Dr. P. P. Mathur Professor & Head Department of Biochemistry & Molecular Biology School of Life Sciences Pondicherry University, Pondicherry India Oct 28, 2006 IMPACT OF ENVIRONMENTAL TOXICANTS ON MALE REPRODUCTION AND PROTECTIVE ROLE OF ANTIOXIDANTS

2 nd APFA, Shanghai, Oct 28, 2006 (Nature, 375, 522, 1995)

(Carlsen et al., Br. Med. J., 305, , 1992) (Adami et al., Int. J. Cancer., 59, 33-38, 1994) 2 nd APFA, Shanghai, Oct 28, 2006

(Aitken et al., Nature, 432, 48-52, 2004) Incidence of testicular cancer has continued to rise whereas incidence of cancers of the female reproductive tract has remained constant (ovary, uterus) or has decreased (cervix). 2 nd APFA, Shanghai, Oct 28, 2006

(Aitken et al., Nature, 432, 48-52, 2004) 2 nd APFA, Shanghai, Oct 28, 2006

(Loder, N., Nature, 406, 4, 2000) 2 nd APFA, Shanghai, Oct 28, 2006

ENVIRONMENTAL TOXICANTS   INDUSTRIAL CHEMICALS (eg., polychlorobiphenyls, alkyphenols)   PESTICIDES (eg., DDT, methoxychlor, lindane)   PLASTICIZERS (eg., phthalates)   HEAVY METALS (eg., lead, cadmium) 2 nd APFA, Shanghai, Oct 28, 2006

(Said et al., Fertil. Steril., 82, , 2004) (Agarwal, A. & Prabakaran, S.A., Iranian J. Reprod. Med., 3, 1-8, 2005) 2 nd APFA, Shanghai, Oct 28, 2006

Can environmental toxicants affect the pro-oxidant/ antioxidant balance in male reproductive system? 2 nd APFA, Shanghai, Oct 28, 2006

  Studies were conducted on Methoxychlor, Lindane, Nonylphenol, 2,3,7,8-tetrachlorodibenzo-p-dioxin, Bisphenol A  In vivo studies were carried out using rat as an experimental model   The animals were exposed to differing doses of toxicants for varying duration to assess dose-dependent effects on testicular and epididymal antioxidant system. 2 nd APFA, Shanghai, Oct 28, 2006

  Determination of free radicals in tissues   Superoxide anion ( Prodczasy and Wei, 1988)   Hydrogen peroxide generation assay (Pick and Keisari, 1981)   Determination of antioxidant enzymes in tissues   Superoxide dismutase (Marklund and Maklund, 1974)   Catalase (Claiborne, 1985)   Glutathione reductase (Carlberg and Mannervik, 1975)   Glutathione peroxidase (Mohandas et al., 1984)   Lipid peroxidation (Buege and Aust, 1976) 2 nd APFA, Shanghai, Oct 28, 2006

  Significant alteration in the levels of antioxidant enzymes were observed in the crude homogenate of the testis of rats exposed to varying doses of methoxychlor, nonylphenol, lindane, TCDD and bisphenol A for 45 days. Mitochondrial & microsomal fractions of testis 2 nd APFA, Shanghai, Oct 28, 2006

  Important constituents of the cell and in case of the testis both play a crucial role in steroidogenesis   Mitochondria- production of ATP; therefore any impairment of mitochondrial functioning will result in impairment of tissue functioning. Mitochondrial & microsomal fractions of testis 2 nd APFA, Shanghai, Oct 28, 2006

Effect of environmental toxicants on mitochondrial & microsomal fractions of testis 2 nd APFA, Shanghai, Oct 28, 2006

(Latchoumycandane et al., Toxicology, 171, , 2002)  Exposure of pubertal rats to (  Exposure of pubertal rats to TCDD (1, 10 and 100 ng/ Kg body weight) for 45 days 2 nd APFA, Shanghai, Oct 28, 2006

Effect of TCDD on the levels of lipid peroxidation in mitochondrial & microsomal fractions of rat testis Effect of TCDD on the levels of hydrogen peroxide generation in mitochondrial & microsomal fractions of rat testis (Latchoumycandane et al., Toxicology, 171, , 2002) 2 nd APFA, Shanghai, Oct 28, 2006

( Latchoumycandane, C. & Mathur, P.P., Toxicology, 176, 67-75, 2002)  Exposure of pubertal rats to (1, 10 and 100 mg/ Kg body  Exposure of pubertal rats to methoxychlor (1, 10 and 100 mg/ Kg body weight) weight) for 45 days 2 nd APFA, Shanghai, Oct 28, 2006

Effect of environmental toxicants on steroidogenic enzymes and serum hormonal levels 2 nd APFA, Shanghai, Oct 28, 2006

TCDD-induced alterations in steroidogenic enzymes ParametersControl1 ng10 ng100 ng 3β-hydroxysteroid dehydrogenase ± ± 4.63*45.13 ± 2.68*45.16 ± 3.68* 17β- hydroxysteroid dehydrogenase ± ± ± 3.11*21.81 ± 3.68*  Exposure of pubertal rats to TCDD (1, 10 & 100 ng/ Kg bw) for 45 days 2 nd APFA, Shanghai, Oct 28, 2006

