ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

FUNCTIONAL STUDIES ON THE EFFECTS OF 

2,3,7,8 - TETRACHLORODIBENZO-p-DIOXIN (TCDD) 

ON TESTIS AND EPlDlDYMlS OF RATS 

I HE \IS .SLIB.bf 17'J-E/) 7-0 'TI/?< f'O*VDI(. HEIIR I C ,VI l.ER.S/TI' 

FOR THE DEC;KEE OF 

DOCTOR OF PHILOSOPHY 

IN 

LIFE SCIENCES 

BY 

C. LATCHOUMYCANDANE 

SCHOOL OF LIFE SCIENCES 

PONDICHERRY UNIVERSITY 

PONDICHERRY -605 014 

JUNE - 2002

DECLARATION 

I hereby declare that the work presented in this thesis has been carried out by me 

under the supervision of Dr. P.P. Mathur, Professor, School of Life Sciences, Pondicherry 

University. To the best of my knowledge, no part of this thesis has been submitted for the 

award of a research degree of any other University. 

J 

c. i. Lwd-+'- 

(C. l,A~~CliO~lMY('ANl)ANl~) 

Candidate

ACKNOWLEDGEMENTS 

I wish to express my sincere gratitude to Dr. P. P. Mathur, Professor, School 

of Life Sciences, Pondicheny University, Pondicherry for his invaluable guidance, 

unceasing help, unerring supervision, critical suggestions, encouragement and the 

freedom with which I could work in his laboratory throughout the course of my 

research work. 

I express my sincere gratitude to Dr. E. Vijayan, Professor and Head, School 

of Life Sciences, Pondicherry University, Pondicheny for providing me laboratory 

facilities. My thanks are due to the other faculty members of my department Dr. B. 

Ka~abiran, Dr. A. Ramachandra Reddy and Dr. K. Srikumar, School of Life 

Sciences, Pondicherry University for their encouragement. 

My sincere gratitude to Dr. Stefen Safe, Department of Veterinary Anatomy 

and Public Health, A & M University, Texas, USA for the gift of TCDD without 

which I could not complete my work successfully. 

My sincere thanks to Dr. Dheer Singh, Scientist, National Dairy Research 

Institute, Karnal and Dr. R.R. Manimaran, Department of Pharmacology and 

Therapeutics. McGill University, Montreal. Canada, for their valuahlc suggcslions 

and encouragements. 

1 would also thank Dr. S.C. Parija, Officer in-Charge and other staff of the 

Central Animal House. JIPMER, Pondicherry for providing animals. 

I am highly thankful to Dr. P.P. Mathur, Officer in-Charge and Mr. M. 

Sundaramohan and Ms. V. Amoudha Staff of Bioinformatics Centre, Distributed 

Information Sub-centre, Pondicherry University, Pondicheny for providing me the 

necessary facilities for literature search and other help. 

I sincerely thanks to Ms. K.C. Chitra, Research Scholar, School of Life 

Sciences, Pondicheny University for her continuous help throughout my experiments. 

I also thank Ms. P. Poonkothai, Ms. R. Sujatha, Mr. M. Arul Murugan, Ms. Bindhu

Gangadharan and Ms. V. Bindhu Mol, M. Phil. Scholars, School of Life Scicnces, 

Pondicherry University for their cooperation in the laboratory. 

I would also thank Mr. V. Meibalan, Deputy Librarian, Central Library, 

Pondicherry University and Mr. R. Desingh, Research Scholar, School of Life 

Sciences, Pondicherry University, Pondicherry for their support and help in many 

ways. 

I wish to thank Mr. P. Ramalingam, Mr. K. Veeraragu and Mr. R. 

Jagajeevanram, Office Staff, School of Life Sciences for their cooperation and help in 

all aspects. 

The financial assistance by the Indian Council of Medical Research (ICMR), 

New Delhi in the form of a Senior Research fellowship is deeply acknowledged. 

Above all, I owe a deep sense of gratitude to my parents for their everlasting 

encouragement and support. 

c / Ck4L7 

(C. LATCHOUMYCANDAN :)

TABLE OF CONTENTS 

Page No 

ACKNOWLEDGEMENTS 

LIST OF TABLES 

LIST OF FIGURES 

LIST OF ABBREVIATIONS 

1 INTRODUCTION 

1.1 Description of male reproductive system 

1 .I. 1 Testis 

1 .1,1.1 Spermatogenesis 

1.1.1.2 Hormonal regulation of spermatogenesis 

1.1.1.3 Testicular steroidogenesis 

1.1.1.4 Hormonal regulation of steroidogenesis 

1.1.1.5 Paracrine regulation of testicular functions 

1.1.2 Structure and functions of the epididymis 

1.2 Target organ response to testosterone 

1.3 Effect of environmental contaminants on male reproductive 

system 

1.3.1 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCDD) 

1.3.1.1 Effect of TCDD on various organs 

1.3.1.2 Effect of TCDD on male reproduction 

1.3.1.3 Mechanism of TCDD action 

1.4 Effect of oxidative stress on male reproduction 

2 SCOPE OF THE PRESENT STUDY 

3 MATERIALS AND METHODS 

3.1 Animals 

3.2 Maintenance 

3.3 Chemicals 

i 

iii 

v 

viii

TABLE OF CONTENTS (Continud) 

Handling of TCDD and animals 

Treatments 

Group I : Administration of TCDD to pubertal rats 

Group 11: Administration of TCDD along with vitamin E 

Killing of animals 

Collection of serum 

Collection of tissues 

Organ weights 

Daily sperm production 

Collection of epididymal sperm and sperm function tests 

Sperm viability test 

Epididymal sperm motility 

Epididymal sperm counts 

Histometric studies 

Tubular and lumen diameter measurements 

Quantitative determination of serum hormone levels 

Quantitative determination of serum FSH 

Quantitative determination of serum LH 

Quantitative determination of serum prolactin 

Quantitative determination of serum testosterone 

Quantitative determination of serum estradiol 

Activities of steroidogenic enzymes 

Assay of 3P-hydroxysteroid dehydropnase 

Assay of 17P-hydroxysteroid dehydrogenase 

Determination of nucleic acids and protein contents in testis 

Extraction of nucleic acids fractions 

Determination of DNA 

Page No 

25 

25 

25 

26 

26 

26 

26 

27 

27 

27 

28 

28 

28 

29 

29 

29 

29 

3 1 

32 

34 

35 

36 

36 

37 

37 

37 

38

TABLE OF CONTENTS (Continued) 

Determination of RNA 

Determination of protein 

Subcellular fractionation of testis 

Preparation of tissue homogenates of epididymis 

Preparation of tissue homogenate of kidney 

Determination of free radicals/ reactive oxygen species in tissues 

Determination of superoxide anion 

Determination of nitric oxide 

Hydrogen peroxide generation assay 

Determination of antioxidants in tissues 

Estimation of reduced glutathione 

Estimation of a-tocopherol 

Estimation of ascorbic acid 

Determination of antioxidant enzymes in tissues 

Assay of superoxide dismutase 

Assay of catalase 

Assay of glutathione reductase 

Assay of glutathione peroxidase 

Estimation of lipid peroxidation 

Statistical analysis 

RESULTS 

Administration of TCDD to rats 

Effect of TCDD on body and organ weights 

Effect of TCDD on daily sperm production 

Effect of TCDD on epididymal sperm viability, motility and count 

Effect of TCDD on seminiferous tubular tlnd lunicn dianicters 

Effect of TCDD on serum hormone levels

TABLE OF CONTENTS (Cuntinucul) 

Effect of TCDD on the testicular sleroidogencsis 

Effect of TCDD on nucleic acids and protein contents in testis 

Effect of TCDD on the production of reactive oxygen species in 

the crude homogenate, mitochondrial and ~nicrosome-rich 

fractions of testis of rats 

Effect of TCDD on the levels of antioxidants in the crude 

homogenate, mitochondrial and microsome-rich fractions of testis 

of rats 

Effect of TCDD on the antioxidant enzymes and lipid peroxidation 

in crude homopennte. mitochondrial and microsomc-rich l'ractions 

of testis of rat 

Effect of TCDD on the epididymal sperm and epididymis 

Effect of TCDD on the production of reaclive oxygen species in 

the epididymal sperm of adult rats 

Effect of TCDD on the levels of antioxidants in the epididymal 

sperm of adult rats 

Effect of TCDD on the antioxidant enzymes and lipid peroxidation 

in the epididymal sperm of adult rats 

Effect of TCDD on the production of reactive oxygen spccies in 

the capuc. corpus and cauda epididymides of adult rats 

Effect of TCDD on the antioxidants in the caput, corpus and cauda 

epididymides of adult rats 

4.1 .I 1.6 Effect of TCDD on the antioxidant enzymes and lipid peroxidation 

in the caput. corpus and cauda epididymides of adult rats 

4.1.12 Effects of TCDD on the levels of reactive oxygen species 

production, antioxidants, antioxidant enzymes and lipid 

peroxidation in kidney 

4.2 Co-administration of TCDD and Vitamin E to Rats 

4.2.1 Eflbcl or co-administration oTT


Effect of co-administration of TCDD and vitaniin E on 

seminiferous tubular and lumen diamcter 

Effect of co-administration of TCDD and vitamin E on the levels of 

serum hormones 

Effect of co-administration of TCDD and vitamin E on thc 

testicular steroidogenesis 

Effect of co-administration of TCDD and vitamin E on nucleic 

acids and protein contents of testis 

Effect of co-administration of TCDD and vitamin E on the 

production of reactive oxygen species in the crude homogenate, 

mitochondrial and microsome-rich fractions of testis of rats 


antioxidants in the crude homogenate, mitochondria1 and 

microsome-rich fractions of testis of rats 


antioxidant enzymes and lipid peroxidation in crude homogenate, 

mitochondria1 and microsome-rich fractions of testis of rats 


epididymal sperm of adult rats 

Effect of co-administration of TCDD and vitamin I: on tlic 

production of reactive oxygen species in the epididymal sperm of 

adult rats 


antioxidant in the epididymal sperm of adult rat 


antioxidant enzymes and lipid peroxidation in the epididymal sperm 

of adult rats 


production of reactive oxygen species in the caput, corpus and 

cnudn cpididyniidcs of adnlt rats 

EHbct of co-administration of 'fCDD and vitamin Li on thc 

antioxidants in the caput. corpus and cauda epididyniidcs of adult 

rats 

El'f'ect of co-administration of I'CDD and vitamin li on thc 

antioxidant enzymes and lipid peroxidation in the caput. corpus and 

cauda epididymides of adult rats 

vii 

Page No 

56


viii 


reactive oxygen species production, antioxidants, antioxidant 

enzymes and lipid peroxidation of kidney of adult rats 

DISCUSSION 

Significance of the experimental procedures 

Experimental treatments 

Significance of the parameters studied 

Effect of TCDD on male rats 

Effect of TCDD on body and organ weights 

Effect of TCDD on daily sperm production 

Effect of TCDD on the epididymal sperm viability. motility and 

count 

Effect of TCDD on seminiferous tubular and lumen diameter 

Effect of TCDD on serum hormone levels and steroidogenic 

enzymes in testis 

Effect of TCDD on nucleic acid and protein contcnts in lestis 

Effect of TCDD on antioxidant system in testis 

Effect of TCDD on antioxidant systelii in the epididymal spcrm 

Effect of TCDD on antioxidant systems in the caput, corpus and 

cauda epididymides of rats 

Effect of TCDD on antioxidant system in kidney of rat 

Effect of co-administration of TCDD and vitamin E on male rats 

Effect of co-administration of TCDD and vitamin E on body and 

organ weights 

Effect of co-administration of TCDD and vitamin E on daily sperm 

productio~i, opididyniol sperm viahilily. 111olilily ;111d spcr111 COUlllh 

Effect of co-administration of I'CDD and vitamin C on 

seminiferous tubular and lumen dimcier 

Effect of co-administration of TCDD and vitamin E on serum 

hormone Levels and steroidoyenic cnzymcs in ~cstis

TAULIC OF CON'I'ISN'I'S (C'untinued) 

5.3.5 Effect of co-administration of TCDD and vitamin E on nucleic acid 

and protein contents in testis 

5.3.6 Effect of co-administration of TCDD and vitamin E on antioxidant 

systems in testis, epididymal sperm and epididymis 

5.3.7 Effect of co-administration of TCDD and vitamin E on antioxidant 

system in Kidney 

5.3.8 Epilogue 

6 CONCLUSIONS 

7 REFERENCES 

APPENDIX I 

I'REPARATION OF REAGENTS 

APPENDIX 2 

LIST OF PUBLICATIONS OF THE CANDIDATE

LIST OF TABLES 

Page No. 

Table la 

Table lb 

Table 2 

Tablc 3 

'Table 4 

Table 5 

Table 6 

'fablc 7a 

Table 7b 

Table 7c 

Table 8a. 

Table 8b 

Effect of TCDD on the body weight and weights of tlic 

testis. epididymis, kidney and accessory sex organs of adult 

male rats 

Effect of TCDD on the body weight and weights of the 

testis, epididyrnis, kidney and accessory sex organs @er 

100 g body weight) of adult male rats 

Effect of TCDD on the daily sperm production. epididymal 

sperm viability, sperm motility and sperm cou~its in adult 

male rats 

Effect of TCDD on the seminiferous tubular and lumcn 

diameter of testis of adult male rats 

Effect of TCDD on the serum hormone levels in adult male 

rats 

Effect of TCDD on the activities of steroidogenic enzymes 

in rat testis 

Effect of TCDD on the DNA, RNA and protein contents in 

testis of adult male rats 

Effect of TCDD on the production of superoxide anion in 

the crude homogenate, mitochondrial and microsome-rich 

fractions of testis of adult rats 

Effect of TCDD on the production of nitric oxide in the 

crude homogenate, mitochondrial and microsome-rich 

fractions of testis of adult rats 

Effect of TCDD on the hydrogen perox~de generation in 


fractions of rat testis 

Effect of TCDD on the level of glulnthiolic in thc crudc 

homogenate, mitochondrial and microsome-rich liactiol~?; 

of rat testis 

Effect of TCDD on the level of a-tocopherol in the crude 

homogenate, mitochondrial and microsome-rich fractions 

of rat testis

xi 

LIST OF TABLES (Continued) 

I'agc No. 

Table 8c 

Table 9a 

Table 9b 

Table 9c 

Table 9d 

Table 10 

Effect of TCDD on the levels of ascorbic acid in the crude 

homogenate. mitochondrial and microsome-rich fractions 

of rat testis 

Effect of TCDD on the activity of superoxide dismutase in 

the cmde homogenate, mitochondrial and microsome-rich 


Effect of TCDD on the activity of catalase in the crude 

homogenafe, mitochondrial and microsome-rich fractions 

of rat testis 

Effect of TCDD on the activity of glutathione reductase in 



Effect of TCDD on the activity of glutathione peroxidase in 



Effect of TCDD on the level of lipid peroxidation in the 

crude homogenate, mitochondrial and microsonie-rich 


Effect of TCDD on the production of s~lpcroxidc anion. 

nitric oxide and hydroycn pcl.oxide ill tlic epididym~ll 


Table 12 

Table 13 

Table 14 

Table 15 

Table 16a 

Effect of TCDD on the levels of antioxidants in the 


Effect of TCDD on the activities of antioxidant enzymes 

and the level of lipid peroxidation in the epididymal sperm 

of adult rats 

Effect of TCDD on the production of superoxide anion, 

nitric oxide and hydrogen peroxide in the epididymides of 

adult rats 

Effect of TCDD on the levels antioxidants in tho 



and the level of lipid peroxidation in the caput epididyniis 

of adult rats

LIST OF TABLES (Continued) 

Table 16b 

Table 16c 

Table 17a 

Table 17b 

Table 17c 

Table 18a 

Table 18b 

Table 19 

Table 20 

Table 21 

'l'ablc 22 

Table 23 


and the level of lipid peroxidation in the corpus epididymis 

of adult rats 


and the level of lipid peroxidation in the cauda epididymis 

of adult rats 

Effect of TCDD on the production of superoxide anion, 

nitric oxide and hydrogen peroxide in the kidney of adult 

male rats 

Effect of TCDD on the levels of antioxidants in the kidney 

of adult male rats 

Effect of TCDD on the antioxidant enzymes and lipid 

peroxidation in the kidney of adult rats 


body weight and weights of the testis, epididymis, kidney 

and accessory sex organs of adult male rats 


body weight and weights of the testis, epididymis, kidney 

and accessory sex organs (per 100 g body wciyht) of adult 

male rats 


daily sperm production, epididyrnal sperm viability, sperm 

motility and sperm counts in adult male rats 


seminiferous tubular and lumen diameter of testis of adult 

male rats 


serum hormone levels in adult male rats 

Effecl of co-adniinislration of TCDD and vitamin II on thc 

activities of steroidogcnic enzymes of rut [tstis 


DNA, RNA and protein contents in testis of adult male rats

Table 24a 

Table 24b 

Table 24c 

Table 25a 

Table 25b 

Table 25c 

Table 26a 

Table 26b 

Table 26c 

Table 26d 

Table 27 

LIST OF TABLES (Cuntinued) 

Page No. 


production of superoxide anion in the crude homopenate. 

mitochondrial and microsome-rich liuclions ol' tcstis of 

adult rats 8 1 


production of nitric oxide in the crude homogenate, 

mitochondrial and microsome-rich fractions of testis of 

adult rats 82 


hydrogen peroxide generation in the crude homogenate, 

mitochondria1 and microsome-rich fractions of rat testis 82 


level of glutathione in the crude homogenate, 



level of vitamin E in the crude homogenate, mitochondrial 

and microsome-rich fractions of rat testis 83 


levels of ascorbic acid in the crude homogenate, 

mitochondrial and microsome-rich fractions of rat testis 


activity of superoxide dismutase in the crude homogenate, 



activity of catalase in the crude hornogenate, mitochondrial 

and microsome-rich fractions of rat testis 

Effect of co-administration of TCDD and vitamin E on thc 

activity of glutathione reductase in the crude homogenate. 



activity of glutathione peroxidase in the crude homogenate, 



level of lipid peroxidation in the crude homoyenatc. 

mitochondria1 and microsome-rich fractions of rat testis 86

Table 28 

Table 29 

Table 30 

Table 3 1 

'Table 32 

Table 33a 

Table 33b 

Table 33c 

Table 34a 

Table 34b 

Table 34c 

LIS'I' OY 'I'AI5LICS (Cunlinucd) 


production of superoxide anion, nitric oxide and hydrogen 

peroxide in the epididymal sperm of adult rats 


levels of antioxidants in the epididymal sperm of adult rats 


activities of antioxidant enzymes and the level of lipid 

petoxidation in the epididymal sperm of adult rats 



peroxide in the epididymidcs of adult ruts 


levels antioxidants in the epididyrnides of adult rats 

Effect of eo-administration of TCDD and vitamin E on the 


peroxidation in the caput epididymis of adult rats 



peroxidation in the corpus epididymis of adult rats 



peroxidation in the cauda epididymis of adult rats 



peroxide in the kidney of adult rats 


levels of antioxidants in the kidney of adult rats 


antioxidant enzymes and lipid peroxidation in the kidney of 

ndult rats 

i'ayc No.

Fig. 1 

Fig. 2 

Effect of TCDD on the body weight gain of rats 

Correlation between sperm counts and DNA contents of rat 

epididymal sperm 

Body weight changes in the TCDD administered with 

various doses of TCDD 

Fig. 4a 

Fig. 4b 

Effect of TCDD or TCDD and vitamin E on the weight of 

the testis of adult rats 


the epididymis of adult rats 

Effect of 'SCDD or 'SCDD icnd vitamin E on thc wcigllt ol 

the seminal vesicles (intact) of adult rats 

Fig. 4d 

Fig. 4e 

Fig. 4f 

Fig. 5a 

Fig. 5b 

Fig. 6 

Fig. 7a 

Fig. 7b 

Fig. 7c 


the seminal vesicles (empty) of adult rats 


the ventral prostate of adult rats 


the kidney of adult rats 

Effect of TCDD or TCDD and vitamin E on daily sperm 

production and epididymal sperm counts of adult rats 

Effect of TCDD or TCDD and vitamin E on epididymal 

sperm viability and motility of adult rats 

Effect of TCDD or TCDD and vitamin E on seminiferous 

tubular and lumen diameter of adult rats 

Effect of TCDD or TCDD and vitamin E on serum FSH 

levels in adult male rats 

Effect of TCDD or TCDD and vitamin E on serum LH 

levels in adult male rats 

Effect of TCDD or TCDD and vitamin E on serum . 

prolactin levels in adult male rats

Fig. 7d 

I:iy. 7c 

Fig. 8 

Fig. 9 

Fig. IOa 

Fig. lob 

Fig. lla 

Fig. I lb 

Fig. I lc 

Fig. 12a 

Fig. 12b 

LIST OF FIGURES (Continued) 

Effect of TCDD or TCDD and vitamin E on serum 

testosterone levels in adult male rats 

Effect of' 'fCDD or TCDI) and vitaniin li on seru~ii 

estradiol levels in adult male rats 

Effect of TCDD or TCDD and vitamin E on the activities 

of testicular steroidoyenic enzymes of adult male rats 

Effect of TCDD or TCDD and vitamin E on DNA, RNA 

and protein contents in testis of adult male rats 

Effect of TCDD or TCDD and vitamin E on the production 

of superoxide anion in the cmde homogenate. 



of nitric oxide in the crude homogenate, mitochondrial and 

microsome-rich fractions of rat testis 


of hydrogen peroxide in the crude homogenate. 

rnitochondrial and microsome-rich fractions of rat testis 

Effect of TCDD or TCDD and vitamin E on the level of 

glutathione in the crude homogenate, mitochondrial and 


Effect of TCDD or TCDD and vitamin E on the level of a- 

tocopherol in the crude homogenate, mitochondrial and 



ascorbic acid in the crude homogenate, mitochondrial and 


Effect of TCDD or TCDD and vitamin E on the activity of 

superoxide dismutase in the crude homogenate, 



catalase in the crude hornogenate, m~tochondrial and 


Page No. 

119 

120 

121 

122

Fig. 12c 

Fig. 12d 

Fig. 13 

Fig. 14a 

Fig. 14b 

LIST OF FlGURES (Continued) 

Effect of TCDD or TCDD and vitamin E on the activity ol' 

glutathione reductase in the crude homogenate. 



glutathione peroxidase in the crude hornogenate. 



lipid peroxidation in the crude homogenate, mitochondrial 

and microsome-rich fractions of rat testis 


of superoxide anion in the epididymal sperm of adult rats 


of nitric oxide in the epididymal sperm of adult rats 

Page No. 

131 

Fig. 14c 

Fig. 15a 

Fig. 15b 

Fig. 16a 

I:ig. IOh 

Fig. 16c 

Fig. 16d 

Fig. 17 

Fib 18a 

Enict ol'l'CDD or 'l'CL)I) and vitamin I: 011 tlic ~-rrc~duclit~~i 

of hydrogen peroxide in the epididymal sperm ofadult rats 


glutathione in the epididymal sperm of adult rats 


of a-tocopherol in the epididyrnal sperm of adult rats 


superoxide dismutase in the epididymal sperm of adult rats 

EFCcct of TCDD or YCDD and vilaniin 1: on 11ic activity 01' 

catalase in the epididymal sperm ol'adult rats 


glutathione reductase in the epididymal sperm of adult rats 


glutathione peroxidase in the epididymal sperm of adult 

rats 


lipid peroxidation in the epididymal sperm of adult rats 


of superoxide anion in the caput. corpus and cauda 

epididyrnides of adult rats

Fig. 18b 

Fig. 18c 

Fig. 1Ya 

Fig. 19b 

Fig. 20a 

Fig. 2Ob 

Fig. 20c 

Fig. 2Od 

Fig. 21 

Fig. 22a 

Fig. 22b 

Fig. 22c 

Fig. 23a 



of nitric oxide in the caput, corpus and cauda epididymides 

of adult rats 


of hydrogen peroxide in the caput, corpus and cauda 



glutathione in the caput, corpus and cauda epididymides of' 

adult rats 

Effect of TCDD or TCDD and vitamin E on the level of a- 

tocopherol in the caput, corpus and cauda epididymides of 

adult rats 


superoxide dismutasc in the caput, corpus and cauda 



catalase in the caput, corpus and cauda epididymides of 

adult rats 


glutathione reductase in the caput, corpus and cauda 



glutathione peroxidase in the caput, corpus and cauda 


Effect of TCDD or TCDD and vitamin E on the levels of 

lipid peroxidation in the caput. corpus and cauda 


Effect of TCDD or I'CDD and vitamin B on the production 

of superoxide anion in the kidney ofadult rats 


of nitric oxide in the kidney of adult rats 


of hydrogen peroxide in the kidney of adult rats 


glutathione in the kidney of adult rats 

Page No. 

140


Fig. 23b Effect of TCDD or TCDD and vitamin E on the level of a- 

tocopherol in the kidney of adult rats 

Page No. 

151 

Fig. 24a Effect of TCDD or 'I'CDD and vitamin C on the uclivity 01' 

superoxide dismutase in the kidney of adult rats 

Fig. 24b 

Fig. 24c 

Fig. 24d 

Fig. 25 


catalase in the kidney of adult rats 


glutathione reductase in the kidney of adult rats 


glutathione peroxidase in the kidney of adult rats 


lipid peroxidation in the kidney of adult rats

LIST OF ABBREVIATIONS 

ABP 

Ah 

AhR 

ATPase 

BSA 

C 

CAMP 

CYP 

DDT 

DHT 

DNA 

DRE 

EDTA 

ELlSA 

ER 

ERKO 

FSH 

g 

R 

h 

GTP 

hCG 

HCH 

HRP 

IGF 

Ke 

L 

androgen binding protein 

aryl hydrocarbon 

aryl hydrocarbon receptor 

adenosine triphosphatase 

bovine serum albumin 

centigrade 

cyclic adenosine mono-phosphate 

cytochrome P450 

dichlorodiphenyl-trichloroethane 

dihydrotestosterone 

deoxyribonucleic acid 

dioxin response element 

ethylenediaminetetraacetic acid 

enzyme-linked immunosorbant assay 

estrogen receptor 

estrogen receptor knockout 

follicle stimulating hormone 

gram 

acceleration due to gravity 

hour 

guanosine tri-phosphate 

human chorianic gonadotrophin 

hexachlorocyclohexane 

horseradish peroxidasc 

insulin-like growth factor 

kilogram 

litre

LH 

M 

PL 

mg 

mRNA 

N AD 

NADH 

NADP 

NADPH 

nrn 

YO 

PCA 

PCB 

PCDD 

PCDF 

PModS 

I'U PA 

SD 

RNA 

ROS 

rPm 

TBA 

TCA 

'I'CL)l> 

TMB 

XRE 

luteinizing hormone 

molar 

microlitre 

milligram 

messenger ribonucleic acid 

nicotinamide adenine dinucleotide 

nicotinamide adenine dinucleotide (reduced) 

nicotinamide adenine dinucleotide phosphate 

nicotinamide adenine dinucleotide phosphate 

(reduced form) 

nanometre 

percentage 

perchloric acid 

polychlorinated biphenyls 

polychlorinated dibenzo-p-dioxins 

polychlorinated dibenzofurans 

peritubular modulatory substance 

polyunsaturi~~ud fiit~y acid?; 

standard deviation 

ribonucleic acid 

reactive oxygen species 

revolutions per minute 

thiobarbituric acid 

trichloroacetic acid 

2,3.7.B-Lctrachlorodihcnzo-/~-dic1xi1i 

tetramethylbenzidine 

xenobiotic rrsponsc elenlcnl

1 INTRODUCTION 

1.1 Description of male reproductive system 

Male reproductive system comprises a pair of testes, epididymides and 

accessory sex glands. Testes are encapsulated ovoid organs consisting of seminiferous 

tubules separated by interstitial tissue. Testis has two main functions, production of 

spermatozoa, which transmit male's gene to embryo and male sex hormone 

testosterone, which plays an important role in maintaining spcrnlatoycncsis. isccssory 

sex organs and secondary sexual characters. 

The posterior border of testis is associated with epididymis and with spermatic 

cord, the latter incorporating ductus deferens. together with neurovascular structures 

running to and from the middle and upper ends of posterior border of testis (de 

Kretser, 1982). Epididymis is a single highly convoluted duct, closely applied to the 

surface of the testis extending from the anterior to the posterior pole of testis. 

Epididymis acts as storage place for the spermatozoa where they acquire motility and 

capacity to fertilize the ova. 

Seminal vesicles are paired, bag-shaped glands and the internal surface 

consists of intricate system of folds to form irregular diverticula. Seminal vesicles 

secrete a viscous fluid, which is expelled along with sperm. it contains several 

essential nutrients which are required by the sperm for their fertilizing ability. 

Ventral prostate is a bilobed structure situated ventral to urethra. It has 

numerous small ducts through which the secretions are discharged directly into the 

urethra. The secretions are rich in nutrients and serve as lubricant for the semen 

(Setchell o ul., 1994). 

