54745480 Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527534 Chitra KC, Sujatha R, Latchoumycandane C, Mathur PP (2001) Eect of lindane on antioxidant enzymes in epididymis and epididymal sperm of adult rats. Asian J Androl 3:205208 Chitra KC, Latchoumycandane C, Mathur PP (2002) Induction of oxidative stress in the epididymal sperm of rat after exposure to nonylphenol. Arch Toxicol 76:545551 Cooper TG (1999) Epididymis. In: Knobil E, Neil JD (eds) Encyclopedia of reproduction, vol 2. Academic Press, San Diego, pp 117 El-Sabeawy F, Wang S, Overstreet J, Miller M, Lasley B, Enan E (1998) Treatment of rats during pubertal development with 2,3,7,8-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. Toxicol Appl Pharmacol 150:427442 Enan E, El-Sabeawy F, Overstreet J, Matsumura F, Lasley B (1998) Mechanisms of gender-specic TCDD-induced toxicity in guinea pig adipose tissue. Reprod Toxicol 12:357369 Faqi AS, Dalsenter PR, Ligensa A, Merker HJ, Chahoud I (1997) Eect on male fertility and testis concentrations of low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in ospring of rats exposed during pregnancy and lactation. Teratology 56:403 Gray LE Jr, Kelce WR, Monosson E, Ostby JS, 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 ospring with normal androgenic status. Toxicol Appl Pharmacol 131:108118 Gray LE, Ostby JS, Kelce WR (1997) A dose-response analysis of the reproductive eects of a single gestational dose of 2,3,7,8tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat ospring. Toxicol Appl Pharmacol 146:1120 Griveau JF, Lannou DL (1997) Reactive oxygen species and human spermatozoa: physiology and pathophysiology. Int J Androl 20:6169 Hassoun EA, Li F, Abushaban A, Stohs SJ (2001) Production of superoxide anion, lipid peroxidation and DNA damage in the hepatic and brain tissues of rats after subchronic exposure to mixtures of TCDD and its congeners. J Appl Toxicol 21:211219 Hess RA (1998) Eects of environmental toxicants on the eerent ducts, epididymis and fertility. J Reprod Fertil 53:24759 Ichikawa T, Oeda T, Ohmori H, Schill WB (1999) Reactive oxygen species inuence the acrosome reaction but not acrosin activity in human spermatozoa. Int J Androl 22:3742 Klinefelter GR, Hess RA (1998) Toxicology of the male excurrent ducts and accessory sex glands. In: Korach KS (ed) Reproductive and developmental toxicology. Marcel Decker, New York, pp 553591 Kobayashi T, Miyazaki T, Natori M, Nozawa S (1991) Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Hum Reprod 6:987991 Kono Y, Fridovich I (1982) Superoxide radical inhibits catalase. J Biol Chem 257:57515754 Latchoumycandane C, Mathur PP (2002a) Eect of vitamin E on reactive oxygen species mediated 2,3,7,8-tetrachlorodibenzop-dioxin toxicity in rat testis. J Appl Toxicol 22:345351 Latchoumycandane C, Mathur PP (2002b) Eect of methoxychlor on the antioxidant system of mitochondrial and microsome-rich fractions of rat testis. Toxicology 176:6775 Latchoumycandane C, Mathur PP (2002c) Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor. Arch Toxicol 76:692 698 Latchoumycandane C, Chitra KC, Mathur PP (2002a) The eect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the antioxidant system in mitochondrial and microsomal fractions of rat testis. Toxicology 171:127135 Latchoumycandane C, Chitra KC, Mathur PP (2002b) Induction of oxidative stress in the rat epididymal sperm after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Toxicol 76:113 118 Latchoumycandane C, Chitra KC, Mathur PP (2002c) The eect of methoxychlor on the epididymal antioxidant system of adult rats. Reprod Toxicol 16:161172 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275 Mably TA, Moore RW, Peterson RE (1992) In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzop-dioxin. Toxicol Appl Pharmacol 114:97107 Marklund S, Marklund G (1974) Involvement of superoxide anion radical in autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469474 Mohammadpour H, Murray WJ, Stohs JJ (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced lipid peroxidation in genetically responsive and non-responsive mice. Arch Environ Contam Toxicol 17:645650 Paglia DE, Valentine WN (1967) Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158169 Pick E, Keisari Y (1981) Superoxide anion and H2O2 production by chemically elicited peritoneal macrophages-induction by multiple nonphagocytic stimuli. Cell Immunol 59:301318 Safe S (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol Ther 67:247281 Safe S (2001) Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol Lett 120:17 Senft AP, Dalton TP, Bebert DW, Genter MB, Hutchinson RJ, Shertzer HG (2002) Dioxin increases reactive oxygen production in mouse liver mitochondria. Toxicol Appl Pharmacol 178:1521 Sharpe RM (1993) Declining sperm counts in men is there an endocrine cause? J Endocrinol 136:357360 Sinet PM, Garber P (1981) Inactivation of human CuZn superoxide dismutase during exposure to O2) and H2O2. Arch Biochem Biophys 212:411416 Skene SA, Dewhurst IC, Greenberg M (1989) Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans: the risk to human health. A review. Hum Toxicol 8:177203 Stohs SJ (1990) Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Free Radic Biol Med 9:7990 Sujatha R, Chitra KC, Latchoumycandane C, Mathur PP (2001) Eect of lindane on testicular antioxidant system and steroidogenic enzymes in adult rats. Asian J Androl 3:135138 Williams GM, Iatropoulos (2001) Principles of testing for carcinogenic activity. In: Hayes AW (ed) Principles and methods of toxicology. Raven Press, New York, pp 9591000