ParametersControl5 mg 3β-hydroxysteroid dehydrogenase ± ± 2.49* 17β-hydroxysteroid dehydrogenase ± ± 5.99* Lindane-induced alterations in steroidogenic enzymes  Exposure of adult rats to lindane (5mg/ Kg body weight/ day) for 30 days ( Sujatha et al., Asain J. Androl., 3, , 2001) 2 nd APFA, Shanghai, Oct 28, 2006

Lindane-induced alterations in serum hormonal levels 2 nd APFA, Shanghai, Oct 28, 2006

Lindane-induced alterations in serum hormonal levels 2 nd APFA, Shanghai, Oct 28, 2006

Protective role of Vitamin E on toxicant -induced oxidative stress in testis 2 nd APFA, Shanghai, Oct 28, 2006

(Latchoumycandane, C. & Mathur, P.P., J. Appl. Toxicol., 22, , 2002)  Exposure of pubertal rats to TCDD (1, 10 & 100 ng/ Kg bw for 45 days) with & without supplementation of vitamin E (20 mg/ Kg bw) 2 nd APFA, Shanghai, Oct 28, 2006

NONYLPHENOLNONYLPHENOL Reduction in the activities of SOD & Glutathione reductase + Increase in levels of lipid peroxidation No changes in the activities of antioxidant enzymes 1, 10 and 100 µg/ Kg bw 45 days Vit E (Based on Chitra, K.C. & Mathur, P.P., Ind. J. Exp. Biol., 42, , 2004) 2 nd APFA, Shanghai, Oct 28, 2006

Effect of environmental toxicants on epididymis 2 nd APFA, Shanghai, Oct 28, 2006

Effect of lindane on the activity of GPx in various regions of epididymis Effect of lindane on the activity of SOD in various regions of epididymis (Saradha, B. & Mathur, P.P., Environ. Toxicol. Pharmacol., 22, 90-96, 2006)  Exposure of adult male rats to lindane (1, 5, 50 mg/ kg body weight per day) for 45 days 2 nd APFA, Shanghai, Oct 28, 2006

Effect of lindane on the activity of catalase in various regions of epididymis Effect of lindane on the levels of lipid peroxidation in various regions of epididymis  Similar changes in the activities of antioxidant enzymes were observed in pubertal rats following exposure to methoxychlor (1, 10 & 100 mg/ Kg bw) for 45 days (Saradha, B. & Mathur, P.P., Environ. Toxicol. Pharmacol., 22, 90-96, 2006) 2 nd APFA, Shanghai, Oct 28, 2006

Effect of environmental toxicants on epididymal sperm 2 nd APFA, Shanghai, Oct 28, 2006

Effect of bishenol A on epididymal sperm motility of rats Effect of bishenol A on epididymal sperm count of rats (Chitra, et al., Toxicology, 185, , 2003)  Exposure of pubertal rats to bisphenol A (0.2, 2, 20 µg/ Kg body weight) for 45 days 2 nd APFA, Shanghai, Oct 28, 2006

Effect of methoxychlor on goat epididymal sperm in vitro (Gangadharan et al., Asian J. Androl., 3, , 2001) 2 nd APFA, Shanghai, Oct 28, 2006

Effect of bisphenol A on the activity of superoxide dismutase in the epididymal sperm of rats Effect of bisphenol A on the activity of glutathione reductase in the epididymal sperm of rats (Chitra et al., Toxicology., 185, , 2003 ) 2 nd APFA, Shanghai, Oct 28, 2006

Effect of bisphenol A on the levels of lipid peroxidation in the epididymal sperm of rats Effect of bisphenol A on the on the levels of hydrogen peroxide generation in the epididymal sperm of rats (Chitra et al., Toxicology, 185, , 2003) 2 nd APFA, Shanghai, Oct 28, 2006

(Gangadharan et al., Asian J. Androl., 3, , 2001) Effect of methoxychlor on goat epididymal sperm in vitro 2 nd APFA, Shanghai, Oct 28, 2006

Protective effect of vit C on methoxychlor-induced oxidative stress in goat epididymal sperm Co-incubation of sperm with methoxychlor and vitamin C (Gangadharan et al., Asian J. Androl., 3, , 2001) 2 nd APFA, Shanghai, Oct 28, 2006

Environmental toxicants- pseudosubstrate of CYPs of steroidogenic pathway Cytochrome P450 side-chain cleavage (P450scc) and 17α- hydroxylase/ 17,20-lyase (P450c17) Catalyze hydroxylation of the substrate using molecular oxygen and electrons denoted from NADPH Exogenous chemicals (act as pseudosubstrates) interact with substrate-binding site of CYP CYP- pseudosubstrate-O 2 complex oxygen-derived free radicals (due to the inability of the substrate to be hydroxylated) 2 nd APFA, Shanghai, Oct 28, 2006

ANTIOXIDANTROS Impairment of Spermatogenesis, Epididymal sperm maturation Inhibits steroidogenesis Decreased intratesticular testosterone Decreased activity of antioxidant enzymes Increased susceptibility of germ cells to ROS Apoptosis Lipid peroxidation ENVIRONMENTAL TOXICANTS 2 nd APFA, Shanghai, Oct 28, 2006

Acknowledgements Personnel   Latchoumycandane   Chitra K   Sujatha K   Bindhu Gangadharan   Arul Murugan M   Saradha B Financial support  Department of Science and Technology, DST-FIST  Population Council, New York  Indian Council of Medical Research, New Delhi  Lady Tata Memorial Trust, Mumbai 2 nd APFA, Shanghai, Oct 28, 2006