Testis is surrounded by a dense connectiv.: tissue. the tunica albuginea which 

is a tough fibrous capsule enclosing testicular parenchyma. It is composed of three

layers, an outer layer of visceral peritoneum, the tunica vaginalis, the tunica albuginea 

proper and on the inside the tunica vasculosa. Posteriorly funica albuginea is 

thickened and projects into the parenchyma of testis to form mediastenum, a 

honeycomb-like structure, which provides a passageway linking seminiferous tubules 

with efferent ductules and epididymis. 

Testis is made up of seminiferous tubules where the spermatozoa are formed. 

Senliniferous tubules owe their cylindrical shape to multiple layers of cellular and 

extracellular elements collectively known as the lamina propria. tunica propria, or 

peritubular tissue. Seminiferous tubules are two ended. convoluted loops with both 

ends opening into rete testis, through which sperm and fluid produced by the tubules 

pass on their way to excurrent duct systems. There are 30 tubules in rat testis (Dym. 

1976) with a diameter between 50 and 100 pm (Setchell, 1970; Wing and 

Christensen. 1982). Within the seminiferous tubules germ cells and somatic Sertoli 

cells make up the seminiferous epithelium, a complex stratified arrangement of germ 

cells. supported by tall columnar Sertoli cells. which rest upon the basement 

membrane and extend apically toward the lumen of the tubule (de Kretser er rrl. 

1995). 

Germ cells are highly synchronized in their proliferation and development to 

lorm a distinct cellular associations, establishing a precisely coordinated 'cycle of the 

seminiferous epithelium', believed to be regulated in part by Sertoli cells which do 

not divide in adult testis (de Kretser cr ul.. 1998; Orth. 1982). Walls of' the 

seminiferous tubules are composed of four layers of non-cellular materials surrounded 

by a layer of smooth muscle-like or myoid cells which are responsible for peristaltic 

movements of the tubules (Clermont, 1958). Interstitial tissue between seminiferous 

tubules is composed of clusters of Leydig cells, mesenchymal cells. macrophages. 

occasional mast cells, blood and lymph vessels (for review, see Johnson el al, 1999). 

The interstitial tissue is mainly composed of Leydig cells, which are the site of 

testicular steroidogenesis. Leydig cells have an abundance of smooth endoplasmic 

reticulum, many mitochondria with tubular cristae. Golgi complex, centrioles,

lysosome, peroxisome and number of lipid droplets (for review, see Johnson e! ul.. 

1999). Leydig cells are associated with blood vessels (Fawcett et 01.. 1973) or found 

nearer to the walls of the seminiferous tubules (Bergh, 1983). The presences of mast 

cells and macrophages have been reported in the interstitial compartment (Nistal el 

a/., 1984; Christensen et 01.. 1985). Sertoli cells are found immediately inside the 

boundary tissue of the seminiferous tubular and surrounded by undeveloped germ 

cells before puberty. The interstitial and seminiferous tubules compartments are not 

only structurally distinct but are also separated physiologically by cellular barrier, 

which develop during puberty and limit the free exchange of water-soluble materials 

(Johnson and Everitt, 1995). The plasma membranes of Sertoli cells and tight 

junctions between adjacent cells create adluminal compartment of the blood-testis 

barrier. Sertoli cells share the surface of the boundary tissue with the developing 

spermatogonia (Fawcett, 1975). Sertoli cells have intricate cytoskeleton and 

numerous processes, which are responsible for positioning, movement and shaping of 

the spermatogenic cells. Occluding junction between adjacent Senoli cells prevent 

free exchange of molecules from outside of the tubule into the spermatocytes a~d 

spermatids (de Kretser er ol., 1995). Sertoli cells have been reported to perform large 

number of specific functions including secretion of fluid, phagocytosis, maturation, 

release of spermatozoa and synthesis of intratubular androgen binding prorein 

(Chemes, 1986; Clermont el al. 1987). 

Spermatogenesis is the process of gradual transformation of germ cells into 

spermatozoa over an extended period of time within the boundaries of thc 

seminiferous tubules of testis. This process involves cellular proliferation by repeated 

mitotic divisions. duplication of chromosomes. genetic recombination through 

crossover and reduction division by meiosis to produce haploid spermatids, and 

terminal differentiation of sperrnatids into spermatozoa (for review, see de Kretser r/ 

ul.. 2000). Seminiferous tubules contain a large n~rmber of germinal epithelial cells 

called spermatogonia, located in two to three layers along the outer border of the

tubular epithelium and continually proliferate to replenish themselves. At the start of 

spermatogenesis, diploid spermatogonia proliferate producing three sub-populations 

of cells with markedly different destinies. One sub-population of spermatogonia are 

presumably identical to their progenitors and continue to function as stem cells and 

the majority of spermatogonia have been shown to enter a differentiative pathway to 

become spermatozoa (for review, see de Kretser er al., 2000). The stem 

spern~atogonia (type A) are located immediately adjacent to the basement membrane 

of the germinal epithelium. Type A spermatogonia exhibiting fine pale-staining 

nuclear chromatin and type B with coarse granules or more heavily stained chromatin 

associated with nuclear membrane and nucleolus (Clermont, 1972). 

'Type A 

spermatogonia enter a cycle and produce a chain of aligned undifferentiated 

spermatogonia, which differentiate into type A, spermatogonia. These cells undergo 

a sequence of six cell cycles and mitotic divisions resulting in the formation of A2, A>. 

A4 intermediates and finally into slightly more differentiated cells, the type B 

spermatogonia. After several divisions these cells give rise to very large primary 

spcrniatocytes (Steinberger and Steinberger, 1975). 

The primary spermatocytes undergo a long meiotic prophase and subsequently 

form secondary spermatocytes reducing the number of chromosomes (Steinberger and 

Steinberger, 1975; Weinbauer er a/., 1991). In the 2-stage process they dtvide into 

secondary spermatocytes and then spermatids. These spermatids undergo a precise 

process of morphological differentiation to form spermatozoa, which are released a1 a 

specific stage of development from the seminiferous epithelium into the lumen of the 

tubules. 

The temporal successions of all the spermatogenic stages called 

'spermatogenic cycle' have been reported in different species (Johnson rr 01.. 1999). 

The time taken from the division of type A spermatogonia to the release of 

spermatozoa has been reported to be approximately 50 days in rats and 64 days in 

men (Clermont and Harvey, 1965). In the mouse, the total duration of 

spermatogenesis from the stem cell to the mature spermatid is about 34.5 days 

(Monesi, 1965). The mitotic phase of spermatogenesis lasts about 8 days, meiosis 13 

days and spermiogenesis about 13.5 days (Monesi, 1965). The DNA synthesis in

apidly proliferating cells of testis reflects the initial phase ol' sper~iiiltoycncsis 

including spermatogonial proliferation by mitosis and the onset of meiotic prophase in 

prc-leptotcne primary spermatocylcs (Monesi, 1965). ItNA synthesis is highost 

during the mid pachytene phase and the end of the meiotic prophase and mRNA 

specific for structural protein increases during prophase of meiosis (Slaughter el 01.. 

1989). The rate of protein synthesis has been reported to be higher in type A 

spermatogonia than in type B (Monesi, 1965). Toxicants that would alter mitosis or 

differentiation of spermatogonia would be expected to have a major influence on the 

efficiency and total productivity of spermatogenesis. 

1.1.1.2 Hormonal control of spermatogenesis 

There is general agreement that for quantitatively normal spermatogenesis to 

occur, testis requires stimulation by pituitary gonadotrophins such as follicle 

stimulating hormone (FSH) and luteinizing hormone (LH). The progressive rise of 

FSH and LH during sexual maturation has been reported in humans and in other 

animals (for review, see de Kretser et ul., 1998). FSH and LH have been shown to 

stimulate seminiferous tubules and reinstate spermatogenesis in hypophysectomized 

rats (Greep and Fevold, 1937). Synergistic action of FSH and testosterone for the 

maintenance of spermatogenesis has also been reported (Steinberger. 1971). In the 

prepubertal rat, FSH alone is required to maintain testicular growth (Courot el al., 

1970) and synthetic activity of Senoli cells (Means et al., 1976). High intratesticular 

concentrations of testosterone are required for normal spermatogenesis. Testosterone 

has been shown to act by stimulating Senoli cells and receptors for FSH are located 

on the Senoli cells and spermatogonia. FSH has been shown to exert its action via 

CAMP. Injection of FSH is associated with a stimulation of protein kinase activity 

and protein synthesis. FSH has been shown to play a role in stimulating mitotic and 

meiotic DNA synthesis in type B spermatogonia and pre-ieptotene spermatocytes as 

well as in preventing apoptosis of pachytene spermatocytes and round spermatids 

(Henricksen el ul.. 1996: Shetty el ul.. 1996). Mc lachlnn el ol. (1995) detnonstrntod 

that FSH alone could partially restore spermatogenesis in GnRH-immunized rats.

lmn~unization of monkeys against FSH either actively or passively has been shown to 

be associated with disruption of fertility and a decline in sperm concentrations. It has 

been reported that the specific spermatogonial subtypes are affected by withdrawal of 

FSH alone (Moudgal et al., 1997). FSH has been shown to promote spermatogenesis 

and fertility in primates (Moudgal and Sairam, 1998). 

Administration of highly 

purified FSH to GnRH antagonist treated monkeys has been shown to niaintnin 

spermatogonial numbers (Weinbauer el a/., 1991). Actions of FSI-I on Sertoli cells 

from prepubertal animals have been reported biochemically by increased estradiol 

production (Fritz et al., 1976). It has been reported that FSH P-subunit gene knocked 

out mice. rendering them FSH deficient showed that spermatogenesis could proceed 

to completion albeit in the presence of smaller testes (Kumar et al., 1997). The 

binding of LH to Leydig cells in the interstitial compartment of testis has been 

reported to stimulate testosterone synthesis. 

Initiation of spermatogenesis by testosterone and dehydrotestosterone has been 

reported in mouse (Singh el al.. 1995). 

Testosterone alone has been shown to 

maintain spermatogenesis qualitatively in a dose-dependent fashion in monkeys 

(Weinhnuer el (11.. 1988). 

A high concentration of tcstosteronc within thc 

seminiferous tubules LH has been shown to play a role in the initiation and 

maintenance of sperm production in mouse (di Zerega and Sherins, 1980; Matsumota 

and Bremmer. 1987). 

In rodents and primates, by virtue of its local production. 

intratesticular testosterone levels have been shown to be elevated approximately 100 

fold above those in serum (Huhtaniemi er al., 1987). 

A role for estrogens in normal spermatogenesis has also been reported (Hess ef 

ul.. 1997) and at least two major steps have been shown to be influenced by estrogens 

in the seminiferous tubules, stem cell number and spermatid maturation (Hess cr ul., 

1997). It has been shown that in virro multiplicntion of rat gonocytes is controlled by 

growth factors and estradiol, which corresponds well with the presence of ERP in 

these cells (Hess et a!., 2000). 

In estrogen receptor a knockout (ERKOa) mouse 

estrogens are necessary for the achievement of fertility (Hess et al.. 2000).

1.1.1.3 Tcsticular steroidogcnesiv 

Testis secretes a variety of steroids which are synthesized from cholesterol 

which itself can be synthesized from acetatc. Thc principal sccrctory stcn)id product 

in male is testosterone of which 95% is synthesized in the Leydig cells and the 

remainder being produced by the adrenal glands. Cholesterol is derived from two 

sources, consisting of an uptake mechanism by which circulating low-density 

lipoprotein bind to specific receptors on Leydig cells or by internalization. that 

provide n ready source ol'cholcstclol. It1 rat I.cydig cells. receptors lir I~igli-ilc~lsily 

lipoprotein have also been reported (Chen el al., 1980). Cholesterol has been shown 

to get converted into pregnenolone in the testicular tissues by successive oxidations 

under the influence of C20-C22 desmolase complex (Cytochrome P-450) in 

mitochondria in the presence ofNADPH and oxygen (Burstein and Gut, 1971). 

Tcsticular homogenates of rat has been shown to convert pregnenolone more 

to progesterone than that of 17-hydroxypregnenolone. Incubation of labeled 17- 

hydroxypregnenolone has been shown to produce dehydroepiandrosterone instead of 

17-hydroxyprogesterone (for review, see Hall, 1988). Existence of an alternative 

pathway of conversion of 17-hydroxypregnenolone to androstenediol 

withor~t 

involving dehydroepiandrosterone or androstenedione has also been reported 

(Slaunwhite and Burgen, 1965). Formation of A~') ketosteroids such as progesterone. 

17-hydroxyprogesterone, androstenedione and testosterone from A'" P- 

hydroxysteroids in the presence of 3P-hydroxysteroid dehydrogenasel A~~ isomerase 

has been reported (Hall, 1970; Eik-Nes, 1975). Progesterone has been shown to get 

convened into 17-hydroxyprogesterone, androstenedione and testosterone by 17- 

hydroxylase, 17-20 lyase and 17P-hydroxysteroid dehydrogenase respectively (Hall. 

1970, Eik-Nes, 1975). Progesterone has also been shown to get converted into 

17-hydroxyprogesterone, androstenedione and testosterone in the incubation of slices 

or homogenates of testis of hypophysectomized or immature rats treated with human 

chorionic gonadotrophin (hCG) to increase the volume of the interstitial cells 

(Slaunwhite and Samuels, 1956). Testis has the capacity to produce 17p-estradiol,

and this reaction is catalyzed by the enzyme cytochrome P450 aromatase (for review, 

see Hall, 1988; Carreau et al., 1999). This conversion involves a series of reactions 

resulting in hydroxylation at C3. Testis has also the capacity to secrete small amounts 

of dihydrotestosterone and the enzyme catalyzing the conversion of testosterone to 

dihydrotestosterone is 5 a-reductase (Carreau, 2000). 

1.1.1.4 Hormonal regulation of steroidogenesis 

Testosterone production in testis is controlled by LH through the specific 

receptors on the surface of the Leydig cells. The LH receptor has been isolated and 

characterized (de Kretser et al., 1971). Considerable evidence indicates that 

interactions of LH with its receptor activate the CAMP pathway through a GTP 

binding protein (Hall, 1988; Dufau et al.. 1978). Testosterone production has been 

shown to get stimulated when decapsulated testis of adult rats is incubated with LH or 

hCG (Rommerts er al.. 1972). Stimulation of androgen production has been reported 

when isolated Leydig cells of rats are incubated either with LH or hCG (Qazi er al.. 

1974). Increase in the production of testosterone and de novo protein synthesis in 

Leydig cells of adult rats revealed a strong correlation with the dosage of LH (Janszen 

et al., 1978). 

Activity of A' 3P-hydroxysteroid dehydrogenase has been shown to respond to 

hCG in neonatal interstitial cells in culture (Meidan cr a/. 1985). Stimulation of 36- 

hydroxysteroid dehydrogenase activities in testes of i~lirnaturc hypophyscctol~liscd 

rats has been shown by the administration of hCG in vivo (Murono and Payne, 1979). 

Suppression of the activities of 7-hydroxylase and 17P-hydroxysteroid dehydrogenase 

has been reported in the rat testis after administration of hCG or testosterone (Inano el 

rtl.. 1973). lmplanta$ion of testosterone-estradiol filled silastic capsules in rats caused 

a reduction in the volume of smooth endoplasmic reticulum in the Leydig cells and 

the ability of the testis to secrete testosterone (Ewing er al.. 1983). 

Ascorbic acid has been shown to play an important role in the biosynthesis of 

testicular steroids (Luck et a/., 1995). Testosterone could regulate the rnorphogenic

and steroidogenic processes as Leydig and precursor cells have been shown to contain 

receptors for androgen (Verhoeven, 1980). Testosterone secretion in the prepubertal 

rats reduced following treatment with an androgen receptor antagonist (Puwis and 

Hansson, 1978). Androgen receptor mRNA and protein levels are reported to be 

lower in adult rather than immature Leydig cells, which might result from down 

regulation exerted by the pubertal rise in androgen production (Shan and Hardy, 

1992). Prolactin has been shown to increase the number of LH receptors and could 

potentiate steroidogenic effect of LH on Leydig cells and testicular prolactin receptors 

have been shown to be confined to the interstitial tissue of the testis (Johnson and 

Everitt, 1995). Prolactin also increases the uptake of androgen and increases 

Sa-reductase activity (Johnson and Everitt, 1995). 

1.1.1.5 Paracrine regulation of testicular functions 

Leydig cells, Sertoli cells, germ cells and peritubular myoid cells have been 

shown to interact with each other in order to maintain testicular functions. 

Cryptocrine communications are reported between germ cells and Sertoli cells in the 

seminiferous tubules (Funder, 1990). It has been reported that stimulatory effect of 

Sertoli cells on germ cells mediated by diffusible factors and to obtain full expression 

of these stimulation germ cells must be in contact with Sertoli cells (Rivarola er ul., 

1985). Seminiferous tubules have been shown to secrete a meiosis inducing 

substance and its secretion is maximal at stage VIIc-d of the spermatogenic cycle, just 

before the onset of premeiotic DNA synthesis in preleptotene spermatocytes 

(Parvinen, 1982). Regulation of Sertoli cell function by germ cells has been shown in 

in vivo and in virro studies (Saez ef al., 1991). Regulation of FSH and testosterone on 

spermatogenesis is reported to be mediated by Sertoli cells, which contain FSH and 

androgen receptors (Tindall er al., 1977). Sertoli cells synthesize and secrete 

transport proteins, growth factors that are regulated by FSH and also modulate by 

local mechanisms related to the spermatogenic cycles. The nutrient and growth 

factors required for spermatogenesis was shown to be transported from the

extratubular environment or synthesized by the Sertoli cells and delivered to the 

developing germinal cells within the tubule (Skinner, 1987). 

Germ cells require lactate for swival but they are unable to metabolize 

glucose (Jutte el al., 1982), this energy substrate can be supplied by Sertoli cells 

which are able to transform glucose to lactate, a process stimulated by FSH (June er 

al., 1983) and insulin-like growth factor I (IGF-I) (Mita and Hall, 1982). Lactate and 

pyruvate secretions by Sertoli cells have been reported to regulate and maintain 

spermatogenesis (Grootegoed and de Boer, 1989). Degeneration of germ cells has 

been reported due to the absence of proper substrate required for their metabolic 

activiries (June el al.. 1981). It has been reported that human Sertoli cells cultured 

with FSH and testosterone have been reported to produce lactate, estradiol and 

transfemn (Foucault el a[., 1992). 

Sertoli cells have been shown to synthesize and secrete transport proteins and 

growth factors (Bardin el d.. 1988; Ritzen el al.. 1989). which are regulated by FSH. 

Several Sertoli cell secretory products have now been identified which include 

transferrin, androgen binding protein, ceruioplasmin, plasminogen activator, clusterin 

(sulfate glycoprotein-2). a2 macroglobulin, testin sulfated glycoprotein-I, testiburnin 

and others (for review, see Mruk and Cheng, 2000a). The stimulatory effects of 

Sertoli cells on germ cells have been reported which are mediated either by cell-cell 

contacts (Russell, 1980) and! or by diffusible factors (Rivarola er a!.. 1985). Sertoli 

cells have been reported to secrete several growth factors including IGF-I (Cailleau el 

01.. 1990), TF-a (Skinner et al.. 1989). TGF-P (Skinner and Moses, 1989). FGF 

(Smith el al., 1989) and seminiferous growth factor (Bellve and Zheng, 1989). Germ 

cells have been shown to regulate biosynthesis of rat Sertoli cell clusterin. a:- 

macroglobulin and testin (Grima et al., 1992). Peritubular cells have been shown to 

enhance the production of a Leydig cell stimulatory factor when co-cultured with 

Senoli cells (Verhoeven and Cailleau. 1986). Peritubular cells have been reportcd 10 

secrete a factor PMod-S, which stimulated secretion of Sertoli cell proteins (Skinner 

el al., 1988). PMod-S production was reported to be under androgen regulation

(Skinner and Fritz. 1985) and provided a potential mode of action for androgens to 

regulate Sertoli cell function indirectly (Skinner, 1987). Supprcssion of scrum 131 I 

concentration has been achieved following inhibin administration to castrated rats 

suggesting the feedback regulation of FSH secretion by inhibin (Robertson el ul.. 

1991). Activin has been shown to stimulate LH release and circulating FSll 

concentration in macaques and rats in vivo (Schwall er al, 1989; McLachlan el al., 

1989). Leydig cells have been shown to secrete oxytocin-like immunoreactive 

material to influence the contractibility of the seminiferous tubules by acting on 

peritubular cells (Worley et al.. 1984). 

Rat germ cells co-cultured with rat Sertoli cells produce an inhibited RNA and 

DNA synthesis of Sertoli cells (Rivarola el al.. 1985) as well as an increased secretion 

of androgen binding protein (ABP). transferrin and inhihin and an inhihition 01' Scrtoli 

cell aromatase activity (Le Magueresse and Jegou, 1988). The number of factors 

implicated in paracrine regulation of testicular functions has been steadily increasing 

and many factors have been shown to be of potential or proven physiological 

significance (for review, see Spiteri-Grech and Nieschlag, 1993; Griswold, 1993). 

1.1.2 Structure and functions of epididymis 

Iipididymis is composed of a single, long. highly coiled duct closcly applied to 

the surface of the testis and embedded in a variahle amount of adiposc tissue of 

epididymal fat pad. It has been reported that mammalian epididymis. histologically 

and functionally, can be divided into initial and middle segments where sperm 

maturation takes place and a terminal segment, which subserves the function of sperm 

storage (for review. see Robaire er 01.. 2000). Most abundant cell type found in the 

epididymal epithelium is the principal cell, which has prominent stereocilia that 

extend into the lumen. In rats, principal cells constitute 80% of the total epithelial cell 

population in the initial segment and this number gradually decreases to 65% in the 

cauda epididymis (Robairr and Hermo. 1988; Jones. 1999). Large numbers of 

proteins arc secreted in the epididymis and the anterior par? of it has been shown to be

the most active in protein secretion in rats and boer (Brooks, 1981; Syntin cl ul., 

1996). High levels of several proteins that mediate antioxidant action are found in 

epithelial cells and in epididymal fluid, which may protect epithelial and/ or sperm 

cells against oxidative stress from exposure to endogenous reactive oxygen species or 

exogenous toxic substances (Hinton er al., 1995; Jones, 1999). Expression of 

secretory antioxidant factors has been shown to be region specific, indicating that the 

need for antioxidant enzymes may vary along the epididymis. Some of the other 

major proteins secreted by the epididymal epithelium possess carrier functions for thc 

transport of retinol (Porter er ul., 1985) or of cholesterol (Baker el ul., 1993; KirchhofS 

cr 01.. 1998). 

Epididymis has been shown to be dependent on androgens secreted into the 

peripheral circulation as well as other factors that are secreted directly into the 

epididymal lumen from the seminiferous tubules. rete testis and efferent ducts (for 

review. see Robaire er 01.. 2000). There is a time dependent dramatic weight loss of 

the epididymis after androgen withdrawal and has been attributed to the loss of 

spermatozoa and fluid from the lumen of the epididymis and in part due to changes in 

the epithelium of the tissue (Klinefelter and Hess, 1998). Apoptotic cell death after 

orchidectomy has been shown to appear first in the epithelium of the initial segment 

ol' the rpididymis and subsequently in more distal segments (for review, src Robaire 

rr ul.. 2000). The main androgen responsible for maintaining epididymal structure 

and functions is the 5 a-reduced metabolites of testosterone. dihydrotestostcronc 

(DHT). The rate-limiting enzyme in the pathway leading from testosterone to its 5 a- 

reduced metabolites is 4-ene steroid 5 a-reductase (Hermo er a/., 1988). In the rat. 

the levels of dihydrotestosterone have been shown to be approximately 10 fold higher 

than those of testosterone in the caput and corpus epididymis, but the levels of these 

androgens are similar in the cauda epididymis (Turner cr at.. 1984). l'estosteronc 

entering the epididymis via the lumen is associated with transport of androgen binding 

protein (ABP) from seminiferous tubule to the epididymis (Danzo, 1995). In rat. 

epithelium in cauda epididymis is characterized by the presence of numerous clear 

cells. These cells have been shown to be responsible for the phagocytosis of

cytoplasmic droplets shed from sperm dwing epididymal transit (Henno er 01.. 1988). 

The journey from the testis to the cauda epididymis has been shown to take 

approximately 4 days in rats (Klinefelter and Hess, 1998). 

Estrogen has also been shown to play an important role in fluid reabsorption in 

efferent ductulcs and epididymis of rats (Hess et a[., 1997; Hess et al., 2000). Effect 

of estrogen on the epididymis can be viewed either as indirect or direct. since 

estradiol is a potent suppressor of LH secretion. Administration of estradiol results in 

a decrease in gonadotrophins and consequently in a shutdown of Leydig cell function, 

and hence androgen production. The presence of aromatase activity has been reported 

in spermatozoa entering the epididymis (Janulis et al., 1998) and of estrogen receptors 

in epididymal principal cells (Hess et al., 1997). Estrogen has been shown to cause 

reduction in epididymal sperm number and motility in adult male rats (Kaneto er al., 

1999). 

1.2 Target organ response to testosterone 

Accessory sex organs in male have been reported to be androgen dependent 

and thus reflect the availability of androgens (Hunt el al., 1978). Weights of the 

accessory sex organs in castrated male rats have been widely used as a bioassay for 

androgenic and antiandrogenic compounds (Neumann and Steinbeck, 1974). 

Weights. sizes, cytological structures and mitotic activity of the seminal vesicles of 

mouse were established as indicators of bioassay for androgenic hormones (Deanesly 

and Parkes, 1933). Measurement of weights of the accessory sex organs in intact rats 

has been shown to reflect the estimation of cumulative effect of biologically active 

testosterone over a period (Mathw and Chattopadhyny. 1982). 

The sensitivity of the accessory sex organs varied from organ lo orgun 21s 

shown by the requirement of androgen for seminal vesicles being 3 times more than 

that of prostate (Moore and Gallagher. 1930; Burkhart. 1942). The maintenance of 

structural and functional integrity of prostate gland has ken reported to be dependent 

upon the amount of circulating androgen pr$nccd by the testis (Butler and Schode,

1958). A rapid fall in serum testosterone levels following bilateral orchidectomy has 

been shown to caw prostate involution (Bruchovsky cl ul., 1975; Lee, 1981 ). Whilc 

replacement of androgen exogenously has been to stimulate the prostate growth 

(Bruchovsky er 01.. 1975). 

A remarkable increase in total weight, contents of water, inorganic ions and 

metabolic activity of seminal vesicles was induced by subcutaneous injection of 

testosterone propionate to castrate rats (Rudolph and Samuel, 1949). Kidneys were 

shown to have potential to respond to androgen stimulation for growth and metabolic 

activities similar to accessory sex organs (Kochakian, 1977). Kidneys of the castrated 

mice decreased in size by 30 to 50% in comparison to that of the intact animals. 

Treatment with testosterone propionate restored the weights of the kidney to normal 

range in the rats treated with cisplatin (Malarvizhi and Mathur, 1996). Testosterone 

has been shown to stimulate compensatory hypertrophy of the kidney and ameliorate 

renal dysfunction (de Ruiter, 1981). Androgen has been shown to produce a two to 

five fold increase in the specific activity of the enzyme in mouse kidney (Bardin el 

01. 1978). 

1.3 Effect of environmental contaminants on male reproductive system 

A large number of environmental contaminants have been shown to induce 

reproductive abnormalities both in wildlife and humans (Carlsen el al., 1992; Shape, 

1993). Mean sperm counts in men have been shown to decline in the past fifty years 

(Carlsen el 01.. 1992). It has been hypothesized that environmental exposure to 

synthetic estrogenic chem~cals nnd related endocrine octivc compounds may bc 

rcsponsible for a global decrease in spcrnm counts and decreased male rcproduclivc 

capacity (Fisher el at., 1999; Safe. 2000). The organochlorine compounds comprise 

dichlorodiphenyl-trichloroethane (DD'T) and metabolites. hcxacl~lorocyclol~exill~c 

(HCH). polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs) 

and polychlorinated dibcnzo-p-dioxins (PCDDs). Due to their lipophilicity they 

preferentially bio-accumulate in the adipose. tissue. The presence of' DI)T and

metabolites. HCH, PCBs and PCDDs has been reported in the fluids of human 

reproductive tract, such as seminal fluid, cervical niucus and I'olliculi~r fluid (li~r 

review, see Pflieger-Bruss and Schill, 2000). 

Changes in male reproduction of 

wildlife by environmental contaminants involve feminization, demasculinization. 

reduced fertility, reduced hatchability, reduced viability of the offspring, impaired 

hormone secretion or activity and altered sexual behavior (for review, see Colborn et 

al.. 1993). 

A large number of chemicals, mainly pesticides, currently being used and 

industrial waste have been implicated as environmental contaminants possessing 

estrogenic andl or antiandrogenic properties (for review, see Colborn et al., 1993; 

Gray er 01.. 2001). 

Long-term exposure to small amounts of organochlorine 

contaminants leads to accumulation of considerable burdens in animal and human 

tissues (Calabrese, 1982). Neonatal exposure of rats to methoxychlor or DDf has not 

been shown to affect male reproductive organ weights in adulthood (for review, see 

Gray el a/., 2001). Male mice exposed to methoxychlor or DDT neither induced 

epididymal cysts which are found frequently in mice exposed to synthetic estrogen 

diethylstilbestrol (McLachlan, 1989). However, exposure throughout gestation and 

lactat~on to methoxychlor in rodents resulted in slightly smaller testes and 

epididymides and in lowered sperm counts in male offspring than in controls (Gray el 

01.. 1989; Gray er ol., 1992). Chlordane has been shown to disturb spermatogenesis 

and caused dose-related damage to the testes of mice. A fungicide vinclozolin has 

been shown to disrupt sexual differentiation of male rats (Gray rr ul., 1994) while ye1 

another fungicide benomyl caused a reduction in the number of spermatocytes and 

produced multinucleated germ cells (Hcss cr ul.. 1991). Endosul'nn at thc dosc 01' I 

mg/ Kg body weighcl day and lindane at the dose of 5 mL?/ Kg body weight have been 

shown lo alter the weights of the testis and accessory sex organs in malo rats (('hitrn 

er 01.. 1999; Sujatha el a/., 2001; Chitra er a/., 2001). Administration of methoxychlor 

in adult rats has been shown to decrease weights of the accessory sex organs. 

epididyn~sl sperm counts and motility (Latchoumycandane el ul.. 2002a). Induction 

of oxidative stress in testis. epididymal sperm ind various regions of the epididymis

have also been reported in the rats administered with estrogenic organochlori~lc 

pesticides, lindane and methoxychlor (Sujatha e! a[., 2001: Chitra el al., 2001; 

Latchoumycandane et al., 2002a. b). Methoxychlor has been reported to induce 

oxidative stress in mitochondrial and microsome-rich fractions of rat testis 

(Latchoumycandane and Mathur, 2002). 

1.3.1 23,7,8-Tctmchlorodibenzo-pain (TCDD) 

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), prototypical member of 

halogenated aromatic hydrocarbons, is one of the polychlorinated dibenzo-p-dioxins 

which arc formed as by-products in a number of industrial processes and during 

combustion of indusmal waste. TCDD has been identified as an environmental 

contaminant in the production of chlorinated phenols and their derived products as 

well as other chlorinated industrial compounds. These compounds have also been 

detected as by-products in sewage sludge, pulp and paper mill effluents. in diverse 

high-temperature industrial processes, and by-products of incineration of diverse 

organic materials including wood and coal. TCDD is stable to both chemical and 

thermal degradation and this stability contributes to its persistence in the environment. 

This compound is also lipophilic and environmental residues preferentially 

bioaccumulate in the food chain and is routinely detected in fish, wildlife. human 

adipose tissue, serum and milk (Rappe, 1991). The widespread human exposure and 

environmental contamination of TCDD has spurred research on the biochemical and 

toxic responses and mechanism of action elicited in animal models, mammalian cells 

in culture and in exposed human populations. 

TCDD is one of the most toxic members of polychlorinated dibenzo-p-dioxins 

and has been extensively used as a prototype to investigate various activities of thesc 

compounds and their m~rhanism 01' action (1.ucic.r o (11.. 1093; Sak. IOVS). I'hc 

diverse spectmm of biochemical and toxic responses elicited by TCDD and related 

compounds include a wasting syndrome. immunosuppression. hepatotoxicity and 

porphyria, carcinogenic and anticarcinoge-dc activities, hypo- and hyperplastic

clrects. reproductive and developmental toxicity, tumour production i~ctivity. 

neurotoxicity, chloracne and other dermal effects and the disruption of several 

endocrine-response pathways (Safe, 2001). TCDD has been shown to inhibit 

estradiol induced responses (Safe, 1995). uterine wet weight increase, peroxidase 

activity, progesterone receptor binding, epidermal growth factor receptor binding 

(Safe c.1 01.. 1998). TCDD has also been reported to induce drug-metabolizing 

enzymes, modulate expression of many other genest gene products (Safe, 2001). 

TCDD has been shown to increase the respiratory tract cancer in female but not in 

male rats (Kociba er al.. 1976). 

1.3.1.1 Effect of TCDD on various organs 

TCDD has been reported to alter a variety of organ systems in different 

species. Loss of body weight is a characteristic sign observed in most animals given a 

sub-lethal dose of TCDD. The weight loss usually manifests itself within a few days 

after exposure and results in a substantial reduction of the adipose (Peterson el ol., 

1984) and muscle tissues (Max and Silbergeld, 1987). With sub-lethal doses of 

TCDD, a dose-dependent decrease in body weight gain occurs (Kociba el ul.. 1976). 

The greatest species-specific differences in toxicity concern pathological 

alterations in the liver of the TCDD treated rats. Lethal doses to guinea pigs do not 

result in liver damage, which is comparable to the liver lesions described in rabbits 

and rats or to liver changes observed in mice (McCOM~~I el a/.. 1978; Moore rr ul.. 

1989; Turner and Collins, 1983). Thymic atrophy has been found in all animal 

species given lethal doses of TCDD. Treatment of animals with TCDD inhihits the 

bone marrow hemopoiesis in mice, both in vrvo and in virro, by directly altering the 

colony growth efficiency of stem cells (Chastain and Pazdernik. 1985; I.ustcr cr 111.. 

I V85). Among other signs and symptoms hepitic porphyrio. licmorrliagcs in vurious 

organs. lesticular atrophy, reduced prostate weight, reduced uterine weight. increased 

thyroid weight. lesions of the adreliul glands. inhibited bonc marrow hc~ni~topoi~sis. 

decreased serum albumin and increased serum triglycerides and free €any acids.

Effect of TCDD on heart muscle has also been reported in guinea pigs and rats 

(Brewster et al., 1987; Canga et al., 1988). 

1.3.1.2 Effect of TCDD on male reproduction 

TCDD decreases testis and accessory sex organ weights, causes abnormal 

testicular morphology, decreases spennatogenesis and reduces fertility when given to 

adult animals in doses sufficient to reduce feed intake andl or body weight. Some of 

the effects have been reported in chicken, rhesus monkeys, rats, guinea pigs, and mice 

treated with overtly toxic doses of TCDD (Allen and Lalich, 1962; Khera and 

Ruddick, 1973; Kociba er al., 1976; Chahoud er al., 1989; Moore et al., 1989). 

Impaired division and/ or increased attrition of germ cells during the conversion of 

leptotene spermatocytes to spermatowa and/ or by a concomitant reduction in Sertoli 

cell number have been reported in the rats treated with TCDD (Mably et al., 1992). 

Effects of TCDD on spermatogenesis are characterized by loss of germ cells, the 

appearance of degenerating spermatocytes and immature spermatozoa within the 

lumen of the seminiferous tubules and a reduction in the number of tubules containing 

mature spermatozoa in various species (Allen and Lalich, 1962; Chahoud rr a/., 

1989). 

Prenatal exposure to TCDD has been shown to interfere with fetal 

development at doses lower than those causing overt toxicity in adult animals. 

Exposure to TCDD during development produces alterations in the reproductive 

systems of the developing pups, delayed puberty and reduced sperm counts in males 

and malformations in the external genitalia of females (Hurst rr al.. 2000). In urero 

and lactational exposure of male rats to TCDD profoundly altered ontoyeny of the 

reproductive system and caused reduction in the anogenital distance, delayed testis 

descent and reduction in the weights of the testis, epididymis and accessory sex 

organs throughout sexual development. The number of sperm in the epididymis gets 

reduced in the TCDD treated rats (Faqi er al., 1997). Effects of TCDD on the male 

reproductive system are believed to be due to an androgenic deficiency (Gray el ul.,

1997). This deficiency is characterized in adult rats by decreased plasma testostcronc 

and DHT concentrations, unaltered plasma LH concentrations and unchanged plasn~a 

clearance of androgens and LH (Moore er al., 1985; Moore el ul., 1989). The doserelated 

reduction in plasma testosterone and DHT concentrations along with the 

weights of the seminal vesicles and ventral prostate weights in rats has been reported 

(Moore er a/., 1985). In contrast, TCDD did not affect accessory sex organ weights in 

castrated adult rats implanted with either testosterone or DHT containing capsules 

(Bookstaff er al., 1990). Trophic responsiveness of the seminal vesicles and ventral 

prostate to testosterone and DHT has been shown to remain unaffected by 

postpubertal TCDD treatment TCDD could increase responsiveness of the pituitary to 

these androgens without affecting the responsiveness of the accessory sex organs 

(Gray er al., 1997). TCDD has been shown to increase epididyrnal transit rate of 

sperm through the cauda epididymis of rat primarily due to decreased transit rate ol' 

sperm through the caput and corpus epididymis (Wilker er al., 1996). 

1.3.1.3 Mechanism of TCDD action 

TCDD has been shown to act through aryl hydrocarbon receptor (AhR). 

AhR has been shown to modulate the expression of a number of phase I and phase I1 

enzymes responsible for detoxification1 bioactivation of a variety of lipophilic 

compounds (Hankinson, 1995). This modulation has been shown to be accomplished 

through ligand AhR complex interactions with specific DNA response elements called 

dioxin-response elements or xenobiotic-response elements (Safe, 1995). A number of 

genes, which are involved in the oxidative metabolism, havc been shown to be 

induced by the AhR including CYPIA1, CYPl A2 and CYPl Bl (Spink el 01.. 1990; 

Spink ef al., 1994; Safe. 1995; Hankinson. 1995). TCDD has been shown to induce 

the production of reactive oxygen species thereby causing oxidative stress in multiple 

tissucs through AhR activation or AhR mediated xanthine oxidascl santhinc 

dehydrogenase activity (Stohs, 1990: Safe, 2001; Sugihara o 01.. 2001). Thc 

mechanism of TCDD mediated reactive oxygen species production has been proposed 

to involve cytochrome P450s (Park el ol., 1996;.

1.4 Effect of oxidative stress on mvlc reproduction 

Reactive oxygen species (ROS) have been shown as an important part of thc 

defense mechanisms against infection, but excessive generation of free radicals have 

been shown to damage tissues (Garg and Bansal, 2000). Reactive oxygen and 

reactive nitrogen species have been shown to play a significant role in male 

reproduction (for review, see Sikka, 2001). ROS have been shown to be formed in 

both physiological and pathological conditions in human spermatozoa due to their 

high reactivity they may interact with biomolecules inducing oxidative stress 

(Ichikawa el 01.. 1999). which severely impair male reproduction as indicated by 

reduction in fertility (for review, see Sikka, 2001). In the male genital tract, 

spermatozoa and leukocytes including neutrophils and macrophages have been shown 

to generate ROS which involved in the regulation of sperm function. Excessive 

production of ROS has been shown to cause oxidative stress in sperm (Ochsendorf, 

1999). 

Induction of the activities of intracellular oxidases in the testicular tissue or 

spermatozoa have been shown to result in the production of nanomolar levels of 

superoxide anion resulting in direct interaction with signalling mechanisms (Aitken 

er 01.. 1989). Semen samples from infertile patients have been shown to contain high 

superoxide anion generation. Prolonged inhibition of sperm mitochondria1 function 

by the ROS have been suggested as the cause of inhibition of sperm motility (Aitken 

er 01.. 1991; Kessopoulou er ol., 1992). Sperm viability could be improved by 

addition of catalase to the medium, implying that in their steady state these cells 

generate hydrogen peroxide, which can be deleterious to their survival (Mac Leod. 

1943). 

t-lydropen peroxide has been shown to k n stnhlr rcnctlvc oxygen sprcics with 

high biological diffusion properties and has been reported to transvrrsc thc plasma 

and nuclear membranes causing DNA adduct formation (for review. see Sikka. 2001). 

Peroxide derived ROS has been shown to react with transition metals such as iron 

present in biological systems and affect signaling or tissue injury processes (Peltola el

01.. 1996). Expression of extracellular superoxidc dismutasc has bccn rcportcd in the 

Sertoli cells of rat testis (Mruk and Cheng, 2000b). Mitochondria1 respiration has 

been shown to be the main biological source of superoxide anion radicals under 

physiological conditions (for review, see Chance er a/. 1979). 

Antioxidants have been described as substances that either directly or 

indirectly protect cells against adverse effects of xenobiotics, drugs, carcinogens and 

toxic radicals reactions (Halliwell, 1995). Vitamin C (ascorbic acid), vitamin E (a 

tocopherol), vitamin A, p-carotene, glutathione, superoxide dismutases, catalase, 

glutathione peroxidasel reductase, nitric oxide synthase have been classified as 

antioxidants (Krishna er a/.. 1996; Halliwell, 1999). The antioxidant properties of 

ascorbic acid have been shown to enable it to protect tissues from reactive oxygen 

species (Luck ef a/., 1995). Ascorbic acid has been shown to scavenge free radicals 

caused as a result of environmental pollution and cellular metabolism (Dawson er 01.. 

1990). Vitamin E has been shown to function as a chain breaking antioxidant in the 

cell membrane and plasma lipoproteins (Halliwell. 1999). Lipid peroxidation has 

been shown to be initiated by hydroxy- (OH). alkoxy- (RO), and peroxy- (ROO) 

radicals (Tramer el al., 1998). Cell membranes, which are structurally made up of 

large amounts of PUFA has been shown to be highly susceptible to oxidative attack 

and consequently changes in membrane fluidity, permeability. and cellular metabolic 

functions (Lenzi el 01.. 1994). Co-administration of vitamin E with mercuric chloride 

has been shown to prevent the reduction of epididymal sperm counts, motility and 

morphology as well as succinic dehydrogenase, ATPase, sialic acid. ascorbic acid and 

glutathione (Rao and Sharrna 2001).

2 SCOPE OF THE PRESENT STUDY 

There has been increasing awareness of the possible effects of environmental 

contaminants on male reproduction. 

Several studies have shown that male 

reproductive system is getting deteriorated during the last Tew decades resulting in 

reduced sperm counts and increased testicular and prostate cancer in men. In the 

present studies the effects of one of the most potent environmental contaminants, 

2,3,7,8-tetrachlorodibe11~0-p-dioxin (TCDD), were studied on male reproductive 

functions using rat as an experimental model. TCDD is formed as an unwanted by- 

product in the manufacture of chlorinated hydrocarbons and has also been shown to 

be formed in incineration processes, paper and pulp bleachings, emission from steel 

foundries and motor vehicles. The lipophilicity and low rate of metabolism leads to 

its accumulation and persistence in adipose tissues. Involvement of aryl hydrocarbon 

receptor has been proposed to explain for the toxicity of TCDD and its congeners. 

However. the nature and mechanism of action of TCDD on male reproduction 

remains unclear. Recently many environmental contaminants have been reported lo 

disturb the prooxidant and antioxidant balance of the cells by generating reactive 

oxygen species and oxygen free radicals thereby inducing oxidative stress. which has 

been shown to impair male reproduction. The present studies were undertaken to 

evaluate the effect of TCDD on rhe antioxidant system of testis and epididymis of 

rats. Funher. w-administration of antioxidant such as vitamin E was evaluated for its 

protective effect against TCDD induced toxicity. 

In the present studies the body weight of the rats were recorded to monitor the 

general health status and weight of the testis along with daily sperm production. 

dianleters of seminiferous tubules iu~d lumen werc taken to assess maintenance ot' 

testicular functions of rats. The weights of epididymis, seminal vesicles and ventral 

prostate along with kidney were taken to assess the bioavailability and/ or production

of androgen and the cumulative effect of androgenic activity. Epididymal sperm 

count, viability and motility were studied in order to assess the epididymal function. 

Serum levels of testosterone and estradiol were measured along with the 

activities of 3P-hydroxysteroid dehydro~enasc and 17P-hydroxystcroid 

dehydrogenax in testis in order to assess the status of testicular steroidogensis. 

Serum levels of FSH, LH and prolactin were determined to evaluate the hormonal 

status after TCDD exposure. 

Production of superoxide anion, nitric oxide and hydrogen peroxide were 

determined in testis as well as in the epididymal sperm and epididymis in order to 

assess the levels of reactive oxygen species in tissues. Determination of the levels of 

glutathione, a-tocopherol, ascorbic acid and the activities of superoxide dismutase, 

catalase, glutathione rcductase and glutathione peroxidase in testis, epididymal sperm 

and epididymis were determined to assess the antioxidant system. Co-administration 

of TCDD and vitamin E was done in order to assess if vitamin E provides any 

protective effects against TCDD-induced toxicity.

3 MATERIALS AND METHODS 

3.1 Animals 

Male rats of Wistar strain obtained from the Central Animal House, Jawaharlal 

Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry 

were used in the study. 

3.2 Maintenance 

Animals were housed in polypropylene cages ( l8x 10"x 8") lined with paddy 

husk. under a well regulated light and dark (l2h: 12h) scheduled. Rats were fed with 

pelleted food (Kamedhenu Agencies. Bangalore, India) and water ad libitum. Growth 

of'the animals was monitored regularly and animals displaying poor growth rate were 

discarded from the experiments. 

3.3 Chemicals 

2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. Stefen Safe, 

Department of Veterinary Anatomy and Public Health. A & M University, Texas. 

USA. Trichloroacetic acid, dehydrocpiandrosterone and androsterone 3, 17-dione 

were purchased from Sigma Chemical Company, MO, USA. Thiobarbituric acid and 

malondialdehyde were obtained from E-Merck, Germany. 

For hormone assays. 

ELSA kits were obtained from Diagnostic Systems Laboratories, Inc. Webster, 

Texas, USA. Ham's F12 medium, vitamin E (DL-tocopherol, 99%), horseradish 

peroxidase (HRP), niwtinamide adenine dinucleotide (NAD), nicotinamide adenine 

dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate 

WADP), bovine =rum albumin (BSA), deoxyribonucleic acid (DNA). ribonucleic 

acid (RNA) and 2,6dichlorophenol indophenol (DCPIP) were obtained from Himedia 

Laboratory Pvt. Ltd., Mumbai, India. Nicotinamide adenine dinucleotide phosphate 

reduced (NADPH) and N-(I-naphthyl) ethylene diamine di-hydrochloride were

obtained from Sisco Research Laboratories Pvt. Ltd.. Mumbai, India. Glutathione 

reduced and glutathione oxidized were obtained from Sd. fine Chem Ltd. Mumbai. 

All other chemicals were of analytical grade and obtained from local commercial 

sources. 

3.4 Handling of TCDD and animals 

All materials contaminated with TCDD were treated as hazardous waste. 

Gloves, goggles. mask and laboratory coats were used while handling the powdered 

material and subsequent solutions. The animals were maintained and handled 

according to the guidelines from the Indian Council of Medical Research, New Delhi. 

3.5 Treatments 

The experiments were carried out in pubertal (postnatal days 45) male rats. In 

each experiments, corresponding groups of control animals, treated with vehicle alone 

were maintained and killed along with the treated rats. The treatments to various 

groups of rats are described below. 

3.5.1 Group I: Admhlatration of TCDD to pubertal rats 

TCDD was dissolved in acetone and olive oil (see Appendix 1.1) in the ratio 

of 1 :I9 and administered orally for 45 days. For the administration of TCDD, the 

pipette tip was gently placed just inside the mouth and the dosing solution was then 

carefully expelled from the tip allowing the animal to lick the compound from the tip. 

Corresponding groups of animals were administered with equal volume of vehicle 

alone and served as control. 

Subgroup I : Administration of TCDD at the dose of 1 nd Kg body weight/ 

day. 

Subgroup I1 : Adminiseation of TCDD at the dose of 10 ng/ Kg body weight/ 

by.

Subgroup III: Administration of TCDD at the dose of 100 ng/ Kg body 

weight1 day. 

3.5.2 Group 11: Administration of TCDD along with vitamin E 

Vitamin E was dissolved in olive oil (see Appendix 1 .I) at the dose level of 20 

mg/ Kg body weight and administered orally to different groups of rats along with 

various doses of TCDD. Co-administration of vitamin E and TCDD was done by the 

methods as described previously (see Section 3.4.1). Corresponding groups of control 

animals were administered with equal volume of vitamin E. 

Subgroup I : AdminisIration of vitamin E along with TCDD at the dose of 1 

ng/ Kg body weight/ day. 

Subgroup I1 : Administration of vitamin E along with TCDD at the dose of 

10 ng/ Kg body weight/ day. 

Subgroup 111: Administration of vilamin E along with TCDD at the dosc of 

100 ng/ Kg body weight/ day. 

3.6 Killing of animals 

AfIer 24 h of the last treatment the rats were weighed and killed using overdosage 

of anesthetic ether. 

3.7 Collection of serum 

Blood samples were collected in glass centrifuge tubes either by cardiac 

puncture method or decapitation and were kept at 4'C for 12 h and then centrifuged at 

ZOO0 g for 15 min. Serum samples werc collected and stored at - 20°C i111til IISC~. 

3.8 Collection of tissues 

Kidney. testes and accessory sex organs (seminal vesicles and ventral prostate) 

werc dissated out and washed in cold normal saline. The epididymides were 

dissected out and washed in normal saline at mom temperaturn.

3.9 Organ weights 

Kidney, testes, epididymides and accessory sex organs wcre clcarcd from thc 

adhering tissues and weighed to the nearest milligram on Ohaus Precision Standard 

electronic balance. Testes and epididymides were processed for various experiments 

and the details are given in the following sections. 

3.10 Daily sperm production 

Daily sperm production was determined in testis of rats by the method of 

Blazak rr a]. (1993). Testis was weighed, decapsulated and homogenized in 50 mL of 

ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100 using 

polytron homogenizer (Polytron PT 3000, Kinematica AG) with a speed of 5 for 60 

sec as was used by Sharpe er al. (1995). The homogenate was allowed to settle for 1 

min and then gently mixed and a 10 mL aliquot was collected into a glass vial and 

stored on ice. AAer thorough mixing of each sample, number of sperni heads were 

counted in four chambers of an improved Neubauer type hernocytometer. Number of 

sperm produced per gram of tenicular tissue per day were calculated according to the 

following formula: average count of sperm heads from four chambers x square factor 

(which is 5) x hemocytometer factor (which is 10') x dilution factor (which is 50) 

divided by testis weight (g) and the time (days) during spermatogenesis that these 

cells are resistant to homogenization (which is 4.61 days for rats according to Sharpe 

el a/. (1995). The daily sperm production was done in duplicate and the data were 

expressed as mean * SD for four d~fferent rats in each group. 

3.1 1 Collection of epididymal sperm and sperm function tests 

Epididymal sperm were collected by the method of Gray er al. (1989). The 

sperm collected from left epididymis were used for the determination of sperni 

viability. sperm motility and sperm counts. The sperm collected from right 

epididymis were used for biochemical assays. The epididyrnides collected from 

experimental and control rats were washed in normal saline. Epididyrnis was

choppcd with the help of sharp razor blude in 5 mL ol' Ilam's 1:12 medium and 

incubated for 5 min at 35'C. After several washings in the Ham's F12 medium. 

sperm collected in the medium were used for various experiments. 

3.11.1 Sperm viability test 

Sperm viability test was done by the method as described in the WHO 

Laboratory Manual (1999). An aliquot of 100 pL of epididymal sperm was mixed 

with I00 pL of 0.5% eosin solution. A drop ofthe ubovc mixturc wils put on a n1icl.oslide 

covered with a cover slip and examined after 30 sec at 200 x with a help of light 

microscope. Randomly two hundred spermatozoa were counted. The livc 

spermatozoa were unstained and dead cells were stained. The sperm viability was 

expressed in percentage as the number of viable sperm of the total sperm counted. 

3.11.2 Epididymal sperm motility 

Motility of epididymal sperm was evaluated by the method as described by 

Linder et a/. (1986) and Cooke el al. (1991). A small incision was made in the cauda 

epididymis using a sharp razor. The fluid, which oozed out from the cauda 

epididymis was taken by using a pipene tip and diiuted to 2 mL with Ham's F12 

medium at 3S°C. An aliquot of this solution was placed in a hemocytometer and 

counted for motile and non-motile sperm. First non-motile sperm were counted 

followed by motile sperm. Epididymal sperm motility was expressed as the 

percentage of motile sperm of the total sperm counted. 

3.1 1.3 Epididymal sperm counts 

Epididymal sperm were counted by the method as described in the WHO 

Laboratory Manual (1999). An aliquot of 5 111. of epididyn~al sperm was diluted with 

95 pL of diluents (see Appendix 1.2). A coverslip was placed on the counting 

chambers of the improved Neubauer type hemocytometer.

Approximately 10 FL of thoroughly mixed diluted specimen transferred to 

each of the counting chambers of the hemocytometer and allowed to stand for 5 min 

in a humid chamber to prevent drying out. Sperm cells sedimented during this time 

and were then counted with the help of light microscope at 200 x. Complete 

spermatowa, head with tail. were counted. 

3.1 2 Histometric studies 

The testicular tissue was fixed in Bouin's fixative immediately after isolation. 

After dehydration in alcoholic series and cleaning in xylol, the tissue were embedded 

in paraffin wax. Sectiow were made with 5 pn thickness. The sections were stained 

with hematoxylin-eosin and examined under a light microscope. 

3.12.1 Tubular nod lumen diameter measurements 

The diameter of the seminiferous tubule and lumen were measured with 

eyepiece graticules that have been calibrated with a stage micrometer. Only tubules 

that appeared round were considered for tubular and lumen diameter measurements. 

3.13 Quantitative determination of serum hormone levels 

Serum levels of follicle stimulating hormone (FSH), luteiniziny hormone 

(LH), prolactin. testosterone and esrradiol were determined by enzyme linked 

imrnunosorbant assay (ELISA) using kits from Diagnostic Systems Laboratories, Inc. 

Webstcr, Texas, USA. The assays were done strictly according to the procedure 

given along with the kits 

3.13.1 Quantitative determination of serum FSH 

Serum levels of FSH were determined strictly according to the procedure 

given along with kit. In principle FSH ELlSA kit is an enzymatically amplified 'twostep' 

sandwich-type immunoassay. In the assay, standards. controls and experimental 

serum samples are incubated in microtitration wells, which have been coated with

anti-FSH antibody. After incubation and washing, the wells are treated with another 

anti-FSH detection antibody labeled with the enzyme horseradish peroxidase (HRP). 

After a second incubation and washing step, the wells are incubated with the substrate 

tetramcthylbenzidine (TMB). 

An acidic stopping solution is then added and the 

degree of enzymatic turnover of the substrate is determined by absorbance 

measurement at 450 nm. The absorbance measured is directly proportional to the 

concentration of FSH present in the serum samples. A set of FSH standards is used to 

plot a standard curve of absorbance versus FSH concentration from which FSH 

concentrations in the experimental serum samples can be calculated. 

Before starting the assay all the reagents and serum samples were brought to 

room temperature and were thoroughly mixed. 

The assay consisted of standards 

containing concentrations of approximately 0, 1.5, 4.5, 15.45 and 150 mIU/ mL FSH 

(see Appendix 1.3), and controls containing low (Level 1) and high (Level 11) 

concentrations of FSH and experimental serum samples. 

All the assays were 

performed in duplicate and internal and external quality controls were maintained. 

The microtitration strips were marked and 100 pL of the standard. controls 

and experimental serum samples were added to the appropriate wells and incubated 

while shaking at a fast speed (500 to 700 rpm) on an orbital microplate shaker (P- 

Multi~ope ELISA Reader) for 60 min at room temperature. 

Antibody-Enzyme 

Conjugate solution (100 pL) was added to each well and incubated while shaking at a 

fast speed on an orbital microplate shaker for 30 min at room temperature. The wells 

were aspirated and washed five times with the wash solution using an automatic 

microplate washer (Labsystems Well Wash 4). The microtitration plates were dried 

by invening on absorbent material. TMB chromogen solution (100 pL) was added to 

each well and incubated while shaking at a fast speed (500 - 700 rpm) on an orbital 

microplate shaker for 10 min at room temperature. Stopping solution, 0.2 M sulfuric 

acid, (100 a) was added to each well and absorbance of the solution in the wells was 

read within 30 min using a microplate reader (P-Multiscope ELISA Reader) set at 450 

nm. Mean absorbances for standard, control and experimental serum samples were

calculated. Using a linear-linear gtaph paper mean absorbance readings were plotted 

Tor each of the standards along the y-axis versus the FSH concentrations in mllJ1 ml, 

along the x-axis. The best fitting curve through the mean of the duplicate points was 

drawn. The FSH concentrations of the controls and experimental serum samples were 

determined 'om the standard curve by matching their mean absorbance readings with 

the corresponding FSH concentrations. 

3.13.2 Quantitative determination of serum LH 

Serum levels of LH were determined strictly according to the procedure given 

along with kit. 

sandwich-type immunoassay. 

In principle LH ELISA is an enzymatically amplified 'one step' 

In the assay, standards, controls and experimental 

serum samples are incubated with anti-LH antibody in microtitration wells, which 

have been coated with another anti-LH antibody. After incubation and washing, the 

wells are incubated with the substrate tetramethylbenzidine (TMB). 

An acidic 

stopping solution is then added and the degree of enzymatic turnover of the substrate 

is determined by wavelength absorbance measurement at 450 nm. The absorbance 

measured is directly proportional to the concentration of LH present in the serum. A 

set of LH standards is used to plot a standard curve of absorbance versus LH 

concentration from which the LH concentrations in the experimental serum sanlples 

can be calculated. 

Before starting the assay all the reagents and experimental serum samples 

were brought to the mom temperature and were thoroughly mixed. 

The assay 

consisted of standards containing LH concentrations of approximately 1, 3, 10, 30 and 

100 mlU1 mL [see Appendix 1.4) and controls containing low (Level 1) and high 

(Level 11) concentrations of LH and experimental serum samples. All the assays were 

pcrlbr~i~cd in duplicutc and inlcrnal and cx~crn:~l quality controls wcrc mai~ilai~iuil 

The microtitration strips were marked and 50 pL of the standard, controls and 

experimental serum samples were added to the appropriate wells. Antibody-Enzyme 

Conjugate solution (100 pL.) was then added to each well and incubated. shaking at a

lust sped (500 to 700 rpm) on an orbital microplate shakcr Ibr LJO min at room 

temperature. The wells were aspirated and washed five times with the wash solution 

using an automatic microplate washer. 

The microtitration plates were dried by 

inverting plate on absorbent material. TMB chromogen solution (100 pL) was added 

to each well and incubated. shaking at a fast speed on an orbital microplate shaker, for 

10 min at room temperature. Stopping solution, 0.2 M sulfuric acid, (100 pL) was 

added to each well and absorbance of the solution in the wells was read within 30 min 

using a microplate reader set at 450 nm. 

Mean absorbances for standard, control and experimental serum samples were 

calculated. Using a linear-linear graph paper mean absorbance readings were plotted 

for each of the LH concentrations in mIU/ rnL in x-axis versus along the LH standards 

in y-axis. The best fitting curve through the mean of the duplicate points were drawn. 

The LH concentrations of the controls and experimental serum samples were 

determined from the standard curve by matching their mean absorbance readings with 

the corresponding LH concentrations. 

3.13.3 Quantitative determination of serum prolactin 

Serum levels of prolactin were determined strictly according to the procedure 

given along with kit. In principle prolactin ELlSA is an enzymatically amplified 

'two-step' sandwich-type immunoassay. In the assay, standards, controls and 

experimental serum samples are incubated in microtitration wells, which have been 

coated with anti-Prolactin antibody. AAer incubation and washing, the wells are 

treated with another anti-Prolactin detection antibody labeled with the enzyme 

hotseradish peroxide (HRP). After a second incubation and washing step, the wells 

are incubated with the substrate tetrarnethylbcnzidine (TMB). An acidic stopping 

solution is then added and degree of enzymatic turnover of the substrate is determined 

by the wavelength absorbance measurement at 450 nm. The absorbance measured is 

directly proportional to the concentration of prolactin present in the serum. A set of 

prolactin standards is used to plot a standard curve of absorbance versus prolactin

concentration from which the prolactin concentrations in the expcrinicntal serum 

samples can be calculated. Before starting the assay all the rcagents and cxpcrinicntnl 

serum samples were brought to room temperature and were thoroughly mixed. The 

assay consists of standards containing 0, 2, 6. 20. 60 and 180 ng prolactin1 mL (see 

Appendix 1.5) and controls containing low (Level I) and high (Level 11) 

concentrations of prolactin and experimental serum samples. All the assays were 

performed in duplicate and internal and external quality controls were maintained. 

The microtitmtion strips were marked and 25 pL of the standard, controls and 

unknowns were added to the appropriate wells. Added 100 pL of assay buffer and 

the wells were incubated while shaking at a fast speed (500 to 700 rpm) on an orbital 

microplate shaker for 60 min at room temperature. The wells were aspirated and 

washed five times with the wash solution using an automatic microplate washer. The 

microtitre plates were dried by inverting the plate on absorbent material. Antibody- 

Enzyme Conjugate solution (100 pL) was added to each well. The wells were 

incubated while shaking at a fast speed on an orbital microplate shaker for 60 min at 

room temperature. The wells were aspirated and washed five times with the wash 

solution using an automatic microplate washer and then dried on absorbent material. 

TMB chromogcn solution (100 pL) was added to each well. The wells were 

~ncubated while shaking at a fast speed on an orbital microplate shaker, for 10 min at 

room temperature. Stopping solution, 0.2 M sulfuric acid. (100 pL) was added to 

each well and absorbance of the solution in the wells was read within 30 niin using a 

microplate reader set at 450 nm. Mean absorbances for standard, control and 

experimental xrum samples were calculated. Using a linear-linear graph paper. nican 

absorbance read~ngs were plotted Ibr each ofthc standards along the y-axis versus the 

prolactin concentrations in ng/ mL along the x-axis. The best-fitting curve through 

the mean of the duplicate poinu was drawn. The prolactin concentrations of the 

controls and experimental serum samples were determined from the standard curve by 

matching their mean absorbance readings with the corresponding prolactin 

concentrations.

3.13.4 Quantitative determinntiun uf serum testustcrunc 

The serum levels of testosterone were determined strictly according to the 

procedure given along with kit. In principle testosterone ELlSA follows the basic 

principle of enzyme immunoassay where there is competition between an unlabeled 

antigen and enzyme-labeled antigen bound to the antibody binding sites. The amount 

of enzyme-labeled antigen bound to the antibody is inversely proportional to the 

concentration of the unlabeled analyte present. Unbound materials are removed by 

decanting and washing the wells. The absorbance measured is inversely proportional 

to the concentration of testosterone present in the serwn. A set of testosterone 

standards is used to plot a standard curve absorbance versus testosterone 

concentration from which the testosterone concentrations in the experimental serum 

samples can be calculated. 

Before starting the assay all the reagents and experimental serum samples 

were brought to room temperature and were thoroughly mixed. The assay consists of 

standards containing 0, 0.1, 0.5, 2.5, 5, 10 and 25 ng testosterone1 mL (see Appendix 

1.6) and controls containing low (Level I) and high (Level 11) concentrations of 

testosterone and experimental serum samples. All the assays were performed in 

duplicate and internal and external quality controls were maintained. The 

microtitration strips were marked and 50 pL of the standard, controls and 

experimental serum samples were added to the appropriate wells. Enzyme Conjugate 

solution (100 pL) and Testosterone-Antiserum (100 bL) were added to each well. 

The wells were covered and incubated while shaking at a fast speed on an orbital 

microplate shaker for 60 min at room temperature. The wells were aspirated and 

washed five times with the wash solution using an automatic microplate washer. The 

microtitre plates were dried by inverting on absorbent material. TMB chromogen 

solution (100 &) was added to each well. The wells were incubated while shaking at 

a fast speed on an orbital microplate shaker for 10 min at room temperature. Stopping 

solution. 0.2 M sulfuric acid. (100 pL) was added to each well and absorhance of the 

solution in the wells was read within 30 min using a microplate reader set at 450 nm.

Mean absorbances for each standard, control and experimental serum samples 

were calculated. Using a linear-linear graph paper mean absorbance readings were 

plottcd fnr each of the standards along the y-axis versus the testosterone 

concentrations in ny/ mL along the x-axis. The best-fitting curve through the mean of 

the duplicate points was drawn. The testosterone concentrations of the controls and 

experimental serum samples were determined from the standard curve by matching 

their mean absorbance readings with the corresponding testosterone concentrations. 

3.13.5 Quantitative determination of serum estradiol 

The serum levels of estradiol were determined strictly according to the 

procedure given along with kit. 

binding enzyme immunoassay format. 

In principle estradiol ELISA is the competitive 

In the assay, standards, controls and 

cspcr~~ncntal serum samples are incubated with biotin-labeled estradiol and rabbit 

ant]-estradlol antiserum in microtitration wells where the labeled and biotin-labeled 

antigens compete for a limited number of anti-estradiol binding sites. 

After 

~ncubation and washing, the wells are incubated with streptavidin-Horseradish 

perosidase (HRPO). which binds to the biotinylated estradiol. 

The unbound 

atreptavidin-HRPO is washed. followed by incubation with the substrate 

tetramethylbenzidine (TMB). An acidic stopping solution is then added and degree of 

enzymatic turnover of the substrate is determined by the wavelength absorbance 

measurement at 450 nm. Before starting the assay all the reagents and experimental 

serum samples were brought to room temperature and were thoroughly mixed. The 

assay consists of standards containing 0, 20. 50. 250. 750, 2000 and 6000 pg/ mL (see 

Appendix 1.7). controls containing low (level I) and high (Level 11) concentrations of 

estradiol. All the assays were performed in duplicate. The microtitration strips were 

marked and 50 pL of each standard, controls and experimental serum samples were 

addcd to the appropriate wells. 

Estradiol-Biotin Conjugate solution (100 PL) was 

added to each well. The wells were incubated while shaking at a fast speed on an 

orbital microplate shaker for 60 min at room temperatun. The wells were aspkred 

and washed five times with the wash solution using an automatic microplate washer.

The plates were dried by inverting on absorbent material. Streptavidin-Enzyme 

Conjugate Solution (200 pL) was added to each well and incubated while shaking at a 

fast speed on an orbital microplate shaker, for 30 min at room temperature. The wells 

were aspirated and washed five times with the wash solution using an automatic 

microplate washer. The plates were dried by inverting on the absorbent material. 

TMB chromogen solution (100 pL) was added to each well. The wells were 

incubated while shaking at a fast speed on an orbital microplate shaker, for 10 min at 

room temperature. Stopping solution, 0.2 M sulfuric acid, (100 pL) was added to 

each well and absorbance of the solution in the wells was read within 30 min using a 

microplate reader set at 450 mn. The mean absorbance for each standard, control and 

experimental senun samples were calculated. Using a linear-linear graph paper mean 

absorbance readings were plotted for each of the standards along the y-axis versus the 

estradiol concentrations in pg/ mL along the x-axis. The best-fitting curvc through the 

mean of the duplicate points was drawn. The estradiol concentrations of the controls 

and cxpcrimcntal serum samples wcrc determined Ikon1 tho standard cilrvc hy 

matching their mean absorbance readings with the corresponding estradiol 

concentrations. 

3.14 Activities of steroidogenic enzymes 

The activities of 3P-hydroxysteroid dehydrogenase and I7P-hydroxysteroid 

dehydrogenase were assayed in testis after necessary standardization experiments. A 

5% testicular homogenate was prepared in cold normal saline and centrifuged at 800 g 

for 20 min at 4°C. The supernatant was used for assaying steroidogenic enzymes in 

testis. 

3.14.1 Assay of 3p-bydroxysteroid dehydrogenase 

The activity of 3p-hydroxysteroid dehydrogenase (EC 1.1.1.5 1) was 

determined by the method as described by Bergmeyer (I 974). The reaction mixture 

contained 600 pL of 100 pM pyrophosphate buffer @H 9.0). 200 pL of 0.5 ph4

NAD. 100 pM dehydroepiandrosterone (see Appendix 1.8) and 2 rnL of distilled 

water. 

An aliquot of 100 pL enzyme extract was added and vortexed. Blank was 

prepared simultaneously by adding all the reagents except the enzyme extract. 

Absorbance was measured at 340 nm against blank at 20 sec intervals for 5 min on a 

Systronics Spectrophotometer (Systronics UV Spectrophotometer 119). The activity 

of enzyme was expressed as mole of NAD converted to NADHI mini rng protein. 

3.14.2 Assay of 17p-hydroxysteroid dehydrogenase 

The activity of I7p-hydroxysteroid dehydrogenase (EC 1.1.1.51) was 

determined by the method of Bergrneyer (1974). The reaction mixture contained 600 

pL of 100 pM pyrophosphate buffer (pH 9.0). 200 pL of 0.5 pM NADPH, 100 pL 0.8 

pM 4-androstene 3.17-dione (see Appendix 1.9) and 2 mL of distilled water. 

An aliquot of 100 pL enzyme extract was added and vortexed. Blank was 


Absorbance was measured immediately at 340 nm against blank at 20 sec intervals for 

5 min on a Systronics Spectrophotometer. The activity of enzyme was expressed as 

nnlolc of NADPH converted to NADPI minl mg protein. 

3.15 Determination of nucleic acids and protein contents in testis 

I~c~crriiinntio~is of deoxyrihonuclcic acid (DNA). ribonucleic acid (RNA) and 

pr\>iclll \\CI.C curnerl uul using eslablishcd nrc\hods as described below. 

3.15.1 Extraction of nucleic acids fractions 

Extraction of nucleic acids from testicular homogenate was carried out by a 

modified method of Schneider (1957). Approximately. I00 mg of decapsulated testis 

W;IS 

wcighud and homogunized in normal saline with the help of glass tenon 

I1o1iioycnizcr. An illiquot of 4 ml. homoyenate was mixed with 2.5 ml. of 10% 

chilled Iricliloro;~cctic acid ('I'CA) and allowed to stand for 4OC for 60 min and wns

centrifuged at 3,500 g for I5 min on a Remi R8C Centrifuge. The supernatant was 

discarded and the pellet was resuspended in 2.5 mL of 10% TCA. It was then 

centrifuged at 3,500 g for 15 min. The supernatant was discarded and the pellet was 

resuspended in 5 mL of 95% ethanol. Extraction of lipids was completed with 

vortexing in 3:l ethanol ether mixture for 5 min and then centrifugation at 3,500 g for 

I5 min. 

The pellet was re-extracted with 5 mL of 95% ethanol. The supernatants were 

discarded. The pellet was resuspended in 3 mL of 5% perchloric acid (PCA) and was 

heated for I5 min in a hot water bath at 90°C. After cooling the tubes were 

centrifuged at 3.500 g for 15 min. The extraction of nucleic acids was completed by 

resuspending the pellet in 5% PCA and centrifugation at 3,500 g for 10 min. The 

supcrnalant fluids wcre pooled and were used for DNA and RNA determinations. 

The residual pellet was dissolved in 0.1 N sodium hydroxide after suitable dilution. 

3.15.2 Determination of DNA 

DNA was determined by diphenylamine colour reaction following the method 

of Burton (1956). The tubes containing 1 mL nucleic acid extract, 2 mL of IN 

perchloric acid and 2 mL of diphenylamine reagent (see Appendix 1 .lo) were kept in 

a boiling water bath for 20 min. The samples were cooled and the colour developed 

was read at 600 nm on a Systronics Spectrophotometer. A standard curve was 

prepared by using known concentrations of calf thymus DNA. 

3.15.3 Determination of RNA 

RNA was determined by orcinol reaction method (Schneider, 1957). The 

tubes containing 2 mL nucieic acid extract and 3 mL of orcinol reagent (see Appendix 

I. I I) wcre kept in a boiling water bath for 20 nlin. The samples were cooled and the 

colour developed was read at 665 nm on a Sysuonics Spectrophotometer. A standard 

curvc was prcparcd by using known concentrations of yeast RNA.

3.15.4 Determination of protein 

Protein contents were determined according to the method of Lowry el ul. 

(1951). The pellet obtained after the extraction of nucieic acid was dissolved in 10 

mL of 0.1 N sodium hydroxide solution (see Appendix 1.12). An aliquot of 0.1 mL of 

the extract was taken and was made up to I mL with distilled water. Alkaline copper 

reagent, 5 rnL, was added to the tubes containing protein extract and then vortexed, 

they were allowed to stand for 10 min at room temperature. 

Folin-Ciocalteau reagent IN was added to the above samples, vortexed and 

allowed to stand for 20 min in dark. The optical density was read at 610 nm in a 

Systronics Spectrophotometer. A standard calibration cwe was prepared using 

different concentrations of bovine serum albumin. 

3.16 Subcellular fractionation of testis 

Mitochondrial and microsome-rich fractions of testis were obtained by the 

method of differential centrifugation as described by Chainy e/ a/. (1997). Briefly, a 

20% (w/ V) homogenate was prepared in ice-cold 0.25 M sucrose solution (see 

Appendix 1.13) with the help of a motor-driven glass teflon homogenizer (Potter 

Elvehjem type, Rerni). In order to obtain nuclear pellet the homogenate was 

centrifuged at 1000 g for 10 min at 4°C. 

Mitochondrinl pellets was oblaincd by ccntril'uging post-nuclcar supcrn:it;lnt at 

10.000 g for 10 min at 4'C. Microsome-rich fraction was prepared by calcium 

chloride (CaCIz) sedimentation method (Kamath and Narayan. 1972). Postmitochondria1 

supernatant was diluted with ice-cold CaC12 (I M) so that the final 

concentration of CaC12 was 0.8 M. Incubated at 4°C was done for 10 min with 

occasional stirring. The sample was then centrifuged at 10,000 g for 10 rnin at 4°C 

and the microsome-rich pellet was obtained and dissolved in 0.25 M sucrose solution 

(I mg protein1 0. l mL).

3.17 Preparation of tiasue humogenates of cpididymia 

Epididymis was washed several times in normal saline in order to remove 

maximum number of sperm attached to the epididymal parenchyma. The caput, 

corpus and cauda regions of the epididymides were separated and homogenized 

separately in cold normal saline with the help of glass teflon homogenizer. The 

homogenates were centrifuged at 800 g for 20 min at 4°C. The supernatants were 

collected and used for various biochemical analyses. 

3.18 Preparation of tissue homogenate of kidney 

Kidney was homogenized in normal saline with the help of glass teflon 

homogenizer. The homogenate was centrifuged in a refrigerated centrifuge at the 

speed of 800 g for 20 min at 4'C. The supernatant was used for the biochemical 

analyses. 

3.19 Determination of free radicals/ reactive oxygen species in tissues 

Production of free radicals such as superoxide anion, nitric oxide and 

hydrogen peroxide were determined by the standard methods as described below. 

3.19.1 Determination ofsuperoxidc anion 

Superoxide anion production was measured by the method of Prodczasy and 

Wei (1988) which is based on the rrduction of iodonitrotetrazolium violet (INT). The 

reaction mixtures contained 750 pL of the tissue homogenate and equal volumes of 

4.9 mM INT, 0.3 mM EDTA and 0.92 mM sodium carbonate (see Appendix 1.14). 

The pH of the mixture was adjusted to 10.2. Blank was prepared simultaneously by 

adding all the reagents except the tissue extract. The reaction mixture was incubated 

for 15 min at room temperature. The reaction was terminated by placing the tubes in 

a boiling water bath for 1 min and then cooled to room temperature. After cooling 

absorbance was measured at 505 nrn against blank.

3.19.2 Determination of nitric oxide 

Production of nitric oxide was assayed by measuring the accumulation of 

nitrite/ nitrate in tissue using Griess reagent as described by Green el a/. (1982). The 

assay mixture contained I mL of tissue supernatant and I mL of Griess reagent (see 

Appcndix 1.15). After a I0 min incubation at room temperature absorbance was read 

at 550 nm in Systronics Spectropbotometer. A standard curve was prepared by using 

known concentrations of sodium nitrite. 

3.19.3 Hydrogen peroxide generation assay 

Hydrogen peroxide generation was assayed by the method of Pick and Keisari 

(198 1). The reaction mixture contained 1.641 mL phosphate buffer (50 mM, pH 7.6), 

54 pL of horseradish peroxidase (8.5 units/ mL), 30 pL of 0.28 nM phenol red, 5.5 

nM of 165 LLL of dextrose (see Appendix 1.16) and 600 pL of enzyme source. Blank 

was prepared simultaneously by adding all the reagents except the tissue extract. The 

reaction mixture was ~ncubated at 35OC for 30 rnin. The reaction was terminated by 

addition of 60 pL of I0 N sodium hydroxide. Absorbance was read at 610 m against 

a reagent blank on a Systronics Spectrophotometer. For preparation of standard 

curve, known amounts of hydrogen peroxide and all the above reagents except 

enzyme source were incubated for 30 min at 35°C and then 60 pL of 10 N sodium 

hydroxide was added and optical density was read at 610 nm. 

3.20 Determination of antioxidants in tissues 

Estimations of antioxidants such as reduced glutathione, a-tocopherol and 

ascorbic acid were done by the methods as described below. 

3.20.1 Estimation of reduced glutathione 

Total reduced glutathione content was measured by the method of Moron ef 

a/. (1979). To 0.5 mL of tissue homogenate 2 mL of 5% trichloro acetic acid waa 

added and mixed well. It was centrifuged at 800 g for 10 min and 2 mL of the

supcrnutant was taken. Hlunk was prcpurod simultaneously by addilly ull tho rcuycnls 

except the tissue extract. To this 1 mL of Ellman's reagent and 4 mL of 0.3 M 

disodium hydrogen phosphate (see Appendix 1.17) were added. l'hc yellow colour 

developed was read at 412 nm on a Systronics Spectrophotometer against blank. A 

series of standards, reduced glutathione were treated in a similar manner along with a 

blank. 

3.20.2 Estimation of a-tocopherol 

Tissue a-tocopherol was estimated by the method of Desai (1984):The assay 

mixture contained 0.5 mL of tissue homogenate, 2 rnL of petroleum ether and 1.6 mL 

of ethanol. Blank was prepared simultaneously by adding all the reagents except the 

enzyme extract. The assay mixture was mixed and centrifuged at 800 g for 10 min. 

To the supernatant, 0.2 mL of 2.2'-dipyridyl solution (see Appendix 1.18) was added, 

mixed well and kept in the dark for 5 min. An intense red colour was developed and 

read at 520 nm on a Systronics Spectrophotometer against blank. Various 

concentrations of a-tocopherol were taken and treated similarly along with a blank. 

The colour in the aqueous layer was read at 520 nrn and used for standard. 

3.20.3 Estimation of ascorbic acid 

Ascorbic acid was assayed by the method of Fisher (1962). Approximately, 

100 mg of testis was taken in 10 mL of cold 2.5% metaphosphoric acid and 

homogenized with the help of glass teflon homogenizr. The homogenate was 

centrifuged at 800 g for 20 min at 4OC. The reaction mixture contained 1 mL of 

supernatant (diluted with 2.5% metaphosphoric acid) and I mL of 2.6-dichlorophenol 

indophenol acetate solution (see Appendix 1.19). Blank was prepared sin~ultaneously 

by adding all the reagents except the tissue extract. The optical density was measured 

in a within 30 xc at 520 nm in a Systronics Spectrophotometer. A standard 

calibration curve was prepared using different concentrations of standard ascorbic 

acid.

3.21 Determination of antioxidant enzymes in tissues 

Activities of antioxidant enzymes such as superoxide dismutase, catalase, 

glutathione reductase and glutathione peroxidase were assayed by the methods as 

described below. 

3.21.1 Assay of superoxide dismutase 

Superoxide dismutase (EC 1. IS. 1.1) was assayed by the method of Marklund 

and Marklund (1974). The assay mixture contained 2.4 mL of SO mM tris HCI buffer 

cont;iininy I mM EDTA (pH 7.6). 300 pL of 0.2 mM pyrogallol (see Appendix 1.20) 

and 100 ILL enzyme source. A blank solution was prepared similarly by adding all the 

reagents except the enzyme source. Increase in absorbance was measured at 420 nm 

against blank at 10 sec intervals for 3 min on Systronics Spectrophotometer. 

3.21.2 Assay of catalase 

Catalase (EC. 1.11.1.6) was assayed by the method of Claibome (1985). The 

assay mixture contained 2.40 mL of phosphate buffer (50 mM, pH 7.0). 10 pL of 19 

mM hydrogen peroxide (see Appendix 1.21) and 50 pL enzyme source. Blank was 


Decrease in absorbance was measured at 240 nm against blank at 10 sec intervals for 

3 min on a Systronics Spectrophotometer. 

3.21.3 Assay of glutathione reductnse 

The activity of glutathione reductase (EC. 1 h.4.2) was assayed by the method 

of Carlberg and Mannervik (1975). The assay mixture contained 1.75 mL of 

phosphate buffer (100 mM, pH 7.6). 100 pL of 200 mM NADPH, 100 pL of 10 mM 

EDTA (see Appendix 1.22). SO pL of 20 mM glutathione oxidized and SO pL enzyme 

source. Blank was prepared simultaneously by adding all the reagents except the 

enzyme extract. The extinction coefficient of NADPH was measured at 340 nm 

against blank at 10 sec intervals for 3 min on a Systronics Specwphotomet~.

3.21.4 Assay of glutathione peroxidase 

Glutathione peroxidase (EC. 1.1 1.1.9) was assayed by the method of 

Mohandas rt al. (1984). The assay mixture contained 1.59 mL of phosphate buffer 

(100 mM, pH 7.6). 100 pL of 10 mM EDTA, 100 pL of sodium azidc, 50 pL of 

glutathione reductase, 100 pL of glutathione reduced, I00 pL of 200 mM NADPH, I0 

pL of hydrogen peroxide (see Appendix 1.23) and 10 pL enzyme source. Blank was 

prepared simultanwusly by adding all the reagents except the enzyme extract. The 

extinction coefficient of NADPH was measured at 340 nm against blank containing 

all the components except the enzyme at 10 sec intervals for 3 min on a Systronics 

Spectrophotometer. 

3.22 Estimation of lipid peroxidation 

A break-down product of lipid peroxidation thiobarbituric acid reactive 

substance (TBARS) was measured by the method of Buege and Aust (1976). Briefly, 

the stock solution contained equal volumes of trichloroacetic acid 15% (w/ v) in 0.25 

N hydrochloric acid and 2-thiobarbituric acid 0.37% (wl v) in 0.25 N hydrochloric 

acid (see Appendix 1.24). 1 mL of the tissue test samples and 2 mL of stock reagent 

were mixed in a screw-capped centrifuge tube, vortexed and heated for 15 niin in a 

boiling water bath. Blank was prepared simultaneously by adding all the reagents 

except the enzyme exrract. After cooling on ice the precipitate was removed by 

centrifugation at 1000 g for 15 min and absorbance of the supernatant was measured 

at 532 nm against blank. A standard curve was constructed extrapolating the amount 

of malondialdchyde to the measured absorbance 

3.23 Statistical analysis 

The data in the experimental groups of rats were obtained from the individual 

rat, which received identical treatments. All other biochemical estimations were 

carried out in duplicate. The significance of the results has been analyzed using one-

way nnalysis of variance (ANOVA) followed by Duncan's Multiple Ranyc 'I'est. 'I'hc 

P values of less than 0.05 were considered as significant against control.

4 RESULTS 

4.1 Administration of TCDD to rats 

TCDD was administered orally at the doses of 1, 10 and 100 ng/ Kg body 

weight/ day to pubertal rats for 45 days. Corresponding groups of control animals 

were maintained and received equal volumes of vehicle only. The animals were 

killed after 24 h of the last treatment. 

4.1.1 Effect of TCDD on body and organ weights 

Administration of TCDD did not show any significant change in the body 

weight of the rats at the end of the treatments in all the experimental groups as 

compared to the corr'esponding groups of control animals (Fig. 1; Table la). 

The weight of the testis decreased significantly In rats administered with 

TCDD as compared to the corresponding groups of control animals (Table la). The 

wciyht of the testis when expressed relative to the body weight also decreased 

significantly in the rats treated with TCDD as compared to the corresponding groups 

of control animals (Table 1 b). 

The weight of the epididymis decreased significantly in rats treated with 

TCDD as compared to the corresponding groups of control rats (Table la). The 

weight of the epididymis when expressed relative to the body weight also decreased 

significantly in the rats treated with TCDD with respect to the corresponding groups 

of control nnininls (Table I b). 

Administration of TCDD decreased significantly in thc weight of the scminal 

vesicles with respect to the cornspanding groups of convol animals (Table la). The 

weight of the seminal vesicles when expressed relative to the body weight also 

decreased significantly in the rats treated with TCDD as compared to the 

Corresponding groups of control animals (Table I b).

The weight of the venlral proshte showcd a siynilicunt reduction in the 'I'C1)U 

treated rats as compared to the corresponding groups of control animals (Table la). 

The weight of the ventral prostate when expressed relative to the body weight also 

decreased significantly in the rats treated with TCDD as compared to the 

corresponding groups of control animals (Table I b). 

The weight of the kidney did not show any significant change in the TCDDtreated 

rats as compared to the corresponding groups of control animals (Table la). 

The weight of the kidney when expressed relative to the body weight did not decrease 

in the rats treated with TCDD as compared to the corresponding groups of control 

animals (Table I b). 

4.1.2 Effect of TCDD on daily sperm production 

Administration of TCDD decreased the daily sperm production in a dosedependent 

manner with respect to the corresponding group of control animals 

(Table 2). 

4.1.3 Effect of TCDD on epididymal sperm viability, motility and counts 

The epididymal sperm viability did not show any significant change in the 

'I'CDD treated rats as compared to the corresponding groups of control animals 

(Table 2). Administration of TCDD decreased epididymal sperm motility in a dosedependent 

manner as compared to the corresponding group of control animals 

(Table 2). The epididymal sperm counts decreased significantly in a dose dependent 

manner in the TCDD treated rats as compared to the corresponding group of control 

animals (Table 2). 

4.1.4 Effect of TCDD on seminiferous tubular and lumen diameter 

The diameter of the seminiferous tubules was noted to be decreased 

significantly in the rats after administration of TCDD with respect to the 

corresponding group of control animals (Table 3). The lumen diameter of the

seminiferous tubules was found to be decreased significantly in rats allcr 'I'CDL) 

treatment as compared to the corresponding group of control animals (Table 3). 

4.1.5 Effect of TCDD on serum hormone levels 

Administration of TCDD did not show any significant change in the level of 

serum FSH in the rats as compared to the corresponding group of control rats (Table 

4). The levels of serum LH remained unchanged in rats administered with TCDD 

with respect to the corresponding group of control animals (Table 4). The levels of 

serum prolactin decreased significantly in rats administered with TCDD as compared 

to the corresponding group of control animals (Table 4). 

The levels of serum testosterone decreased significantly in rats administered 

with TCDD as compared to the corresponding groups of control animals ('l'ablc 4). 

The levels of serum estradiol decreased significantly in rats administered with TCDD 

as compared to the corresponding group of control animals (Table 4). 

4.1.6 Effect of TCDD on the testicular steroidogenic enzymes 

The activity of 3p-hydroxysteroid dehydrogenase decreased in a dose- 

dependent manner in the rats treated with TCDD with respect to the corresponding 

groups of control animals (Tahlc 5). 

l'hc activity of 17P-hydroxystcroid 

dehydrogenase decreased in a dose-dependent manner in the rats treated with TCDD 

as compared to the corresponding g~oups of control animals (Table 5). 

4.1.7 Effect of TCDD on nucleic acids and protein contents of testis 

The DNA content of testis decreased significantly in a dose-dependent manner 

in the rats after administration of TCDD as compared to the corresponding group of 

control animals (Table 6). Administration of TCDD also decreased the RNA content 

of testis in a dose-dependent manner as compared to the corresponding group of 

control animals (Table 6). The protein content of the testis decreased in the rats after

administration of TCDD in a dosc-dcpcndcnt manner as comparcd to 111~. 

corresponding group of control animals (Table 6). 

4.1.8 Effect of TCDD on the production of reactive oxygen species in the crude 

homogenate, mitochondrial and microsome-rich fractions of testis 

Administration of TCDD caused a significant increase in the production of 

superoxide anion in crude homogenate as well as in mitochondrial and microsomerich 

fractions of rat testis as compared to the corresponding groups of control animals 

(Table 7a). 

Administration of TCDD caused a significant increase in the levels of nitric 

oxide in crude homogenate as well as in mitochondrial and microsome-rich fractions 

of rat testis as compared to the corresponding groups of control animals (Table 7b). 

Administration of TCDD caused a significant increase in the generation of 

hydrogen peroxide in crude homogenate as well as in mitochondrial and microsomerich 


(Table 7c). 

4.1.9 Effect of TCDD on the levels of antioxidants in the crude homogenate, 

mitochondrial and microsome-rich fractions of testis 

Administration of TCDD caused a significant decrease in the level of reduced 

glutathione in the crude homogenate as well as in the mitochondrial and microsome- 

r~ch fractions of rat testis as compared to the corresponding groups of control animals 

(Table 8a). 

Administration of TCDD caused a significant decrease in the level of a- 

tocopherol in the crude homogenate as well as in the mitochondrial and microsome- 

rich fractions of rat testis as compared to the corresponding groups of control animals 

(Table 8b). 

Administration of TCDD caused a significant increase in the level of ascorbic 

acid in crude homogenate as well as in the mitochondrial and microsome-rich


(Table 812). 

4.1.10 Effect of TCDD on the antioxidant enzymes and lipid pcroxidation in 

crude homogenate, mitochondrial and microsome-rich fractions of testis 

The activity of superoxide dismutase decreased significantly in the crude 

homogenate as well as in the mitochondrial and microsome-rich fractions of rat testis 

as compared to the corresponding group of control animals (Table 9a). 

The activity of catalase decreased significantly in the crude homogenate as 

well as in the mitochondrial and microsome-rich fractions of rat testis as compared to 

the corresponding group of control animals (Table 9b). 

The activity of glutathione reductase decreased significantly in the crude 


as compared to the corresponding group of control animals (Table 9c). 

The activity of glutathione peroxidase decreased significantly in the crude 


as compared to the corresponding group of control animals (Table 9d). 

The level of lipid peroxidation increased significantly in the crude homogenate 

as well as in the mitochondria1 and microsome-rich fractions of rat testis as compared 

to the corresponding group of control animals (Table 10). 

4.1.1 1 Effcct of TCDD on the cpididymnl spcrm and cpididymis 

002661. 

A positive correlation (r = 0.95; n = 24) was observed between sperm counts 

and DNA content in the epididymal sperm (Fig. 2). The DNA contents were routinely 

used as an indicator of sperm counts. The production of reactive oxygen species. 

levels of antioxidants, activities of antioxidant enzymes and lipid peroxidation were 

expressed both in terms of protein and DNA.

Fig. 2. Correiation between sperm counts and DNA contents of rat epididymal 

sperm (14.95; n= 24) 

Sperm counts (x 10')

4.1.11.1 Effect of TCDD on the production of rcactivc oxygen spccics in the 

epididymal sperm 

Administration of TCDD caused a significant increase in the production of 

superoxide anion nitric oxide and hydrogen peroxide in the epididymal sperm of 

adult rats as compared to the corresponding groups of control animals (Table 1 1 ). 

4.1.11.2 Effect of TCDD on the levels of antioxidants in the epididymal sperm 

Administration of TCDD caused a significant decrease in the level of 

reduced glutathione and a-tocopherol in the epididymal sperm of adult rats with 

respect to the corresponding groups of control animals (Table 12). 

4.1.11.3 Effect of TCDD on the antioxidant enzymes and lipid peroxidation in 

the epididymal sperm 

The activities of superoxide dismutase, catalase, glutathione reductase and 

glutathione peroxidase decreased significantly in the epididymal sperm of the treated 

animals as cornpared to the corresponding group of control animals (Table 13). 

The level of lipid peroxidation increased significantly in the epididymal 

sperm of treated rats as compared to the corresponding group of control animals 

(Table 13). 

4.1.11.4 Effect of TCDD on the production of reactive oxygen species in the 

caput, corpus and cauda epididymides 

Administration of TCDD caused significant increase in the production of 

superoxide anion. nitric oxide and hydrogen peroxide in the caput. corpus and cauda 

epididynlides of the treated rats as compared to thc corresponding groups of control 

animals (Table 14).

4.1.11.5 Effect of TCDD on the antioxidants in the caput, corpus and cauda 

epididymides 

Administration of TCDD caused significant decrease in the levels of 

reduced glutathione and a-tocopherol in the caput, corpus and cauda regions of the 

epididymis of the treated rats with respect to the corresponding groups of control 

animals (Table 15). 

4.1.11.6 Effect of TCDD on the antioxidant enzymes and lipid peroxidation in 

the cnput, corpus and caudaepididymides 

The activity of superoxide dismutase, catalase, glutathione reductase and 

glutathione peroxidase decreased significantly in the caput, corpus and cauda regions 

of the epididymis of the treated rats as compared to the corresponding groups of 

control animals (Table 16a, 16b & 16c). The level of lipid peroxidation increased 

significantly in all the regions of the epididymis of the treated rats as compared to the 

corresponding groups of control animals (Table 16a. 16b & 16c). 

4.1.12 Effect of TCDD on the levels of reactive oxygen species production, 

antioxidants, antioxidant enzymes and lipid peroxidation of kidney 

Administration of TCDD at the dose of 1 and 10 ngl Kg body weight did not 

cause any significant change in the levels of superoxide anion, nitric oxide. hydrogen 

peroxide, glutathione, a-tocopherol, superoxide dismutase. catalase. glutathione 

reductase, glutathione peroxidase and lipid peroxidation as compared to the 

corresponding groups of control animals (Table 17a to 17c) while administration of 

TCDD at the dose of 100 ngl Kg body we~ght caused significant increase in the 

production of superoxide anion. nitric oxide. hydrogen peroxidu and 

lipid 

peroxidation and decreased in the levels of' antioxidant system in the kidney as 

compared to the corresponding groups of co~itrol animals (Table 17a to 17c).

4.2 Co-administration of TCDD and vitamin E to rats 

TCDD (1, 10, and 100 ng/ Kg body weight1 days) and vitamin E (20 mg/ Kg 

body weight/ day) were administered orally to pubertal rats for 45 days. 

Corresponding groups of animals received equal volumes of vehicle only (vitamin E 

dissolved in olive oil) and served as control. The animals were killed after 24 h of the 

last treatment. 

4.2.1 EfYect of co-administration of TCDD and vitamin E on the body and 

organ weights 

Administration of TCDD and vitamin E did not show any significant changes 

in the body weight of rats in all the experimental groups as compared to the 

corresponding groups of control animals (Table 18a). 

The weight of the testis, epididymis, seminal vesicles and ventral prostate did 

not alter significantly in rats administered with TCDD along with vitamin E as 

compared to the corresponding groups of control rats (Table 18a). The weights of the 

testis. epididymis and accessory sex organs when expressed relative to the body 

weights also showed no significant changes in the rats treated with TCDD and 

vitamin E as compared to the corresponding groups of control animals (Table 18b). 

The weight of the kidney did not show any significant changes in the TCDD 

and vitamin E treated rats as compared to the corresponding groups of control animals 

(Table 18a). The weight of the kidney when expressed relative to the body weight did 

not decrease in the rats treated with TCDD and vitamin E as compared to the 

corresponding groups of control animals (Table 18b). 

4.2.2 Effect of co-adminiaimtion of TCDD and vitamin E on the daily sperm 

production 

Administration of TCDD decreased daily sperm production in a dose 

dependent manner as compared to the corresponding group of control animals (Table 

19).

4.23 Effect of co-administration of TCDD and vitamin E on the epididymal 

sperm viability. motility and counts 

The epididymal sperm viability, motility and counts did not change 

significantly in the rats treated with TCDD and vitamin E as compared to the 

corresponding groups of control animals (Table 19). 

4.2.4 Effect of co-administration of TCDD and vitamin E on seminiferous 

tubular and lumen diameter 

The seminiferous tubules and lumen diameters remained unchanged in the rats 

after co-administration of TCDD and vitamin E as compared to the corresponding 

groups of control animals (Table 20). 

4.2.5 Effect of co-administration of TCDD and vitamin E on the levels of serum 

hormones 

Co-administration of TCDD and vitamin E did not significantly change in the 

levels of serum FSH. LH, prolactin, testosterone and estradiol in the rats as compared 

ro [he corresponding groups of control animals (Table 21 ). 

4.2.6 Effect of co-administration of TCDD and vitamin E on the testicular 

steroidogcnic emmes 

The activities of 3P-hydroxysteroid dehydrogenase and 17P-hydroxysteroid 

dehydrogenase did not show any significant change in the rats treated with TCDD and 

vitamin E as compared to the corresponding groups of control animals (Table 22). 

4.2.7 Effect of co-administration of TCDD and vitamin E on nueleic acids sncl 

protein contents of testis 

Co-ndministr~tion ol' I'CDI) and vilanii~~ I: did 1101 show any siynilica~i~ 

change in DNA. RNA and protein contents of testis as compared to the corresponding 

groups of control animals (Table 23).

4.2.8 Effect of co-administration of TCDD and vitamin E on the production of 

reactive oxygen species in the crude homogenate, mitochondrial and 

microsome-rich fractiom of testis 

Co-administration of TCDD and vitamin E did not show any significant 

change in the production of superoxide anion, nitric oxide and hydrogen peroxide in 

crude testicular homogenate as well as in mitochondrial and microsome-rich fractions 

of rat testis as compared to the corresponding groups of control animals (Table 24a; 

24b & 24~). 

4.2.9 Effect of co-administration of TCDD and vitamin E on the levels of 

antioxidant in the crude homogenate, mitochondrial and microsome-rich 

fractions of testis 


change in the levels of reduced glutathione, a-tocopherol and ascorbic acid in the 

crude testicular homogenate as well as in mitochondrial and niicrosome-rich fractions 

of rat testis as compared to the corresponding groups ot' control animals (Table 25a; 

25b & 25c). 

4.2.10 Effect of co-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in crude homogenate, mitochondrial and 

microsome-rich fractions of testis 


change in the activities of superoxide dismutase, catalase, glutathione reductase and 

glutathione peroxidase as well as the levels of lipid peroxidation in the crude 

t~sticular homoycnatc. mitochondrial and microso~nc-riel1 lii~ctil~ns 01' r;11 tcsti?, as 

comparcd to the corresponding groups vl'contrul animals ('lhblc 26a (o 20d LY: 27). 

4.2.11 Effect of co-administration of TCDD and vitamin E on epididymal sperm 

Rats were treated with TCDD and vitamin E in order to see the effect of co- 

administration on the epididymal sperm of adult rats.

4.2.11.1 Effect of co-administration of TCDD on the production of rcvctive 

oxygen rpeciea In the epididymal sperm 

Co-administration of TCDD along with vitamin E did not show any 

significant change in the production of superoxide anion, nitric oxide and hydrogen 

peroxide in the epididymal sperm of adult rats as compared to the corresponding 

groups of control animals (Table 28). 

4.2.11.2 Effect of co-administration of TCDD and vitamin E on the levels of 

antioxidants in the epididymal sperm 


change in the levels of reduced glutathione and a-tocopherol in the epididymal sperm 

of adult rats as compared to the corresponding groups of control animals (Table 29). 

4.2.11.3 Effeet of eo-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in the epididymal sperm 


glutathione peroxidase remained unchanged in the epididymal sperm of rats treated 

with TCDD and vitamin E as compared to the corresponding groups of control 

animals (Table 30). The levels of lipid peroxidation also remained unchanged in the 

rpididymal sperm of the animals administered with TCDD and vitamin E as 

compared to the corresponding groups of control animals (Table 30). 

4.2.11.4 EfTect of co-administration of TCDD and vitamin E on the production 

of reactive oxygen spccics in the euput, corpus and eaudn cpididyntides 



all the regions of the epididymis of adult rats as compared to the corresponding 

groups of control animals (Table 31).

4.2.11.5 Effect of co-administration of TCDD and vitamin E on the antioxidants 

in the caput. corpus and cauda epididyrnideo 


change in the levels of reduced glutathione and a-tocopherol in all regions of the 

epididymis of adult rats with respect to the corresponding groups of control animals 

(Table 32). 

4.2.1 1.6 Effect of co-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in the caput, corpus and cauda 

epididymides 


glutathione peroxidase as well as the levels of lipid peroxidation did not alter 

significantly in the caput, corpus and cauda regions of the epididymis of rats treated 

with TCDD and vitamin E when compared to the corresponding group of control 

animals (Table 33a; 33b & 33c). 

4.2.12 Effect of co-administration of TCDD and vitamin E on the levels of 

reactive oxygen species production, antioxidants, antioxidant enzymes 

and lipid peroxidation in Kidney 



the kidney of adult rats as compared to the corresponding groups of control animals 

(Table 34a). Administration of TCDD along with vitamin E did not cause any 

significant changc in the levcls of roduccd gluti~thic)nc and cr-Iocoplicrol in Ihc Lidncy 

ofadult rats with respect to the corresponding groups of control animals (.!'able 34b). 


glutathione peroxidase and the level of lipid peroxidation of the kidney did not show 

any significant change in the rats administered with TCDD and vitamin E as 

compared to the comsponding group of control animals (Table 34c).

Table la. Effect of TCDD on the body weight and weights of the testis, epididylnis, 

kidney and accessory sex organs of adult male rats 

TCDDI Kg body weight 

Control I nS I0 ng I00 ng 

Body weight 116i 2.82 115i 1.36 1141 1.60 114+ 1.86 

Testis 964 + 3.86 936 f 9.97' 922 + 2.89' 92 1 5 7.08* 

Epididyln~s 414 f 2.63 393 * 5.20. 388 f 4.23' 386+ 4.00' 

Seminal vesicles 

Intact 

Empty 

Ventral prostate 1935 2.66 180f 1.83. 179i 2.50' 175 t 3.55. 

Kidney weight 578 i 8.2 1 590 i 7.26 586 f 9.38 540 f 6.40' 

Body weights are expressed in gram and tissue weights are expressed in mg 

The data are expressed as meanfSD for six animals per group 

'pc0.05 against the control group

Table I b. Effect of TCDD on the weights ofthe testis, epididymis, kidney and accessory 

sex organs of adult male rats 

TCDDl Kg body weight 

Control I nfi I0 ng 100 11g 

Testis 828 f 8.69 809 f 5.45. 807 i 8.93. 805 f 2.28. 

Epididymis 355 f 7.14 339i 6.92' 339 t 3.31. 337 + 6.87. 


Intact 

Emptr 

Ventral prostate 165f 2.78 155t3.31' 156i3.20* 1521 2.85' 

Kidney 489 i 6.1 1 498 t 8.62 480 f 6.32 443 * 4.81 ' 

Tissue weights are expressed in mg/ loop body weight 

The data arc expressed as mean f SD for six aninield dose 

*p

Table 2. Effcct of TCDD on the daily sperm production, epididymal sperm viability, 

sperm motility and sperm counts in adult male rats 

TCDDi Kg body weight 

Control I ng I0 ng 100 ng 

Testis 

Daily sperm 

Production (x 10" 22.19 + 2.67 15.67 i 2.65. 13.65 f 2.19. 13.10' 3.16* 

Epididymul Sperm 

Sperm viability (%) 95.50 + 1.51 94.16 f 2.56 95.83 i 2.27 92.50 * 2.16 

Sperm motility (%) 93.66 * 2.16 74.83 i 3.54. 57.83 i 3.48' 46.16 * 3.41. 

Sperm counts (x 10') 8.20 i 0.14 7.51 * 0.15* 6.36 f 0. I2* 5.3 1 f 0.15* 

The data are expressed as mean i SD for six animals1 dose 

'p

Table 4. Effect of TCDD on the serum hormone levels in adult cnulc rals 


Control 1 ng I0 ng I00 ng 

FSH' 14.40f 2.10 13.62 f 2.92 14.50i 1.73 13.001 1.41 

'ny/ mL; bPd mL 

The data arc expressed as mean f SD for six animals/ dose 

*p

Table 6. Effect of TCDD on the DNA, RNA and protein contents in testis 


Control I ng I0 II~ I00 ng 

DNA 

RNA 

Protein 

The values are expressed in mgl g tissue wet weight 

The data are expressed as mean t SD for six animals1 dose 

*p

Table 7b. Effect of TCDD on the production of nitric oxide in the crude ho~noyenute, 

mitochondrial and microsome-rich fractions of' rat testis 


Control I ng 10 ng I00 ng 

Crude homogenate 2.38 f 0.10 3.61 * 0.08* 3.72 rt 0.04' 4.77 f 0.06. 

Mitochondrial fractions 1.43 f 0.1 1 2.33 f 0.108 2.79 f 0.14' 3.10 1 0.42' 

Microsome-rich 

fractions 2.41 f 0.77 2.81 1 0.20* 3.01 10.14' 3.16* 0.72* 

The values are expressed in mmole/ min/ mg protein 

The data are expressed as mean i SD for six animals1 dose 

'pcO.05 against the control 

Table 7c. Effect of TCDD on the hydrogen peroxide generation in crude Iiomogenate, 




Crudc Iromoyenatc 18.70 f 2.78 22.16 r 1.26. 74.1 7 + 2.88' 76.4 1 i 1 .St)* 

Mitochondrial fractions 15.73 i 2.12 18.08 f 1.21' 19.87 i 1.14' 21.76 r 1.93' 


fractions 12.68 f 2.01 16.31 22.41' 17.37 f 1.98. 19.45 i 1.89* 

The values are expressed in nmold minl mg protein 

The data are expressed as mean * SD for six animals1 dose 

*p

Table 8a. Effect of TCDD on the levels of glutathione in the crude homogenate, 




Crude homogenate 336 5 7.66 297 + 8.33* 275 i 6.88. 267 i 7.57' 

Mitochondrial fractions 197 f 5.57 169 f 9.29' 159 i 8.01* 153 5 9.06* 


fractions 158 * 4.26 135 f 9.87' 13Oi 3.01' 126 f 5.75. 

The values arc expressed in nmolel nig protein 

The data arc expressed as mean i SD for six animals! dose 

*pc0.05 against the control 

Table 8b. Effect of TCDD on the levels of a-tocopherol in the crude homogenate, 



Control I ng I0 ng I00 ng 

Crude homogenate 1.32 f 0.08 0.95 i 0.08. 0.78 f 0.04. 0.76 r 0.02' 

Mitochondrial fractions 0.90 f 0.02 0.74 t 0.03' 0.66 f 0.06. 0.56 k 0.03' 


fractions 0.66 f 0.04 0.48 f 0.05' 0.38 f 0.02' 0.35 * 0.03' 

The data arc expressed in pg/ mg protein 

The data an expressed as inmean f SD for six animals1 dose 

*p

Table 8c. Effect of TCDD on the levels of ascorbic acid in the crude homogenate, 

mitochondria1 and microsome-rich fractions of rat testis 



Crude homogenate 3.55 f 0.16 2.71 f 0.1 I* 2.18 + 0.13* 1.63 + 0.10' 

Mitochondrial fractions 2.15 f 0.15 1.92 f 0.17* 1.74 f 0.14' 1.62 f 0.15. 


fractions 1.96f 0.17 1.54f 0.11* 1.37f 0.10' 1.80k 0.18. 

The data are expressed in mg/ g tissue wet weight 

The data are cxpressed as mean * SD for six animals/ dose 

*p

Table 9b. 

Effect of TCDD on the activities of catalase in the crude homogenate. 



Control 1 ng 10 ng 100 ng 

Crude homogenate 0.871 * 0.06 0.696 + 0.05' 0.670 + 0.05' 0.630 3. 0.05' 

Mitochondr~sl fractions 0.718 + 0.04 0.628 f 0.05' 0.567 i 0.04* 0.509 * 0.03- 


fractions 0.401 * 0.04 0.356 1 0.08' 0.321 + 0.01. 0.309 k 0.01' 

The values are expressed in pmole/ mini mg protein 

The data arc expressed as mean * SD for six animals/ dose 

*p

Table 9d. 

Effect of TCDD on the activities of glutathione peroxidase in crude 

homogenate, mitochondrial and microsome-rich fractions of rat testis 

- - 



Crude homogenate 46.35 f 3.57 41.45 f 3.13' 41.57 f 2.93' 40.55 f 4.77' 

Mitochondrial fractions 3 1.3 1 f 2.48 25.46 i 4.1 1 24.68 f 3.26* 2 1.14 + 3.98' 


fractions 26.11 f 4.68 20.13 *3.41* 19.64k 1.96. 19.01 i 2.01; 

The values are expressed in nmolel min/ mg protein 

The data arc expressed as mean f SD for six animals1 dose 

*pc0.05 against the control group 

Table 

10. Effect of TCDD on the lipid peroxidation in crude homogenate, 


TCDD/ Kg body weight 

Crude homogenate 2.47 f 0.16 3.40 f 0.29' 3.60 f 0.37. 4.10 f 0.15* 

Mitochondrial fractions 2.01 f 0.78 2.97 f 0.29' 3.41 * 0.5 I* 3.67 f 0.561 


fractions 1.20 f 0.22 1.79 f 0.27. 2.07 * 0.23' 2.1 1 f 0.41 

The values arc expressed in pnoleJ mg protein 

The data arc expressed as mean f SD for six animals/ dose 

'pX0.05 against the control group

Table I I. Effect of TCDD on the production of superoxide anion, nitric oxide and 

hydrogen peroxide in the epididyrnal spenn of adult rats. 



Superoxide aniona 

(mg protein) 1.93 f 0.18 2.78 f 0.47. 2.91 + 0.14' 3.14 f 0.24" 

(rng DNA) 1.41 f 0.04 2.07f O.lOC 2.47f 0.16. 2.91 f 0.31. 

Nitric oxideb 

(~ng protein) 1.46i0.22 2.01 i 0.16" 2.6910.1 I* ?.99f0.06* 

(mg DNA) 1.14 f 0.1 l 1.90 f 0.17' 2.40 k 0.27* 2.68 ~t 0.13' 

H202 generationC 

(mg protein) 20.80f 1.96 40.70 + 3.1 I* 46.10f 3.82. 55.30 f 2.32' 

(rng DNA) 16.81fl.18 25.42f 1.33; 31.16+1.60* 36.87i2.88' 

' nmoll min/ rng protein or DNA; mmol/ min/ mg protein or DNA; nmol H202 

generatedl min/ rng protein or DNA; The data are expressed as mean f SD for six 

animals! dose; *p

Table 13. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the 




Superoxide dismutase' 

(mg protein) 22.405 1.43 20.30i2.85' 18.50i 1.51. 16.90k 1.57' 

(mg DNA) 18.08+ 0.61 13.64+ 1.14' 12.48i 0.72* 11.385 1.22* 

catalaseb 

(ing protein) 2.49f 0.13 2.33 i 0.15' 2.305 0.14' 2.03 i0.13' 

(mg DNA) 2.01 i 0.05 1.45 i 0.06. 1.44 + 0.08* 1.35 + 0.05' 

Glutathione reductase' 

(mg protein) 40.00* 2.17 31.20f 2.41. 28.20i 2.49* 27.10f 1.57' 

(mg DNA) 32.41 f 0.86 19.46i 0.53. 20.l8i 0.83' 18.07i 0.76* 

Glutathione peroxidase' 

(mg protein) 71.20i 3.87 64.50i 4.88' 56.605 4.41. 48,OOi 3.97. 

(mg DNA) 57.58 i 1.52 40.17 i 1.05' 38.24 i 0.88. 31.94 + 1.79. 

Lipid peroxidatton' 

(trig protein) 

2.17 i 0.20 4.24 i0.418 4.80f 0.38' 6.08+ 0.20' 

(mg DNA) 1.75 + 0.82 2.W+ 0.13. 3.24i 0.08' 4.05i 0.12. 

nlnol/ tninl mg protein or DNA 

pmoll minl rng protein or DNA 

moll mg protein or DNA 

The data are expressed as meaniSD for six animals! dose 

*p

Table 14. Effect of TCDD on the production of superoxide anion, nitric oxide and 

hydrogen peroxide in the caput, corpus and cauda epididyrnides of adult rats. 



Cuput cl~ididyrrri.~ 

Superoxide anion' 2.01 i 0.41 2.1 1 1 0.1 I* 2.91 f 0.17. 3.14 * 0.31- 

Nitric oxideb 1.46-t 0.13 2.66-t 0.41. 2.71 f 0.34' 2.93 * 0.23* 

Hydrogen peroxideb 11.52 * 1.66 19.98 f 3.31' 36.25 + 2.05' 75.42 k 5.7IL 

Superoxide anion0 1.31 i 0.30 2.49* 0.10' 2.71 f 0.19' 3.01 i 0.17. 

Nitric oxideb 1.36* 0.21 2.01 f 0.18. 2.46-t 0.41' 2..91 i 0.14' 

Hydrogen peroxideb 20.50 f 1.68 37.70 13.87' 58.25 2 6.17: 109.6 i 19.3' 

Superoxide anionn 2.07 + 0.2 I 2.71 + 0.26. 2.97 i 0.40. 3.29 i 0.14. 

Nirric oxideb 1.91 -t 0.33 2.61 + 0.40. 2.93 + 0.17' 3.40* 0.16. 

tlydrogen peroxideb 15.76 f 1.40 20.11 i 1.42, 31.86 i 3.43. 52.8 + 5.28* 

nrnoll mid rng protein; mmoll mid mg protein 

nrnol Hz02 generatedl mid mp protein 

The number of animals per group is six 

'pc0.05 against the control group

Table 15. Effect of TCDD on the levels of antioxidants in the caput, corpus and cauda 


TCDDJ Kg body weight 

Control I ng 10 ng 100 ng 

Capur epididymis 

Glutathione* 143 f 6.83 127 r 8.11. 121 t 4.21. 116 r 3.98. 

a-Tocopherolb 0.84i 0.06 0.71 f 0.12: 0.70+ 0.16. 0.69i 0.09. 

Corpus epidrdymb 

Glutathione' 140 i 7.23 120 t 6.26* 117 c 6.93' 113 i 4.90' 

a-Tocopherolb 0.82 i 0.1 1 0.74 + 0.06* 0.72 f 0.08' 0.70 t 0.06' 

" nmoll min/ mg protein; pmol/ min/ mg protein 

'I'he data are expressed as nieanfSD for six aninlalsl dosc 

*p

Tablel6a. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the 

caput cpididymis 

TCDD/ Kg body weight 

Control 1 ng 10 ny 100 ng 

Supcroxide disrnutase' 31.98 f 2.18 26.00 f 1.85' 20.13 f 1.57' 17.97 i 2.29' 

catalaseb 5.18f0.61 4.07+0.21* 3.78f 0.22- 3.65+0.27' 

Glutathionc reductasen 39.91 f 2.99 28.34 + 1.68' 23.22 + 1.93* 21.33 f 4.41' 

Glutathione pcroxidase'61.43 f 3.61 40.05 f 2.38. 30.87 + 1 .45* 27.07 2 3.40* 

Lipid peroxidation' 2.45 i 0.13 3.05 2 0.27. 4.02 * 0.25. 7.56 + 0.76* 

" nmoll mid mg protein; pmoll rnin / mg protein; ' bmol/ mg protein 

The data arc expressed as meaniSD for six animals/ dose 

*pcO.05 against the control group 

Table 16b. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the 

corpus cpididy~nis 

TCDDi Kg body weight 

Supcroxide disrnutase' 41.06 i 2.44 33.14 + 2.44' 30.39 f 1.8 1 * 24.23k 3.68' 

Catalaseb 4.1 7 f 0.31 3.80 f 0.29' 3.43 i 0.35. 3.21f 0.34* 

Glutathione rcductasea 37.48 i 2.40 30.08 + 2.17' 27.50 f 2.63. 23.88f 5.13' 

Glutathione pcroxidase'73.22 f 5.82 52.12 f 4.08. 44.94 + 4.66. 36.47i 6.86* 

Lipid peroxidation' 1.91 * 0.18 2.82 f 0.34. 3.34 i 0.37. 4.88~ 0.52' 

nmoll mid mg protein; pol/ min I mg protein; ' pmol/ rng protein 

Thc data arc expressed as rncanfSD for six animals/ dose 

*p

Table 16c. Effcct of TCDD on the antioxidant enzymes and lipid peroxidation in the 

cauda epididyrnis 

TCDD! Kg body weight 


Superoxide dismutase' 45.78 i 2.64 36.57 * 2.53' 30.22 i 3.26. 26.56 f 2.36' 

Catalaseb 3.17i0.16 2.76f 0.22' 2.72i0.23' 1.80+0.12* 

Glutathione reduce' 42.69 * 2.62 32.67 + 2.59' 28.35 f 2.53' 15.36 f 1.86. 

Glutathione peroxidase'45.44 f 2.34 40.71 i 2.1 I* 34.81 f 2.53* 29.17 1t 2.52' 

Lipid peroxidationC 2.21 5 0.19 3.20 2 0.34* 4.05 + 0.39' 5.94 f 0.42* 

a nmol/ rnin.1 mg protein; pmol/ min 1 rng protein; pmolf mg protein 

The data are expressed as meaniSD for six animals! dose 

*p

Table 17b. Effect of TCDD on the levels of antioxidants in the kidney of adult rats 



Glutathione' 161 i 8.11 158 i 6.23 155 i 3.87 133 f 4.18* 

a-~oco~herol~ 0.90* 0.12 0.86i 0.17 0.80* 0.11 0.59i 0.10' 

nmoll mg protein; pgl mg protein 

The data are expressed as mcan*SD for six animals/ dose 

'pc0.05 against the control group 

Table 17c. Effect of TCDD on the antioxidant enzyliirs and lipid pcrosidation in lllc 

kidney of adult rats 



Superoxide dismutase' 34.56 I 3.10 35.21 i 2.93 33.27 2 2.24 28.13 '-2.16' 

Catalaseb 4.21 i0.70 4.19i0.38 4.15 k0.48 4.01 t0.18* 

Gli~tathione redmtase" 37.22 * 3.12 36 53 i 2.08 35.18 * 2.65 30.17 t 2.05' 

Cilulatliionc pcroxidoscd56.67 * 4.81 54. I? i 3.10 53.86 + 3.1 1 40.07 i 2.13. 

L~pid peroxidation' 2.1 1 * 0.17 2.16 f. 0.28 2.22 f. 0.30 2.46 i_ 0.25* 

* n~rioll mi"/ mg pmtein; wnoll min 1 m& protein;' bmoll nlg pmtein 

The data arc expressed as mcarutSD for six animals1 dose 

*P

Table 18a. Effect of co-administration of TCDD and vitamin E on the body weight and 

weights of the kidney, testis, epididymis and accessory ssx organs of adult 

male rats 

TCDD and vitamin E 

Control 

Body weight 116 f 2.04 

Testis 991 i 7.32 

Epididymis 413 t 4.56 


Ventral prostate 198 i 2.63 

Kidney 56814.13 

Body weight is expressed in gram and tissue weights are expressed in mg 

The data are expressed as mean + SD for six animalsl dose

Table 18b. Effect of oo-administration of TCDD and vitamin E on the weights of the 

kidney, testis, epididymis and accessory sex organs of adult male rats 


Control lng+20mg 10ng+20mg lOOng+20mg 

Testis 847 f 18.45 845 + 21.67 850+ 16.72 845 + 13.22 

Epididymis 353 * 6.49 348 + 10.14 353 f 7.17 350 f 6.94 


Intact 829+ 16.52 822 t21.99 838 i 18.74 827i 17.23 

Empty 704 + 16.65 696 i 16.96 705 + 15.56 700 f 13.26 

Ventral prostate 169 + 3.97 165 * 6.99 169 f 5.98 :67 f 5.81 

Kidney 467 i 4.87 480 i 3.47 478 ?; 3.12 470 i 2.87 

Tissue weights are expressed in rngl lOOg body weight. 

The data are expi-esd as meant SD for six ansmald dose

Table 21. Effect of co-administration of TCDD and vitamin E on the serum hormone 

lcvels in adult male rats 

-- 


Control 1ngs20mg 10ng+20mg 100ng+20mg 

Testosterone' 2.51 i 0.13 2.48 f 0.08 2.35 f 0.05 2.34 _+ 0.06 

FSH' 15.66f 2.08 14.00k 0.40 15.25 i 1.84 14.63 r 0.30 

Prolactin' 18.33 f 1.52 17.50i 0.57 18.25 i 1.25 19.75 zt 1.21 

'ngl mL; bpg/ mL 

The data are expressed as mean 

SD for six animals/ dose 

Table 22. Effect of co-administration of TCDD and vitamin E on the activities of 

stemidogenic enzymes in testis. 


Cotitrol Ing+20mg lOng+201ng 100ng+20mg 

3-P hydroxysreroid 54.31 2 2.68 56.14 k 3.96 55.63 f 3.01 53.44 + 3.46 

dehydrogenax 

The data arc expressed in nmolel mild mg protein 

The data are expressed and rneaniSD for six animalsi dose

Table 23. Effect of co-administration of TCDD and vitamin E on the DNA, RNA and 

protein contents in testis 


Control Ing+20mg 10ng+20mg 100ng+20mg 

DNA 10.62 t 2.1 1 11.31 f 2.48 10.48 * 1.96 12.1 1 * 2.08 

RNA 5.46 i 1.91 6.07 + 1.34 6.12 f 1.20 5.69 i 1.07 

Protein 8.76 i 0.13 9.1 1 i 1.23 9.41 t 1.40 8.97 i 0.10 

Nucleic acids and protein contents are expressed in mg/ g tissue wet weight 

The data are expressed as mean * SD for six animals! dose 

Table 24a. Effect of co-administration of TCDD and viranlir~ E on the productio~~ ot' 

superoxide anion in the c ~de homogenate, mitochondria1 and microsomerich 



Control lng+20mg lOng+20mg 100ng+20mg 

Crude homogenate 3.76 f 0.12 3.79 i 0.18 3.80 f 0.40 3.79 k 0.27 

Mitochondria1 fractions 2.13 i 0.19 2.21 i 0.12 2.19 f 0.21 2.20 i 0.17 


fractions 1.61 i 0.31 1.62 t 0.27 1.68 i 0.12 1.67 i 0.13 

'l'hc data are cxpresscd in nniold cninl~ng pn)tcin 

The data are expressed as mean f SD for six animals! dose

Table 24b. Effect of co-administration of TCDD and vitamin E on the production of 

nitric oxide in the crude hornogenate, mitochondrial and microsome-rich 


- 


Control 1ng+2Omg IOng+20rng 100ng+20mg 

Crude homogenate 1.7850.ll 1.83f0.16 1.88fO.l2 1.87i0.21 

Mitochondrial fractions 1.37 i 0.16 1.47 i 0.13 1.42 f 0.1 1 1.40 * 0.17 


fractions 1.465 0.42 1.495 0.14 1.53 * 0.13 1.51 f 0.13 

The data are expressed in mmold mint mg protein 

The data are expressed as mean * SD for six animalsi dose 

Table 24c. Effect of co-administration of TCDD and vitamin Eon the hydrogen peroxide 

generation in the crude homogenate, mitochondrial and microsome-rich 



Control I ng+20mg IOng+ZOmg 100ng+2Omg 

Cmdc homogenate 16.16 f 2.17 17.23 5 2.62 17.68 5 2.30 17.00 k 1.96 

Mitochondrial fractions 14.26f 2.41 15.37 f 1.96 14.46 f 1.87 13.19 f 1.91 


fractions 10.62f 1.30 10.78 f 0.96 11.21 f 1.40 12.01 * 1.81 

The data are expressed in nmold minl rng protein 

The data ate cxpmsed as rneanfSD for six animalsi dose

Table 25a. 

Effect of co-administration of TCDD and vitamin E on the level of 

glutathione in the crude homogenate, mitochondria1 and microsome-rich 



Control 1 ng+20mg 10ng+20mg 100ng+20mg 

Crude homogenate 346 i 8.21 340 -+ 7.21 343 * 6.26 349 f 7.62 

Mitochondrial fractions 174 f 2.81 180 i 9.31 176 f 6.14 179 f 5.62 


fractions 149f 3.13 156i 4.87 15Oi 4.91 1572 5.13 

The data arc expressed in nmolel mg protein 

The data are expressed as mcanfSD for six animals/ dose 

Table 2Sb. Effect of co-administration of TCDD and vitamin E on the level of a- 

tocopherol in the crude homogenate, mitochondrial and microsome-rich 


TCDD and vitam~n E 

Control 1 ng+2Omg IOng+20mg 100ng+20mg 

Crude homogenate 1.48 * 0.03 1.36 ;t 0.08 1.44 i 0.06 1.40 i 0.04 

Mitochondrial fractions 0.91 i 0.10 0.92 i 0.09 0.97 i 0.13 0.90 k 0.09 

Microsame-rich 

fractions 0.62 * 0.16 0.63 i 0.10 0.63 i 0.06 0.62 + 0.07 

The data are expressed in pgl mmy protein 

The data are expressed as msanf SD for six animals/ dose

Table 25c. Effect of co-administration of TCDD and vitamin Eon the lcvcls of ascorbic 

acid in the crude homogenate, mitochondria1 and ~nicrosome-rich fractions 

of rat testis 


Control I ng 10 ng 100 ng 

Crude homogenate 3.68 f 0.1 1 3.60 t 0.15 3.63 f 0.16 3.58 r 0.20 

Mitochondrial fractions 2.36 * 0.26 2.23 i 0.19 2.28 f 0.20 2.20 * 0.23 


fractions 1.85f 0.18 1.79f 0.16 1.90k 0.20 1.87i 0.19 

The data are expressed in md g tissue wet weight 

The data are expressed as mean f SD for six animals/ dose 

Table 26a. 

Effect of co-administration of TCDD and vitamin E on the activity of 

superoxide dismumse in the crude hornogenate, mitochondria1 and 



Control I ng+20mg IOng+20mg 100ng+20mg 

Crude hornogenatc 33.14 f 1.68 32.66 f 1.43 34.1 1 i 1.21 33.16 + 1.04 

Mitochondrial fractions 26.98 f 2.75 25.16 2 1.92 24.48 k 1.68 25.13 + 1.88 

Micmsonic-rich 

fractions 21.21 * 1.34 20.68i2.01 21.37i 1.98 20.45 t 1.89 

The data are expressed in nmolc/ mid mg protein 

The data arc expressed as meaniSD for six animals/ dose

Table 26b. Effect of co-administration of TCDD and vitanlin E on thc activity ol'catalasc 

in the crude homogcnate, mitochondrial and microsome-rich fractions of rat 

testis 


Control 1 ng+20mg IOng+20mg 100ng+20mg 

Crude homogenate 0.880 f 0.04 0.879 f 0.01 0.882 f 0.03 0.881 1 0.04 

Mitochondrial fractions 0.630 f 0.1 1 0.627 i 0.1 0 0.63 1 i 0.09 0.63 1 + 0.1 1 

Microsomc-rich 

fractions 0.398 f 0.10 0.395 i 0.02 0.396 + 0.06 0.396 + 0.02 

The data are expressed in pmole/ mid mg protein 

The data are expressed as mcaniSD for six a~~iniald dose 

Table 26c. Effect of co-administration of TCDD and vitamin E on the activity of 

glutathione ductase in the crude homogenate, mitochondria1 and 



Control I ng+20mg 10ng+20mg 100ng+20mg 

Crude homogcnate 45.01 i 1.3 1 44.24 1 1.68 45.43 f 2.01 44.62 1 2.1 1 

Mitochondrial fractions 32.44 * 1.98 33.16 i 1.60 32.64 i 1.01 33.14 i 2.07 

The data are expressed in nmolc/ m id mg protein 

The data are expressed as mcanfSD for six animals1 dose

Table 26d. Effect of co-administration of TCDD and vitamin E on the activity ol' 

glutathiom peroxidase in the c~de homogenate, mitochondrial and 


- - - - 

TCDD and v~tamin E 

Control Ing+20mg 10ng+20mg 100ng+20m6 

Crude homogenate 43.53 * 2.57 44.16 i 1.88 44.67 i 1.61 43.26 i 2.10 

Mitochondrial fractions 30.66 * 2.10 32.14 * 1.8 1 32.66 f 1.66 3 1.26 i 1.07 


fractions 25.11 k3.17 24.42i2.92 26.6212.01 24.141 2.93 

The data am expressed in nmold mid mg protein 

The data am expressed as mean iSD for six animalsl dose 

Table 27. Effect of co-administration of TCDD and vitamin E on the level of lipid 

peroxidation in the crude homogenate, mitochondrial and microsome-rich 



Control I ng+20mg IOng+20mg lOOng+2Omg 

Crude homogenate 2.17 0.16 2.21 * 0.23 2.20 f 1.04 2.18 * 1.21 

Mitochondrial fractions 2.1 1 i 0.21 2.14 i 0.27 2.10 * 0.17 2.1 1 + 0.19 

The data arc expressed in pnole/ minl mg protein 

The data arc expressed as mean* SD for six animals1 dose

'I'able 28. 

Eff'ecl ol' co-admi~listratiott ol' 'I'CVD ettd vitamill I2 otl lhc pi.oductio~t ol' 

suproxide anion, nitric oxide and hydrogen peroxide in the epididymal 



Control 

Superoxide anion' 

(mg protein) 

(mg DNA) 

Nitric oxideb 

(mg protein) 

(mg DNA) 

H102 generationc 

(mg protein) 

(mg DNA) 

' nmoV mid mg protein or DNA; mmoV minl mg protein or DNA; nmol H202 

generated1 mint mg protein or DNA; The data are expressed as meanfSD for six 

animals1 dose 

Table 29. Effect of co-administration of TCDD and vitamiti E on the levels of 

antioxidants in the epididymal sperm of adult rats 


Control Ing+20mg 10ng+20mg 100ng+20mg 

Glutathione' 

(mg protein) 1622 2.18 170 f 2.16 178f 4.10 176f 4.01 

(mg DNA) 146f2.11 153f2.11 154i2.74 145f2.24 

a-tocopherolb 

(mg protein) 0.461 f 0.04 0.477 + 0.12 0.476f 0.14 0.471 i 0.24 

(mg DNA) 0.354 f 0.06 0.366 f 0.28 0.367 f 0.21 0.361 f 0.26 

' nmoV minl mg protein or DNA; Clp/ mg protein or DNA 

The data arc expressed as meaniSD for six mimalsl dose

Table 30. Effect of co-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in the epididylnal sperm oradult rats 


Control 1ng+20mg 10ng+20mg 100ng+20mg 

Superoxide disrnutase' 

(mg protein) 19.84 f 0.89 19.93 f 1.36 20.16 f 1.57 19.81 -t 0.42 

(mg DNA) 16.84 5 0.64 17.01 f 0.76 17.01 f 0.77 17.04 & 0.89 

catalaseb 

(mg protein) 2.12i 0.08 2.13* 0.10 2.151 0.12 2.12f 0.12 

(mg DNA) 1.80 f 0.03 1.82 i 0.03 1.81 * 0.03 1-82 2 0.04 

Glutathione ductase' 

(mg protein) 34.03 i 1.45 33.80f 1.61 34.36 * 1.94 33.54 +- 2.38 

(mg DNA) 28.87* 0.51 28.85 f 0.67 29.01 f 0.45 28.76& 0.96 

Glutathione peroxidase' 

(mg protein) 58.40 f 2.58 58.70 * 2.85 58.96 i 3.19 58.06 + 3.48 

(mg DNA) 49.53i 0.85 50.12* 1.24 49.80f 1.13 49.92i 0.98 

Lipid peroxidation' 

(mg protein) 2.22* 0.16 2.19i 0.16 2.20f 0.19 2.19* 0.18 

(mg DNA) 1.88iO.09 1.87f0.07 1.8620.12 1.87+0.06 

nmold mid mg protein or DNA 

moV mid mg protein or DNA 

pnol rnalondialdehyde produced/ mg protein or DNA 

The data are expressed as mean* SD for six animals1 dose

Table 31. Effect of TCDD on the production of superoxidc anion. nitric oxide and 

hydrogen peroxide in the caput, corpus and cauda epididyunides of adult rats 


Control Ing+20mg IOng+20mg 100ng+20mg 

Capur epididymh 

Superoxide anion' 1.69tt 0.09 1.76i 0.12 1.79k 0.18 1.76 + 0.17 

Nitric oxideb 1.39i 0.19 1.43* 0.09 1.47i 0.16 1.49 i 0.11 

Hydrogen peroxidec 13.43 i 1.40 11.75 i 1.07 11.73 * 1.39 11.47 2 0.73 

Corpus epididymb 

Superoxide aniona 1.71 + 0.11 1.76+ 0.21 1.792 0.13 1.81 + 0.12 

N~tric oxideb 1.372 0.13 1.37+ 0.09 139i 0.08 1.442 0.30 

Hydrogen peroxidec 17.88 + 0.93 15.84 1.41 14.66 + 1.16 13.18 + 1.32 

Coudo epidi+mis 

Superoxide anion' 1.82 + 0.09 1.92 * 0.13 1.87 1 0.36 1.84 i 0.14 

Nitric oxideb 1.29f 0.21 1.37+ 0.09 1.33 1- 0.16 1.35+ 0.13 

Hydrogen peroxidec 15.85 i 0.49 15.22 i 1.08 14.89 i 0.86 14.89 + 0.74 

a nmole/ mini rng protein; mrnolel rnin/ mg protein 

nmol Hz02 generated1 minl mg protein 

The data are expressed as rneanYSD for six animals/ dose

Table 32. 

Effect of caadrninistration of TCDD and vitamin E on the levels of 

antioxidants in the caput, corpus and cauda epididymides of adult rats 


Control 1 ng+20mg 10ng+20mg 100ng+2Omg 

Glutathione' 1675399 15654.90 15826.31 160i4.11 

a-~ocopherol~ 0.83i 0.1 1 0.84i 0.09 0.84t 0.40 0.84i 0.17 

Corpus epididymis 

Glutathione' 149 f 6.14 152 i 2.62 150 i 3.21 151 i 4.26 

a-Tocopherolb 0.84 i 0.16 0.844 2 0.21 0.84 2 0.20 0.82 i 0 I l 

Glutathione' 160 * 5.26 163 i 6.16 166 i 7.11 162 5 7.36 

a-~oco~herol~ 0 85 f 0.1 l 0.86 i 0.26 0.85 + 0.21 0.85 + 0.32 

nmoldmg protein; pg mg protein 

The data are expressed as rneanfSD for six animals/ dose

Table 33a. Effect of co-administration of TCDD and vitamin E on tlic antioxidant 

cnzymcs and lipid pcroxidation in the caput cpididy~iiis 


Superoxide dismutasea 33.82 i 2.35 33.47 f 1.58 33.66 f 2.39 33.84 * 1.78 

catalaseb 2.56i0.17 2.51 i 0.10 2.52f 0.18 2.545 0.15 

Glutathione reductase' 43.46 f 2.82 42.84 f 1.68 43.23 * 2.96 43.49 12.84 

Glutathione peroxidase'64.49 f 4.32 63.53 i 3.09 63.91 k 4.25 64.1 1 f 4.22 

Lipid peroxidatione 2.50 i 0.1 l 2.35 * 0.20 2.30 i 0.14 2.35 f 0.21 

'nmold min/ mg protein; pnold minl rng protein 

' pmole malondialdehyde producedl mg protein 

The data arc expressed as me.aniSD for six animals/ dose 

Table 33b. Effect of co-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in the corpus epididymis 


Control 1ng+20rng IOng+20mg 100ng+20mg 

Superoxide dismutasc' 36.39 * 1.49 37.1 1 f 2.48 36.91 i 3.48 37.03 _+ 2.32 

Catalascb 3.49f 0.18 3.51 i0.17 3.47i0.27 3.45i 0.15 

Glutathione reductasec 32.80 * 1.53 33.20 f 1.66 32.78 It 2.73 23.63 5 1.49 

Glutathione peroxidasec62.36 f 3.74 62.85 f 2.98 62.24 i 4.20 62.14 f 3.52 

Lipid peroxidation' 2.46 f 0.30 2.49 i 0.12 2.60 i 0.29 2.55 + 0.23 

'nmold mint rng protein; wold minl mg protein 

'wnole malondialdehydc produced1 mg protein 

The data arc expressed as rneaniSD for six animals/ dose

Table 33c. Effcct of co-administrution or 'I'CnD and vil;~~nin I:, OII tlw :~~~tiosidi~~~t 

enzymes and lipid peroxidation in the cauda epididymis 


Control Ing+20mg IOng+20mg 100ng+20mg 

Superoxide dismutase' 43.80 * 1.88 44.71 f 2.05 44.64 r 1.57 44.79 i 2.18 

catalaseb 2.99i0.13 3.03 f 0.15 3.01 i0.08 3.02t0.12 

Glutathione reductase' 40.18 * 1.49 40.89 2 2.20 40.6 1 * 1.46 40.75 f 1.68 

Glutathione peroxidasec41.85 * 1.87 42.53 t 2.05 42.45 f 1.39 42.42 f 1.56 

Lipid peroxidationd 1.91 * 0.14 1.85 + 0.19 1.80* 0.15 1.78 i 0.13 

'nmolel m~nl mg protein; pnolel mid mg protein 

' pmole malondialdehyde produced/ mg protein 

The data are expressed as mcaniSD for six animals1 dose 

Table 34a. Effect of co-administration of TCDD and vitamin E on the production of 

supzmxide anion, nitxic oxide and hydrogen peroxide in the kidney of adult 

rats 


Control I ng + 20 mg 10 ng + 20 nig 100 11g + 20 nlg 

Superoxide anion' 2.1 1 i 0.18 2.16 i 0.22 2.09 i 0.1 1 2.13 i 0.21 

Nitric oxideb l.5lO.ll 1.53f 0.15 1.60f 0.17 1.59 f 0.16 

Hydrogenperoxidd 11.12 t2.01 12.76i 1.97 13.70k 2.11 13.51 _+ 1.67 

nmolel mid mg protein; mmoW mint mg protein 

' nmol H201 generated1 mint mg protein 

The data arc expressed as moanfSD for six animals/ dose

Table 34b. Effect of co-administration of TCDD and vitamin E on the levels of 

antioxidants in the kidney of adult rats 

- 


Control I ng+20mg 10ng+20mg 100ng+20mg 

Glutathione' 178 f 5.21 170 i 9.29 188 * 7.63 172 i 8.33 

a-~ocopherol~ 0.93 f 0.16 0.891 0.07 0.80-f 0.15 0.83 ?C 0.20 

nmolelmg protein; & mg protein 

The data arc expressed as mean* SD for six animals/ dose 

Table 34c. Effect of co-administration of TCDD and vitamin E on the antioxidant 

enzymes and lipid peroxidation in the kidney of adult rats 


Control Ing+20rng 10ng+20mg 100ng+20mg 

Superoxide dismutasc' 37.1 1 i 3.18 36.98 t 2.67 37.38 f 3.10 35.13 f 2.23 

Catalascb 2.61 0.23 2.58 i 0.20 2.56k 0.16 2.61 * 0.26 

Glutathione reductare' 42.16 f 3.3 1 40.01 12.56 42.76 i 3.10 40.54 i 3.91 

Glutathione peroxidax'63.18 i 3.12 60.8914.32 60.41 13.88 59.15 3.10 

Lipid peroxidationC 

2.22 i 0.16 2.28 f 0.26 2.31 t 0.19 2.28 ~ 0.18 

'nmole! mid mg protein; tunold minl mg protein 

' wlnolc malondialdehyde pmduccdl I I I protein 

~ 

The data am expressed as mean* SD for six animals1 dose

5 DISCUSSION 

5.1 Significance of the experimental procedures 

Rats were treated orally with graded doses of TCDD and the effects were seen 

on various parameters of male reproductive functions. In order to see the effects of 

vitamin E on TCDD-induced alterations in the testis and epididymis the rats were 

administered with vitamin E along with TCDD. 

5.1.1 Experimental treatments 

TCDD is one of the most potent environmental contaminants, which has been 

shown to alter male reproduction of rats causing reduced fertility. In the present study 

TCDD (1, 10 and 100 ngi Kg body weight) day) was administered orally to 45 days 

old male rats for 45 days in order to see the effects of subchronic exposure of TCDD 

on functional aspects of male reproduction. In the subchronic toxicity test with rats 

exposure usually begins at 6 to 8 weeks of age (US Environmental Protection Agency, 

1998). Rats at postnatal day 45 have been reported to be at the threshold of puberty 

where the first spermatozoa are seen in the lumen of the seminiferous tubules 

(Clermont and Perey, 1957). The duration of the whole process of spermatogenic 

cycle in male rats is approximately 45 days (Clermont and Perey, 1957). In the 

present studies rats were treated from puberty to adulthood through a complete 

spermatogenic cycle. TCDD was administered orally because most of the 

environmental contaminants enter into the body through this route. 

For standardization of doses, rats were treated with seven different doses of 

TCDD for 45 days. The doses ranged from 0 to 1000 ng/ Kg body weight/ day and 3 

animals were used for each dose. The body weight gains were recorded for 45-days. 

Administration of TCDD at the dose levels of 0 to 100 ng/ Kg body weighti day for 

45 days did not show any significant changes in the body weight (Fig. 2 & 3). 

However, adminiseation of TCDD at higher doses decreased body weight of the

animals (Fig. 2) thereby causing systemic toxicity. Thus, we selected doses ranging 

between 1 to 100 ng/ Kg body weight/ days for 45 days. 

Vitamin E is a lipophilic compound most commonly used as an antioxidant. 

Administration of vitamin E at the dose level of 20 rngl Kg body weight/ day is 

considend to be a therapeutic dose. In the present study TCDD (1, 10 and 100 ng/ 

Kg body weight1 day) was administered orally along with vitamin E (20 mgl Kg body 

weight/ day) to the pubertal rats for 45 days in order to see the protective effects. if 

any. exerted by vitamin E supplementation on the TCDD-induced toxicity in male 

reproductive system of rats. The animals were killed after 24 h of the last treatment in 

order to see the effects of TCDD, with or without supplementation of vitamin E on the 

male reproductive system of rats. 

5.1.2 Significance of the parameters studied 

Monitoring body weight of the animals during treatment provides an index of 

the general health status of the animals and such information is important for the 

interpretation of reproductive effects. Depression in body weight or reduction in 

weight gain may reflect a variety of responses. including rejection of feed and water, 

treatment induced anorexia or systemic toxicity. In the present study the weights of 

the animals were recorded to monitor such changes. Weight of the testis is largely 

dependent on the mass of the differentiated spermatogenic cells and it has been used 

as a crude measure of the damage to spermatogenesis (Schlappack el 01.. 1988). A 

strong correlation exists between weight of the testis and number of germ cells (Sinha 

Hikim el 01.. 1989). Reduction in the weight of testis has been shown to occur due to 

the loss of germ cells (Setchell and Galil, 1983). In the present study weight of the 

testis was taken to assess the maintenance of various testicular functions. 

Weights of the accessory sex organs are dependent on the availability of 

androgens, as castration has been shown to reduce the weights due to absence of 

androgens. Incrcase in the availability of testosterone has teen shown to cause 

bpertmphy md consequent increase in the weights of the accessory sex organs.

Measurement of weights of the accessory sex organs in intact male rats has been 

demonstrated to reflect the cumulative effect of biologically active testosterone over a 

period of time (Mathur and Chattopadhyay, 1982). In the present studies weights of 

epididyrnis, seminal vesicles and ventral prostate were taken to assess the 

bioavailability andl or production of androgen and the cumulative effect of 

androgenic activity. Weight of kidneys of the castrated mice decreased by 30 - 50% 

in comparison to that of intact animals and has been reported to be androgen 

dependent. The toxicity threshold varies in different species as well as varies in 

different organs in the same animals. In the present study weight of kidney was taken 

to assess the toxicity threshold between reproductive and non-reproductive tissue. 

Daily sperm production in rats reflects the status of spermatogenesis in control 

and experimental animals. Epididymal sperm viability. epididymal sperm motility 

and sperm count alter when exposed to environmental toxicants. In the present study 

daily sperm production, epididymal sperm viability, motility and count were done in 

order to assess the status of spermatogenesis. 

Histometric studies on testis provide strongest evidence to see the difference 

between control and experimental tissues (Russell rl a/., 1990). 

A positive 

relationship exists between tubular diameter and spermatogenic activity of testis 

(Sinha Hikim ct a/.. 1989). Measurements of tubular diameter have bren reponed to 

discriminate between varying levels of sp.rnmatogcnic damage (Russcll ct

of testosterone, estradiol, FSH, LH and prolactin were determined in extracellular 

fluid to assess any alteration in their availability to target sites. 

The two key enzymes involved in the biosynthetic pathway of testosterone are 

3p-hydroxysteroid dehydrogenase and I7P-hydroxysteroid dehydrogenase. The 

activities of 3P-hydroxysteroid dehydrogenase and 17P-hydroxysteroid 

dehydrogenase have been used to study testicular steroidogenesis of rats in different 

experimental conditions (Ghosh e! a/., 1991; 1995: Sujatha er u/., 2001). It has been 

reported that increased production of reactive oxygen species decreased the activities 

of steroidogenic enzymes in testis of rats (Sujatha el a/.. 2001). Determination of 

activities of 30-hydroxysteroid dehydrogenase and 17P-hydroxysteroid 

dehydrogcnase in testis reflected the status of steroidogenesis. 

Nucleic acid contents in testis vary in different stages of spermatogenesis and 

the amount of DNA in the primary spermatocyte, secondary spermatocyte and 

spermatid has been shown to be present in the ratio of 4: 2: 1 (Davson and Segal, 

1975). Protein and RNA synthesis occur both in somatic cells and in germ cells. In 

the present study quantitative determination of DNA in testis indicated the rate of cell 

division and subsequent maturation. Determination of the levels of FWA and protein 

showed thc resultant effect of intracellular synthetic and breakdown processes. 

The superoxide anion. nitric oxide and hydrogen peroxide are reactive oxygen 

species produced from the cell under normal cellular metabolism. Increased 

production of superoxide anion and hydrogen peroxide under oxidative stress has 

been reported in the reproductive and and non-reproductive tissues of the animals 

(Hassoun el 01.. 2001; Sujatha er al., 2001). Productions of superoxide anion, nitric 

oxide and hydrogen peroxide were determined in testis as well as in the epididyrnal 

sperm and epididymis in order to assess the levels of reactive oxygen species in 

tissues. 

The levels of antioxidants such as glutathione, a-tocopherol and ascorbic acid 

play an imponant role in the maintenance of tissue against reactive oxygen species. 

These antioxidants have been reported to be distributed in the subcellular

compartment of the cells (Chainy el at., 1997; Reddy er at., 1999). Determination of 

the levels of glutathione, a-tocopherol and ascorbic acid reflect the status of 

antioxidants in tissues. 

Superoxide dismutase, catalase, glutathione reductase and glutathione 

peroxidase are antioxidant enzymes distributed in different subcellular fractions of 

testis as well as in the epididymal sperm and epididymis (Sujatha et al., 2001; Chitra 

er a/., 2001; Latchoumycandane et at., 2002a, b, c). Superoxide dismutase is 

considered to be the first line of defense against deleterious effects of oxyradicals in 

cell by catalyzing dismutation of superoxide anion to hydrogen peroxide and 

molecular oxygen. Catalase, glutathione reductase and glutathione peroxidase protect 

cells from highly toxic hydrogen peroxide by catalyzing hydrogen peroxide into water 

and oxygen. In the present studies the activities of superoxide dismutase, catalase, 

glutathione reductase and glutathione peroxidase in testis. epididymal sperm and 

epididymis were determined to assess the activities of the antioxidant enzymes in the 

TCDD-treated rats. 

5.2 Effect of TCDD on male rats 

Effect of TCDD on the body weight, organ weights and reproductive 

parameters of male rats are discussed in the following sections. 

5.2.1 Effect of TCDD on body and organ weights 

Administration ol"l'CDD did not cause any signilicant cha~~gc in h)d) weight 

of rats as compared to the corresponding groups of control animals (Fig. 3; Table la) 

~ndicating that the general me~aboliconditions of the animals wcrc within the nonnal 

range and TCDD at these doses did not cause systemic toxicity. 

Administration of TCDD caused a significant reduction in the weight of the 

testis as compared to the corresponding groups of control animals (Fig. 4a; Table la 

& I b). In the male rats TCDD exposure during adulthood has been shown to decrease 

weight of the testis (Johnson er at., 1992). It has been reported that TCDD induces a

eduction in Leydig cell volume per testis (Wilker, 1996). Reduction in the weight ol' 

the testis could be due to the Loss of germ cells by direct inhibition of TCDD on 

spermatogenesis or an indirect effect through inhibition of testosterone production. 

The present findings are consistent with previous report in which reduction in weight 

of the testis may be due to the loss of germ cells and Sertoli cells in the TCDD-treated 

rats (Mably el al., 1992; Johnson el ul., 1992). 

The weights of epididymis, seminal vesicles and ventral prostate decreased 

significantly in adult rats while weight of the kidney remained unchanged following 

administration of TCDD (Fig. 4b to 4f; Table la & I b). Epididyrnis and accessory 

sex organs require a continuous androgenic stimulation for their normal growth and 

functions (Klinefelter and Hess, 1998). It has been reported that accessory sex organs 

are androgen dependent and thus reflect the availability of androgen over a period of 

time (Mathw and Chanopadhyay. 1982). TCDD has also been reported to reduce 

plasma testosterone and dihydrotestosterone in the rats (Mably er ul., 1992). 

Inhibition on the weights of the epididymis, seminal vesicles and ventral prostate may 

be either due to the reduction in the levels of serum testosterone or a direct action of 

TCDD on the epididymis and accessory sex organs. 'She observed reduction in thu 

weight of accessory sex organs also reflected reduced bioavailability andl or 

production of androgen in the TCDD-treated rats. 

In the present studies 

administration of TCDD at these doses did not affect the weights of the extra- 

reproductive tissues like kidney though the activity of P-gluco~nidase in kidney has 

been demonstrated as testosterone dependent enzyme (Malarvizhi and Mathur, 1996). 

5.2.2 Effect oCTCDD oo daily spcrm production 

Adn>inistntion of TCDD dccrcascd siynilicunlly thc doily spcrm production 

as compared to the corresponding groups of control animals (Fig. 5a: Table 2). Thc 

observed reduction in the daily sperm production also rellects reduction in 

spermatogenesis. The present findings are consistent with the earlier reports in which

the reduction in daily sperm production has been shown in TCDI>-treated rats 

(Sommer el ul., 1996; Faqi el ul., 1997; 'l'hcohald and I'ctorson, 1997). 

5.2.3 Effect of TCDD on epididymal sperm viability, motility and count 

The epididymal sperm viability remained unchanged while the epididymal 

sperm motility decreased significantly in a dose-dependent manner in the animals 

administered with TCDD (Fig. 5a & 5b; Table 2). 

Administration of TCDD decreased epididymal sperm count significantly 

when compared to the corresponding groups of control animals (Fig. Sa; Table 2). 

The present findings are consistent with the earlier report in which the reduction in 

daily sperm production and epididymal sperm count in the rats treated with TCDD 

(Sommer el al.. 1996; Faqi el al., 1997; Theobald and Peterson, 1997). 

5.2.4 Effect of TCDD on seminiferous tubular and lumen diameter 

Diameter of the seminiferous tubular and lumen decrcased signilicantly alicr 

administration of TCDD as compared to the corresponding groups of control animals 

(Fig. 6; Table 3). The reduction in the tubular and lumen diameter of' thc 

seminiferous tubules may be due to a direct action of TCDD on the seminiferous 

tubules or an indirect effect through inhibition of testosterone production. The 

reduction in the tubular diameter has been correlated with decreased spermatogenic 

activity of the testis (Sinha Hikim cl a/., 1989). 

5.2.5 EfTcct of TCDD on the levels of serum horlnoi~cs and stcrc~idogei~ic 

enzymes in testis 

The levels of serum testosterone and rstradiol were found lo be decreased in 

the TCDD-treated rats along with reduction in the activities of 3P-hydroxysteroid 

dehydrogenase and I7p-hydroxysteroid dehydrogenase in testis (Fig. 7a to 7e & 8: 

Table 4 & 5). A decrease in the intratesticular and serum testosterone concentration 

has been reported in rats following administration of TCDD (Wilker. 1996). It has

een reported that TCDD decreases volume of the smooth e~idoplasmic reticulum and 

mitochondria per testis and has been shown to alter testicular steroidogenesis in rats 

and thereby reduce the production of testosterone and estradiol in rats (Wilker, 1996). 

The decrease in the serum testosterone levels in adult rats could be due to the 

diminished responsiveness of Leydig cells to LH and or the direct inhibition of 

testicular steroidogenesis. It has been reported that two of the steroidogenic enzymes 

involved in the conversion of cholesterol to testosterone in Leydig cells are 

cytochrome P450 enzymes (P450scc and P450c17). Molecular oxygen and electrons 

donated from NADPH were used for hydroxylation of the substrate. During normal 

steroidogenesis, ROS (superoxide anion and or hydroxy radical) can be produced by 

electron leakage outside the electron transfer chains (Homsby and Crivello, 1983; 

Hanukoglu el al., 1993), and these free radicals can initiate lipid peroxidation, which 

can inactivate P450 enzymes (Homsby, 1980). The inability of the pseudosubsuate to 

be oxygenated promotes the release of reactive oxygen species (Peltola et al., 1996). 

It has been reported that increased production of reactive oxygen species following 

lindane treatment decreased the activities of steroidogenic enzymes in testis of rats 

(Sujatha el a/.. 2001). 

The levels of serum FSH and LH remained unchanged while the level of 

prolactin decreased significantly in the animals administered with TCDD (Fig. 7c to 

7e; Table 4). This is supported by previous reports that TCDD does not alter serum 

FSH and LH levels in rats (Moore er 01.. 

1989) thereby suggesting that pituitary 

hypofunction may not be a major cause of the initial stages of subchronic TCDD 

toxicity. It has also been suggested that growth retardation in TCDD-treated rats may 

not be the result of a deficiency of growth hormone (GH), alterations in plasma 

conicosterone concentrations may due to altered responsiveness of the adrenal to 

ACTH stimulation rather than to changes in plasma ACTH concentrations. and that 

impaired spermatogenesis may not be associated with a decrease in plasma FSH 

concentrations (Moore el ul., 1989). 

l'hc levels of scruni prolnctili dccrcucd 

significantly in the animals wated with TCDD. Similar changes have also been

eported in the TCDD-treated rats (Moore rt al.. 1989). It has been rcportcd that 

prolactin increases the number of LH receptors and potentiate steroidogenic effect of 

LH on Leydig cells while testicular prolactin receptors have been shown to be 

confined to the interstitial tissue of the testis (Johnson and Everitt, 1995). Similarly 

prolactin has been shown to increase the uptake of androgen and increases 5-a 

reductas activity in the prostate, while testosterone maintains the number of prolactin 

receptors in the prostate. Prolactin has also been shown to potentiate the effect of 

testosterone on accessory sex organs (Mathur and Chattopadhyay, 1986). The 

reduction in the levels of serum testosterone may also be due to decreased serum 

prolactin level in the TCDD-treated rats. 

5.2.6 Effect of TCDD on nucleic acid and protein contents in testis 

Administration of TCDD decreased in DNA, RNA and protein contents of the 

testis as compared to the corresponding groups of control animals (Fig. 9; Table 6). 

The observed reduction in the levels of DNA. RNA and protein in the TCDD-treated 

rats reflects the reduced synthetic and metabolic activities of the testis. 

5.2.7 Effect of TCDD on antioxidant system in testis 

Administration of TCDD increased the production of superoxide anion, nitric 

oxide and hydrogen peroxide in the crude homogenate, mitochondrial and 

microsome-rich fractions of testis of rats when compared to the corresponding groups 

of control animals (Fig. 10a to IOc; Table 7a to 7c). Treatments of rats with TCDD 

decreased the level of antioxidants such as glutathione, a-tocopherol and ascorbic 

acid in the crude homogenate, mitochondrial and microsonle-rich fractions of testis of' 

rats (Fig. 1 la to I Ic; Table 8a to 8c). 

Administration of TCDD decreased the activities of superoxide dismutase. 

camlase, glutathione rcductase and glutathione peroxidase (Fig. 12a to 12d; Table 9a 

to 9d) while the level of lipid peroxidation (Fig. 13; Table 10) increased significantly 

in the crude homogenate, mitochondria1 and microsome-rich fractions of testis of rats

as compared to the corresponding groups of control animals. Increased production of' 

reactive oxygen species. decreased levels of antioxidants as well as the activities of' 

antioxidant enzymes and increased levels of lipid peroxidation reflected that testis are 

In oxidative stress. 

Increased production of superoxide anion in the hepatic and brain tissues of 

rats after exposure to TCDD has been reponed (Hassoun r! ul., 2001). TCDD 

increased the production of reactive oxygen species in mitochondria1 and microsomal 

fractions of rat testis (Latchoumycandane er ul., 2002 c). Nitric oxide has been shown 

to react with superoxide anion to produce peroxynitrite, which actively react with 

glutathione, cysteine, deoxyribose and thiolsl thioethers (Sikka, 2001). It has been 

reported that acute exposure of TCDD in mice decreased the levels of glutathione and 

a-tocopherol in hepatic tissues (Slezack el a[.. 1999). Glutathione is important in the 

regulation of the cellular redox state and a decline in its cellular level in TCDDtreated 

rats is considered to be indicative of increased oxidative stress. The reduction 

in testicular glutathione levels in TCDD-treated rats might be in part attributed to the 

inhibition of glutathione reductase activity, which is responsible for regcneration of 

glutathione from its oxidised form Sikka (2001) showed that glutathione reductase is 

inactivated by superoxide anion. Therefore, reduced superoxide dismutase activity in 

testis might have enhanced flux of superoxide radicals that potentially could damage 

glutathione rcductase and decrease glutathione content in testis. In addition, decrease 

in glutathione level might reflect a direct reaction between glutathione and free 

radicals generated by TCDD. This is consistent with function of glutathione of 

scavenging oxidants by binding to them covalently. Furthermore. a regression of thc 

antioxidant recycling mechanism duc to a decrease in ascorbic acid level in 'l'CI)Vtreated 

rats might contributed to a decline in glutathione level in testis. This is in 

accordance with the position of glutathione at the end of the cycling mechanism. 

which includes vitamin E and vitamin C (Shindo er ul.. 1994). 

Ascorbic acid is the most important free radical scavenger within membranes 

and lipoproteins. Alpha-tocopherol is a well-known chain breaking antioxidant; its

lilnction is to ililcrccpt lipid pcroxyl rudiouls and thcrchy tcr~lii~l~tc lipid pc~.oxid;~livo 

chain reactions. 

The free radical clearing ability of u-tocopherol is due to 

delocalisation of an unpaired electron in its conjugated double bond system. The 

observed reduction in the levels of antioxidants in the testis of rats treated with TCDD 

might be due reduced recycling of antioxidants in tissues. 

Oxidative stress induced by TCDD has been shown to occur primarily in the 

mitochondria followed by microsome-rich fractions (Latchoumycandane el a/., 

2002~). Decreased activities of antioxidant enzymes and increased level of lipid 

peroxidation in the mitochondrial and rnicrosomal fractions have been reported in the 

testis of rats following TCDD treatments (Latchoumycandane el a[., 2002~). 

5.2.8 Effect of TCDD on antioxidant system in the epididymal sperm 


oxide and hydrogen peroxide in the epididymal sperm of rats when compared to the 

corresponding groups of control animals (Fig. 14a to 14c; Table 11). Administration 

of TCDD decreased the level of antioxidants such as glutathione and a-tocopherol in 

the epididyrnal sperm of rats (Fig. 1 Sa & 15b; Table 12). Administration of TCDD 

decreased the activities of superoxide dismutase, catalase, glutathione reductase and 

glutathione peroxidax (Fig. 16a to 16d: Table 13) while the level of lipid 

peroxidation increased significantly in the epididymal sperm of rats as compared to 

the corresponding groups of control animals (Fig. 17; Table 13). 

TCDD has been shown to increase the production of reactive oxygen species 

in the epididymal sperm of rats (Latcho~~~iiycand;~nc ct 111.. 2002h). Incrcilscd 

production of supcroxide anion nnd hydrogen pcroxidc in the cpididymul hpcrtn ct~uld 

reflect the adverse effect of TCDD 011 the generation of reactive oxygen species. l'he 

observed reduction in the level of glutathione and u-tocopherol reflect adverse effect 

of TCDD on the antioxidants in the epididymal sperm of nts. Reduction in the 

activities of antioxidant enzymes and increased levels of lipid peroxidation in the 

epididyrnal sperm has already been reported following TCDD treatment

(Latchuumycandane el ul.. 2002 b). Uccrcasod activitics of antioxidant cnzymcs and 

increased levels of lipid peroxidation in the epididymal sperm could rcveal the 

adverse effect of TCDD on the antioxidant system of rats. 

5.2.9 Effect of TCDD on antioxidant system in the caput, corpus and cauda 

epididymides 


oxide and hydrogen peroxide in the caput, corpus and cauda epididymides of rats 

when compared to the corresponding groups of control animals (Fig. 18a to 18c; 

'Table 14). Administration of TCDD decreased the levels of antioxidants such as 

glutathione and a-tocopherol in the caput, corpus and cauda epididymides of rats 

(Fig. l9a & 19b; Table 15). Administration of TCDD decreased the activities of 

superoxide dismutase. catalase, glutathione reductase and glutathione peroxidase (Fig. 

20a to 2Od; Table 16a to 16c), while the level of lipid peroxidation incroascd 

s~gnificantly in the caput, corpus and cauda epididymides of rats (Fig. 21: Table 16a 

to 16c). Similar observation has also been reported in caput, corpus and cauda 

epididymides of rats foIlowing treatment with lindane (Chitra eta/.. 2001). 

5.2.10 Effect of TCDD on antioxidant system in kidney 

Administration of TCDD at the dose level of 100 ngl Kg body weight! day 

increased the production of superoxide anion, nitric oxide and hydrogen peroxide in 

the kidney of rats when compared to the corresponding groups of control animals 

(Fig. 22a to 22c; Table 17a). TCDD decreased the levels of antioxidants such as 

glutathione and a-tocopherol in the kidney of rats at 100 ng dose level (Fig.23a L 

23b; Table 17b) when compared to the corresponding groups of control animals. 

Administration of TCDD decreased the activities of superoxide dismutase. catalase. 

glutathione reductase and glutathione peroxidase at 100 ng dose level (Fig. 24a to 

24d; Table 17c) while the level of lipid petoxidation increased significantly in the 

kidney of rats (Fig. 25: Table 17c). However, no significant changes were observed

in the animals administered with TCDD at I and 10 ng dose Icvcls. Thc lcvcls of 

some of the antioxidant enzymes were higher in kidney us compared to testis and 

epididymis. Thus the levels of antioxidant mechanisms could be higher in kidney 

which could prevent it from TCDD-induced oxidative stress at low doses. It is also 

possible that toxicity response threshold in terms of oxidative stress is lower in testis 

and epididymis than in kidney since low doses of TCDD could not induce oxidative 

stress in the kidney of rat. 

5.3 Effect of co-administration of TCDD end vitamin E on male rats 

Effects of co-administration of TCDD and vitamin E on various parameters of 

male reproduction were studied and the results are discussed below. 

5.3.1 Effect of co-administration of TCDD and vitamin E on body and organ 

weights 

Co-administration of TCDI) and vitamin E did not show any significant 

change in the body weight of rats as compared to the corresponding groups of control 

an~mals (Table 18a) indicating that the general metabolic conditions of the animals 

were within the normal range and that the administration oT'TCDD along with vitamin 

E did not cause systemic toxicity. 

Administration of TCDD along with vitamin E did not cause any reduction in 

the weight of the testis when compared to the corresponding groups of control 

animals (Fig. 4 a; Table 18a & 18b). It is thus suggested that co-administration of 

vitamin E along with TCDD could impart protective effect against TCDD on the 

weight of the testis. The weight of the epididymis, seminal vesicles and ventral 

prostate did not show any significant dinermces in the rats administered with 'TCDI) 

along with vitamin E when compared to the corresponding groups of control animals 

(Fig. 4b to 4e; Table 18a & lab). The results suggested that co-administration of 

vitamin E along with TCDD could give protection to the epididymis and accessory

sex organs against TCDD. Weight of the kidney remained unchanged in the animals 

co-administered with TCDD and vitamin E (Fig. 4f; Table 18a & 18b). 

5.3.2 Effect of co-administration of TCDD and vitamin E on daily sperm 

production, epididymal sperm viability, motility and sperm count 

The daily sperm production and epididymal sperm viability, motility as well as 

epididymal sperm count remained unchanged in the animals administered with TCDD 

and vitamin E as compared to the corresponding groups of control animals (Fig. Sa & 

Sb; Table 19). It is thus suggested that co-administration of vitamin E along with 

TCDD could impart protective effect against TCDD on the epididymal spenn 

viability and motility as well as daily sperm production and epididymal sperm count. 

5.3.3 Effect of co-administration of TCDD and vitamin E on seminiferous 

tubular and lumen diameter 

Diameter of the seminiferous tubular and lumen did not show any significant 

change in the rats co-administrated with TCDD and vitamin E as compared to the 

corresponding groups of control animals (Fig. 6; Table 20). Thus co-administration 

of vitamin E along with TCDD could impart protective effect against TCDD to 

seminiferous tubules of rat testis. 

5.3.4 Effect of co-administration of TCDD and vitamin E on serum hormone 

levels and steroidogenic enzymes in testis 

The levels of serum testosterone and estradiol did not show any significant 

change in the rats co-administered with TCDD and vitamin E as were found to be 

decreased in the TCDD-treated rats (Fig. 7a to 7e; Table 21). Co-administration of 

TCDD and vitamin E did not cause any significant change the activities of' 3Phydroxysteroid 

dehydrogenase and 17P-hydroxysteroid dchydrogenasc in tcslis as 

compared to the corresponding groups of control animals (Fig. 8; Table 22). It is thus 

suggested that co-administration of vitamin E along with TCDD could impart

protective effects on serum testostcronc and cstradiol lcvcls by prcvcnting tho 

decreased activities of steroidogenic enzymes in testis of TCDD-treated rats. 

The levels of serum FSH and LH remained unchanged in the animals 

administered with TCDD and vitamin E which is similar to TCDD-treated groups. 

However, the levels of serum prolactin did not decrease significantly in the animals 

treated with TCDD and vitamin E as was shown to decrease in the rats treated with 

TCDD alone (Fig. 7a to 7e; Table 21). It is thus suggested that co-administration of 

vitamin E along with TCDD could impart protective effect on serum prolactin. 

53.5 Effect of co-administration of TCDD and vitamin E on nucleic acid and 

protein contents in testis 

Administration of TCDD along with vitamin E did not show any significant 

chanye in the DNA. RNA and protein contents of the testis as was shown to decrease 

~n tlx TCDD-treated rats (Fig. 9; l'ablc 23). It is thus suggcstcd that coadministration 

of vitamin 1:. along with TCIID could inipart protective efl'cct on 

synthetic and metabolic activities of the testis 

5.3.6 Effect of co-administration of TCDD and vitamin E on antioxidant system 

in testis, epididymal sperm and epididymis 

Administration of TCDD along with vitamin E did not show any significant 

chanye in the production of superoxide anion, nitric oxide and hydrogen peroxide in 

the crude homogenate, mitochondrial and microsome-rich fractions of testis of rats as 

well as epididymal sperm and various regions of the epididymis when compared to 

the corresponding groups of control animals (Fig. !Oa to IOc; 'l'ahlc 24a lo 245; I;ig. 

14a to 14c; Table 28 and Fig. 18a to 18c; 'l'ablc 3 I ). 

Treatments of rats with TCDD along with vitamin E did not alter the level of 

antioxidants such as glutathione, a-tocopherol and ascorbic acid in the crude 

homogenate, rnitochondrial and microsome-rich fractions of testis as well as 

epididymal sperm and epididymis of rats (Fig. 1 la to 1 Ic; Table 2Sa to 25c; Fig. 15a

to Fig. 15b; Table 29 and Fig. 19a lo I'if I9b; 'l'ablc 32). Administralion of'1'CL)I) 

along with vitamin E did not show any significant change in the activities of 

superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase (Fig 

12a to IZd, Table 26a to 26d; Fig. 16a to Fig. 16d; Table 30 and Fig. 20a to Fig. 2Od; 

Table 33a to 33c) as well as the level of lipid peroxidation (Fig.13; Table 27; Fig. 17; 

Table 30; and Fig. 21; Table 33a to 33c) in the crude homogenate, mitochondria1 and 

microsome-rich fractions of testis. epididymal sperm and various regions of the 

epididymis of rats as compared to the corresponding groups of control animals. The 

results suggested that vitamin E could protect testis and epididymis against TCDDinduced 

reactive oxygen species mediated toxicity. 

5.3.7 Effect of co-administration of TCDD and vitamin E on antioxidant system 

in kidney 

Administration of TCDD along with vitamin E did not cause any significant 

change In the production of superoxide anion. nitric oxide and hydrogen peroxide in 

the kidney of rats as was shown to decrease in the TCDD-treated rats at 100 ng dose 

level (Fig. 22a to 22c; Table 34a). The levels of glutathione and a-tocopherol in the 

kldney of rats treated with TCDD and vitamin E remained unchanged (Fig.23a to 23b; 

'Table 34b). There were, however, no changes in the levels of antioxidants in TCDD 

alone treated groups at lower doses such as I and I0 ng levels. 

Co-administration of TCDD and vitamin E did not cause any significant 

change in the activities of superoxide dismutase, catalase, glutathione reductase and 

glutathione peroxidase (Fig. 24a to 24d; Table 34c) as well as the level of lipid 

peroxidation in the kidney of rats as compared to the corresponding groups of control 

animals (Fig. 25; Table 34c). However, no significant changes were observed in the 

animals administered with TCDD at 1 and 10 ng dose levels. Low dose of TCDD 

were nor able to induce oxidative stress in kidney of rat thereby suggesting that thc 

antioxidant mechanism is higher in kidney as well as the toxicity threshold is lower in 

testis and epididymis than in kidney in terms of oxidative stress. The results

suygcstccl that vivamin 1: conld imp;lrt p~.otcclivc cI'(Lc~s i~gi~itlst 'I'('I>Il lo 11o11- 

reproductive tissue as well. 

5.4 Epilogue 

On the basis of the foregoing discussion it can be summarized that exposure to 

TCDD increases oxidative stress in rats as evidenced by increase in reactive oxygen 

species, lipid peroxidation and suppression of antioxidant system in testis. epididymis, 

sperm and kidney. The generation of oxidative stress is triggered by low doses of 

TCDD in the male reproductive system as compared to kidney where it is generated 

only at high doses. Thus male reproductive system appears to be more susceptible to 

the damage caused by environmental contaminants like TCDD. Co-administration of 

TCDD along with vitamin E imparts protective effects against TCDD-induced 

reactive oxygen species mediated toxicity in the reproductive and non-reproductive 

tissues. Thus vitamin E could be uscd as a protective measure against TCDD-induced 

toxicity.

Fig.4a. Effect of TCDD or TCDD and vitamin E on the weight of the testis of 

adult rats (mean* SD; *P

Fig.4~. ENect of TCDD or TCDD and vitamin E on the weight of the sen~inal 

vesicles (Intact) of adult rats (meanfSD; *P

Fig.4~. Effect of TCDD or TCDD and vitamin E on the weight of the ventral 

prostate of adult rats (meaniSD; *P

tE 

W 

d 

In 

w 

it 

I 

I 

(,OCX) 3s PUB (,OCX) dsa 

i

Fig. 'la. Effect of TCDD or TCDD and vitamin E on the rerum FSH levds in 

adult male rats (meanfSD; *PcO.05) 

OOng olng ml0ng EelOOng 

TCDD 

TCDD +Vitamin E 

Fig. 7b. Effect oTTCDD or TCDD and vitamin E on the serum LH levels of 

adult rats (meaniSD; *P-=0.05) 

Dong Blng BlOng 01100ng 

TCDD 

TCDD + Vitamin E

Fig. 7c. 

Effect of TCDD or TCDD and vitamin E oa the serum pmlrctin levels 

of adult rats (meanfSD; *P

Fig. 7c. ElTect of TCDD or TCDD and vitamin E 011 the serum estradiol levels of 

adult rats (meanfSD; *PcO.OS) 

TCDD 

TCDD +Vitamin E

u 3 f ~ = m w * N O 

.- 

~Slew aew enssg 6 1 6 ~

Fig.lOa. EBut of TCDD or TCDD and vitamin E on the production of superoxide anion in crude homogenate, mitochondrial and 

microsome-rich fractions of rat tcstis (mcmf SD: *P

Fig.lOb. ENmt of TCDD or TCDD and vitamin Eon the production of nitric oxide in crude homogenate, mitochondrial and 

microsomcrich fractions of rat testis (mcantSD; *P

Filk Effect of TCDD or TCDD and vitamin E on the hydrogen peroxide generation in crude homogenatc, mitocbondrhl m d 

microsome-rich fnctiow of rat tatis (munfSD; *P

Fig. 1Ir Effect of TCDD or TCDD md vitamin E on the 1 4 of ascorbic acid in crude homogenatq mitochondria1 md micmsomc 

rich fractions of rat tcctir (mean+SD; *P

Fig. 14a. Effect of TCDD or TCDD and vitamin E on the production of superoxide 

anion in the epididymal sperm of adult rats (mean *SD; *P

Fig. 14c. Effect of TCDD or TCDD and vitamin E on the hydrogen peroxide 

generation in the epididymai sperm of adult rats 

(mean kSD; *P

Fig. 1Sb. Effect of TCDD or TCDD and vitamin E on the level of a-tocopherol in 

the epididymal sperm of adult rnts (mean *SD; *P

Fig. 16b. Effect of TCDD or TCDD and vitamin E on the activity ofcatalase in tlte 

epididymal sperm of adult rats (mean iSD: *P

Fig.16d. Effect of TCDD or TCDD and vitnn~in E 011 the activity of glulell~iune 

peroxidase in the epididymal sperm of adult rats 

(mean MD; *Pc0.05) 

mg protein mg DNA mg protein mg DNA 

TCDD 

TCDD + Vitamin E 

Fig.17. Effect of TCDD or TCDD and vita~nin E 011 level of lipid geruxid:~liu~~ ill 

the epididymal sperm of adult rats (mean fSD; *P

Fig.18~. EKect of TCDD or TCDD and vitamin Eon the production of hydrogen peroxide in the caput, corpus and 

cauda epididymides of adult rats (mean +SD; *P

Fig. 19a. Effect of TCDD or TCDD and vitamin Eon the lcvd of glutathione in thc caput, corpus md 

cauda cpididymis of adult rats (me~nfSD;*PQ.05) 

TCDD TCDD +Vitamin E TCDD TCDD +Vitamin E TCDD TCDD +Vitamin E 

Caput epididymis Corpus epididymis Cauda epididymis 

142

1 1.2 l4 

Fig. 19b. ENect of TCDD or TCDD and vitamin E on the level ofa-tocopherol in the caput, corpus and 

cauda epididymides of adult rats (mean fSD: *P

60 

Fig. 20a. Effect of TCDD or TCDD and vitamin E on the activity of superoxide dismutasc in the caput, corpus and 

cauda epididymides oladult rats (mean +SD; 'P

Fig. ZOb. Elkt of TCDD or TCDD and vitamin Eon the activity of catalrse in the eaput, corpus md 

uuda cpididymis of adult rats (mean f SD; *P

Fig. 2k. Ekt of TCDD or TCDD and vitamin Eon the activity of glutathione reductase in the caput, corpus and 

cauda epididymids of adult rats (mean fSD: *P

Fig. 21. 

Effect olTCDD or TCDD and vitamin Eon the level of lipid peroxidation in the caput, corpus md audr 

epididymidu of adult rab (mean fSD; 'P

Fig. 22a. EKect of TCDD or TCDD and vitamin E on the production of 

superoxide anion in the kidney of adult rats (meanfSD; *P

Fig. 22c. Effect of TCDD or TCDD and vitamin E on the production of 

hydrogen peroxide in the kidney of adult rats (meanfSD; *P

Fig. 23b. Effect of TCDD or TCDD and vitamin E on the level of a-tocopherol in 

the kidney of adult rats (mean*SD; *P

Fig. 24b. Eflect ofTCDD or TCDD and vitamin E on the activity of catalase in 

the kidney ofadult rats (meanfSD; *P

Fig. 24d. Effect of TCDD or TCDD and vitamin E on the activity of glutathione 

peroxidase in the kidney of adult rats (meanfSD; .P

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Aitken RJ, Irvine DS, Wu FC. Perspective analysis of sperm-oocyte fusion and 

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Obstet Gynccol 1991; 164: 542-551. 

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1.1 Preparation of dosing solution 

APPENDIX 1 

PREPARATION OF REAGENTS 

1.1.1 2.3.7.8-Tetrachlorodibem-p-dioxin (TCDD) 

100 pg of TCDD was dissolved in acetone and olive oil (1:19) and used as 

stock solution. The stock solution was serially diluted and used for treatments. 

1.1.2 Vitamin E 

200 mg of vitamin E was made up to 10 mL with olive oil and stored in amber 

coloured bottle at 4OC. This solution contained 20 mg vitamin E/ mL. 

1.2 Epididymal sperm counts 

1.2.1 Diluents 

The diluents for sperm counts were prepared by the addition of 50 g sodium 

bicarbonate, 10 mL 35% formalin and 0.25 g trypan blue in a volumetric flask 

and made up to a final volume of 1 L with distilled water. 

1.3 Serum FSH 

1.3.1 Anti-FSH-Coated microtitration strips 

One strip holder contained 96 microtitration wells coated with anti-FSH 

antibody. Stored at 2-8'C in the resealable pouch with a desiccant to protect 

from moisture. 

1.3.2 FSH standards 

One vial, 2 mL, labeled A contained 0 mlU1 mL and five vials. 1 mL each, 

labeled B-F, contained concentrations of approximately 1.5, 4.5, 15, 45 and 

150 mIU/ mL FSH (from human pituitary gland) in porcine serum with a nonmercury 

preservative. 

1.3.3 FSH controls 

Two vials, I rnL each, Levels I and 11. contained low and high concentrations 

of FSH (from human pituitary gland) in porcine serum with a non-mercury 

preservative.

1.3.4 Antibody-Enzyme Conjugate solution 

One amber vial, 0.3 mL, contained anti-FSH antibody conjugated to the 

enzyme horse+adish peroxidase in a protein-based buffer with a non-mercury 

preservative. The Antibody-Enzyme Conjugate concentrate was diluted at a 

ratio of 1 part into 50 parts of the assay buffer. 

1.3.5 Tctramethylbenzidine Chromogen solution (TMB) 

One amber bottle, 11 mL contained a solution of TNIB in citrate buffer with 

hydrogen peroxide. 

1.3.6 Wash concentrate 

One bottle, 105 mL contained buffered saline with a nonionic detergent. 

Diluted 10-fold with deionized water prior to use. 

1.4.1 Anti-LH-Coated Microtitration strips 

One strip holder contained 96 microtitration wells coated with anti-LH 

antibody. Stored at 2-8°C in the resealable pouch with a desiccant to protect 


1.4.2 LH standards 

One vial, 1 mL, labeled A contained 0 mlU/ mL and five vials, 0.5 mL each, 

labeled B-F, contained concentrations of approximately I, 3, 10, 30 and 100 

mlU/ mL LH equine serum with a non-mercury preservative. 

1.4.3 LH controls 

Two vials, 0.5 mL each. Levels I and 11, contained low and high 

concentrations of LH in equine serum matrix with a non-preservative. 


One amber vial, 0.3 mL, contained anti-LH antibody conjugated to the enzyme 

horseradish peroxidase in a protein-based buffer with a non-mercury 


ratio of 1 part into 50 parts of the assay buffer. 

1.4.5 Ternethylbemidine Chromogen solution (TMB) 

One amber bottle, 11 mL contained a solution of TMB in citrate buffer with 

hydrogen peroxide.


One bottle, 105 mL contained buffered saline with a nonionic detergent. 

Diluted 10-fold with deionized water prior to use. 

1.5.1 Anti-Prolactin-Coated microtitration strips 

One strip holder contained 96 microtitration wells coated with anti-prolactin 

antibody. Stored at 2-8°C in the resealable pouch with a desiccant to protect 


1.5.2 Prolactin standards 

One vial, labeled A contained 0 ngl rnL and five vials, labeled B-F, contained 

concentrations of approximately 2, 6, 20, 60 and 180 ngl mL prolactin in 

porcine serum with a non-mercury preservative. 

1.5.3 Prolactin serum controls 

Two vials, Levels I and 11, contained low and high concentrations of prolactin 

in porcine serum with a non-mercury preservative. 


One vial, 0.3 mL, contained anti-prolactin antibody conjugated to the enzyme 

horseradish peroxidase in a protein-based (BSA) buffer with a non-mercury 


ratio of 1 part into SO parts of the assay buffer. 

1.5.5 Tetramethylbenzidine Chromogen solution (TMB) 

One amber bottle, 11 mL contained a solution of TMB in citrate buffer with 



One bottle. 60 mL contained buffered saline with a nonionic detergent. Diluted 

25-fold with deionized water prior to use. 

1.6 Serum testosterone 

1.6.1 GARG-Coated microtitmtion strips 

One strip holder contained 96 polystyrene microtitre wells coated with goat 

anti-rabbit 1gG immobilized to the inside wall of each well. Stored at 2-S°C in 

the resealable pouch with a desiccant to protect from moisture.

1.6.2 Testosterone standards 

One vial, 0.5 mL, labeled A contained 0 ng/ mL testosterone and six vials, 0.5 

mL each, labeled B-G, contained concentrations of approximately 0.1. 0.5. 

2.5, 5.0, 10 and 25 ngl mL testosterone in human serum with a non-mercury 

preservative. 

1.6.3 Testosterone controls 

Two viats, 0.5 mL each, Levels I and II, contained low and high 

concentrations of testosterone in human serum with a non-mercury 

preservative. 

1.6.4 Testosterone-Enzyme Conjugate concentrate 

One vial, 0.3 mL, contained testosterone conjugated to horseradish peroxidase 

in a protein-based (BSA) buffer with a non-mercury preservative. The 

Antibody-Enzyme Conjugate concentrate was diluted at a ratio of 1 part into 

50 parts of the assay buffer. 

1.6.5 Testosterone antiserum 

One vial, I I mL, contained rabbit anti-testosterone (polyclonal) serum in 

protein based (BSA) buffer with a non-mercury preservative. 

1.6.6 Tetramethylbenzidine chromogen solution (TMB) 

One amber bottle. I I mL contained a solution of TMB in citrate buffer with 



One bottle, 60 mL contained buffered saline with a nonionic detergent. Diluted 


1.7 Serum estmdiol 

1.7.1 Anti-Esuadiol-Coated microtitration strips 

One strip holder contained 96 polystyrene microtitre wells coated with rabbit 

anti-estradiol IgG immobilized to the inside wall of each well. Stored at 2-8°C 

in the resalable pouch with a desiccant to protect from moisture. 

1.7.2 Esuadiol standards 

One vial, 3 mL, labeled A contained 0 pgl mL and six vials, 1 rnL each, 

labeled B-G, contained concentrations of approximately 20, 50, 250, 750. 

2000 and 6000 pgl mL estradiol in human serum with a non-mercury 

preservative.

1.7.3 Estradiol controls 

Two vials. 1 mL each, Levels I and 11, contained low and high conccntrulions 

of esuadiol in human serum with a non-mercury preservative. 

1.7.4 Estradiol-Biotin Conjugate concentrate 

One vial, 0.3 mL, contained biotinylated estradiol in a protein-based (BSA) 

buffer with a non-mercury preservative. Diluted prior to use with the 

estradiol -biotin conjugate diluent. 

1.7.5 Tetramethylbenzidine Chromogen solution (TMB) 

One amber bonle, 11 mL contained a solution of TMB in citrate buffer with 



One bottle, 60 mL contained buffered saline with a nonionic detergent. Diluted 


1.7.7 Stopping solution 

One vial, 11 mL contained 0.2 M sulfuric acid 

1.8 3P-Hydroxystcroid dchydrugcnasc 

1.36 g of tris was dissolved in 100 mL of distilled water (Solution A) 

0.9 mL of concentrated hydrochloric acid was made up to 100 rnL with 

distilled water (Solution B). 

62.5 mL of solution A and 55.25 mL of solution B were mixed and made up to 

250 mL with distilled water. The pH was adjusted to 7.2 with 1 N sodium 

hydroxide. 

1.8.2 Sodium pyrophosphate buffer (100 pM; pH 9.0) 

13.3 mg of tetrasodium pyrophosphntc was dissolvcd and made up to 500 1111~ 

with distilled water and pH was adjusted to 9.0. 

1.8.3 NAD (0.5 pM) 

1.66 mg of NAD was dissolved in 5 mL of distilled water.

22.91 pg of 4-androstcne 3,17-dione was dissolved in 100 mL of distilled 

water. 

1.9 17p-Hydroxysteroid dehydrogenase 

1.9.1 Sodium pyrophosphate buffer (1 00 pM; pH 9.0) 

13.3 rng of tetrasodium pyrophosphate was dissolved and made up to 500 mL 


1.9.2 NADPH (0.5 mM) 

2.08 mg of NADPH was dissolved in 5 mL of distilled water 

2.86 mg of androstene 3,17-dione was dissolved in I0 mL of 50% ethanol 

1.10 Estimation of deoryribonucleic acids (DNA) 

1.10.1 Perchloric acid (PCA) 

Perchloric acid 16.66 mL (60%) was made upto 100 mL with distilled water. 

1.10.2 Diphenylamine reagent 

1.5 g of diphenylamine reagent was dissolved in 100 rnL of glacial acetic acid. 

To this 1.5 mL of concentrated sulphuric acid was added and stored at 4°C in 

amber coloured bottle. 

To every 20 mL of this reagent, 0.1 mL of 1.6% aqueous acetaldehyde was 

added to potentiate the colour development. 

1.10.3 DNA standard 

2 mg of calf thymus DNA was dissolved in 10 mL of hot 5% trichloroacetic 

acid to give a concentration of 200 pg/ mL and stored at 4°C. 

1.1 1 Estimation of ribonudeic acid (RNA) 

1.1 1 .I Orcinol reagent 

I g of ferric chloride was dissolved in 1 L of concentrated hydrochloric acid 

and 35 mL of 6% orcinol was added.

1.1 1.2 Orcinol(6%) 

6 g of orcinol was dissolved in I00 mL of ethyl alcohol. 

1.1 1.3 RNA standard 

2 mg of RNA was dissolved in 10 mL of distilled water to give a concentration 

of 200 pg/ mL and stored at 4'C. 

1.12 Estimation of protein 

1.12.1 Alkaline copper reagent 

50 mL of reagent A and I mL of reagent B were mixed fresh at the time of 

use. 

Reagent A 

2 g of sodium carbonate was dissolved in and made up to 100 mL with 0.1 N 

sodium hydroxide solution. 

Reagent B 

5 mg of copper sulfate in 1 mL of 4% sodium potassium tartarate. 

1 .I 2.2 Sodium hydroxide solution (0.1 N) 

0.4 g of sodium hydroxide was dissolved in and made up to 100 mL with 

distilled water. 

1.12.3 Sodium potassium tamirate (4%) 

4 g sodium potassium tartarate was dissolved in and made up to 100 mL with 

distilled water. 

1.12.4 Bovine serum albumin (BSA) standard 

100 mg of bovine serum albumin was dissolved in and made up to 100 mL 

with distilled water. This solution was diluted 10 times to obtain a 

concentration of 0.1 mg/ mL. 

1.12.5 Folin Ciocalteau reagent (IN) 

Commercial Folin Ciocalteau reagent was diluted with equal volume of 

distilled water.

1.13 Subcellulnr frnctionation of testis 

1.13.1 Sucrose solution (0.25 M) 

Sucrose 8.55 g was dissolved in 100 mL distilled water. 

1.13.2 Calcium chloride (1 M) 

14.70 g of calcium chloride was dissolved in 100 mL distilled water. 

1.14 Superoxide anion 

1.14.1 Iodonitrotetrazolium (INT; 4.9 mM) 

230.91 mg of INT was dissolved in I00 mL of distilled water. 

1.14.2 Ethylene diarnine tetraacetic acid (EDTA; 0.3 mM) 

11.16 mg of EDTA was dissolved in 100 mL of distilled water. 

1.14.3 Sodium carbonate (0.92 mM) 

9.74 mg of sodium carbonate was dissolved in 100 mL of distilled water. 

1.15 Nitric oxide 

1.1 5.1 Griess reagent 

10% Sulfanilamide was added to 40% phosphoric acid (Solution A). 

1 g of N-[-naphthyllethylenediamine dichloride was diluted to 100 mL with 


Mix equal volume of solution A and B at the time of use. 

1.16 Hydrogen peroxide generation 

I. 16.1 Phosphate buffer (0.05 M; pH 7.6) 

0.89025 g of disodium hydrogen phosphate dihydrate was dissolved in 100 

mL distilled water (Solution A). 

0.69175 g of sodium dihydrogen phosphate monohydrate was dissolved in 100 

mL distilled water. 

Mixed 13 mL of solution A with 87 mL of solution B and made up to 200 mL 

with distilled water and pH was adjusted to 7.6.

1.16.2 Horseradish peroxidase (8 Units1 mg) 

1 mg of 80 units horseradish peroxidase was dissolved in 10 mL of distilled 

water to make 8 units/ ml. 

1.16.3 Phenol red (28 nM) 

354.4 mg of phenol red was dissolved in IrnL of distilled water to give a 

concentration of 1 M. This solution was made up to 1 L to get the 

concentration of lmM, and this solution was made up to 1 L to get the 

concentration of 1 pM. 28 mL of 1pM phenol red was made up to 1L with 

distilled water to get the concentration of 28 nM. 

1.16.4 Dextrose (100 nM) 

180.1 1 mg of dextrose was dissolved in I mL of distilled water and made up 

to 1 L to get the concentration of 1 mM. 1 mL of ImM dextrose solution was 

made up to 1 L with distilled water to get the concentration of 1 pM. 1 mL of 

IpM dextrose solution was dissolved and made up to 100 mL with distilled 

water to get the concentration of 100 nM. 

1.16.5 Sodium hydroxide (10 N) 

40 g of sodium hydroxide pellet was dissolved in 100 mL of distilled water. 

1.17 Glutathione reduced 

1.17.1 Phosphate buffer (0.1 M; pH 7.0) 

1.78 g of disodium hydrogen phosphate dihydrate was dissolved in 100 mL of 

distilled water (Solution A). 

1.38 g of sodium dihydrogen phosphate dihydrate was dissolved in 100 mL of 


Mixed 39 mL of solution A and 61 mL of solution B and made up to 200 rnL 


I. 17.2 Trichloroacetic acid (5%) 

5 mL of 100% TCA was diluted to 100 mL with distilled water. 

1.17.3 Ellman's reagent 

34 mg of dithionitrobis-benzoic acid was mixed in 10 mL of 0.1% sodium 

citrate.

1.17.4 Disodium hydrogen phosphate (0.3 M) 

5.34 g of disodium hydrogen phosphate was dissolved in I00 tnL of distilled 

water. 

1.17.5 Glutathione standard 

100 mg of glutathione reduced in 100 mL distilled water and stored as 

working standard. Stock was diluted to get a concentration of 100 pd mL. 

1 .I 8.1 2,Z'-Dipyridyl solution (0.2%) 

0.2 g of dipyridyl was dissolved in I00 mL of ethanol. 

1 .I 8.2 Ferric chloride solution (0.5%) 

0.5 g of ferric chloride was dissolved in 100 mL of ethanol. 

1.18.3 a-Tocopherol (standard) 

100 mg of DL-tocopherol was dissolved in 100 mL of distilled ethanol. Stock 

solution was diluted to get a concentration of 10 pgl mL. 

1.19 Ascorbic acid 

1.19.1 Metaphosphoric acid (2.5%) 

2.5 g of mctaphosphoric acid was dissolved in 100 mL of distilled water. 

1.19.2 Dichlorophenol indophenol acetate 

7.5 mg of dichlorophenol indophenol was dissolved in 100 mL of distilled 

water at 85OC, filtered hot, cooled and diluted to 250 mL (Solution A). 

4.5309 g of sodium acetate was dissolved in 100 mL of distilled water and pH 

was adjusted to 7.0 with 0.5 M acetic acid (solution B). 

Mixed equal volume of solution A and Solution B at the time of use 

1.19.3 Ascorbic acid (standard) 

100 mg of ascorbic acid was dissolved in 100 mL distilled water and stored as 

working standard. Stock was diluted to get a concentration of 100 pgl mL.

1.20 Superoxide dismutase 

1.20.1 Tris HCI buffer (50 mM; pH 8.2) 

605.7 mg of Tris HCI was dissolved in I00 mL of distilled water (50 mM). To 

this solution 0.0372 g of EDTA was added and pH was adjusted to 8.2. 

12.6 mg of pyrogallol was dissolved in 50 mL of I0 mM HCI. 

1.20.3 10 mM HCI 

0.0416 mL or 41.6 pL of coocentrated HCI was made up to 50 mL in a 

standard flask. 

1.21 Catalase 

1.2 1.1 Phophate buffer (0.0SM; pH 7.0) 

0.89025 g of disodium hydrogen phosphate dihydrate was dissolved in 100 

mL of distilled water (Solution A). 

0.691 75 g of sodium dihydrogen phosphate monohydrate was dissolved in 100 

mL of distilled water (Solution B). 

Mixed 39 mL of solution A with 61 mL of solution B and made upto 200 mL 

with distilled water and then pH was adjusted to 7.0. 

1.21.2 Hydrogen peroxide (0.019M) 

58pL of 30% hydrogen peroxide was made up to 100 mL with distilled water 

and stored in cool and dark place. 

1.22 Glutathione reductnse 

1.22.1 Phosphate buffer (0.1 M, pH 7.6) 

1.78 g of disodium hydrogen phosphate was dissolved in 100 mL of distilled 

water (Solution A). 

13.835 g of sodium dihydrogen phosphate was dissolved in 100 mL of 


13 mL of solution A and 87 mL of solution B was taken and diluted to 200 mL 

and pH was adjusted to 7.6.

1.22.2 NADPH (0.2 pM) 

16.6672 mg of NADPH was dissolved in 10 mL of distilled water 

1.22.3 Glutathione oxidized (2 mM) 

Glutathione oxidized of 12.2526 my was dissolved in 10 mL of distilled water 

(2 mM). 

1.22.4 Ethylenediaminetetraacetic acid (EDTA, 0.01 M) 

37.224 mg of EDTA was dissolved in I0 mL of distilled water. 

1.23 Glutathione peroxidase 

1.23.1 Phosphate buffer (0.05 M. pH 7.0) 

0.89 g of disodium hydrogen phosphate was dissolved in 100 mL of distilled 

water (Solution A). 

0.78005 g of sodium dihydrogen phosphate was dissolved in 100 mL of 


Take 39 mL of solution A and 61 ml of solution B was taken and diluted to 

200 ml and pH was adjusted to 7.6 

1.23.2 Glutathione reductase 

3 1.25 mg of glutathione reductase was dissolved in 5 mL of distilled water. 

1.23.3 EDTA (0.01 M) 

37.224 mg of EDTA was dissolved in 10 mL of distilled water. 

1.23.4 Sodium azide 

6.5 rng of sodium azide was dissolved in 10 mL of distilled water. 

1.23.5 Glutathione (reduced) 

30.733 mg of glutathione (reduced) was dissolved in 10 mL of distilled water. 

1.23.6 NADPH (0.2 pM) 

16.6672 mg of NADPH was dissolved in I0 mL of distilled water. 

1.24 Lipid peroxidation 

1.24.1 15% v/ v trichloro acetic acid (TCA) 

15 mL of 100 % TCA was made up to 100 mL with distilled water.

1.24.2 Thiobarbituric acid (TBA; 0.37% w/ v) 

0.375 mg of TBA was dissolved in 100 mL of distilled water. 

1.24.3 Hydrochoric acid (HCI; 0.25 N) 

1.08 mL of HCI is made upto 50 mL with distilled water. 

1.24.4 TCA: TBA: HCI (1:I:I) 

Equal volumes of the above mentioned 3 solutions were mixed to make a 1 : 1 : 

1 ratio of TCA:TBA:HCI.

APPENDIX 2 

LIST OF PUBLICATIONS OF THE CANDIDATE 

During the course of the study the candidate has published following papers and 

the available reprints/ preprints are being enclosed. 

1 K.C. Chitra, C. Latchoumycandane and P.P. Mathur (1999). Effect of 

endosulfan on the testicular functions of rat. Asian Journal of Andrology 1: 203- 

206. 

2 R. Sujatha, K.C. Chitra C. Latchoumycandane and P.P. Mathur (2001). Effect 

of lindane on the testicular antioxidant system and steroidogenic enzymes in adult 

rats. Asian Journal of Andrology. 3: 135-1 38. 

3 K.C. Chitra, R. Sujatha., C. Latchoumycandane and P.P. Mathur (2001). Effect 

of lindane on the epididymal antioxidant enzymes in adult rats. Asian Journal of 

Andrology. 3: 205-208. 

4 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). The effect of 

2,3,7,8-tetrachlorodibenzo-p-dioxin on the antioxidant system of rat testis 

mitochondrial and microsomal fraction. Toxicology, 171 : 127-35. 

5 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). Induction of 

oxidative stress on the epididymal sperm of rats after exposure to 2,3,7,8- 

tetrachlorodibenzo-dioxin. Archives of Toxicology, 76: 1 13-1 18. 

6 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). The effect of 

methoxychlor on the epididymal antioxidant system of adult male rats. 

Reproductive Toxicology, 16: 16 1-1 72. 

7 C. Latchoumycandane, and P.P. Mathur (2002). Effect of methoxychlor on the 

antioxidant system of mitochondrial and microsome-rich fractions of rat testis. 

Toxicology, in press. 

8 C. Latchoumycandane, and P.P. Mathur (2002). lnduction of oxidative stress in 

testis of rat after short-term exposure to organochlorine pesticide methoxychlor. 

Archives of Toxicology, in press. 

9 K.C. Chitra, C. Latchoumycandane. P.P. Mathur (2002). Effect of nonylphenol 

on the antioxidant system in epididymal sperm of rats. Archives of Toxicologv, in 

press. 

10 C. Latchoumycandane, and P.P. Mathur (2002). Effects of vitamin E on 

reactive oxygen species mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in 

rat testis. Journal of Applied Toxicology, in press.

Chronic effect: of enclosulfan on the testiculalfi~nctions 

of rat 

:lillt: 'To lind out Llic toxic vlicct of endoililiu~ ol! Ulc tusuc~~lnr ionctiw~ 01 puknill rrb. MilJloti~m\j~ns. I~esu11s: In ~rldt)s~~liiu~.lwaI~tl 

;:it&, UICW WR. n IV~UC~~OII ill LhC lWdy wig111 und tlr \wi@~ts ui Ic\ti\lia ;~nd:tccu:.u,ry rcr urgrns, a rl?. 

CIUW in lllc I~~IICUIU i~~ulc and pymvillc wtivitiu, u;d ut d~c lt~liculitr DNA m~d IINA C~III~.CIIIT.I~~O~I\I i~hc~a\ ih~ 

~L.SUCU~Y pmuUI CO~CC~LTJUO~ was slighlly i t m d ; dw spsific xliviiy of leslicu!iu ~t~~iduglnif CIIBIIIC, :i$-Otis~ciuid 

&hyJ~c)gL?n.m ad IIC iluorbic cad level WPC decrciaul, wllich wlr corrtlated wih a dwrcsc in rtciuidoge- 

~tcsis. 1 1 lywucu~ul ~ emynu? ncid phosplwtw rind brwh-LmrJcr cuyntc nlhlillins pho~phnww ~cliviriu wotr; also Jcc:cwd 

in rhc tcrtis of uwtcd mu. Conclusion: In puberl;ii mLs, endosullm (rca.mtt inl~ibik !he wticukd fu~icttuns. 

(Ailinr~ J Arulru! 1YB Dec; 1 : ?a3 - 206) 

llrm is gro\vutg couccnt lhxt cn!'iroilll~rni;ll clii,llli. 

cJr. tdh n~lurdl md IIUII-ntnde, h?vi!lg csux-gmic 

propeny #nay he ~iusii~g n vvicty of rcpmluctive disor- 

4rs in u,ildlifc d IIWMI ~~plaliUIUj. Most of the 

rl;s~~iicd~. \vl;icll u.4 a p-sticides ;ul: noi !tifill) 

rlccti\%-, but av gcirdly toxic lo ltlimj tloll-lwgcl 

spies, incluili$ tnnn &ad oo(her winds. Endosulfm, 

;u~ inusucidc ol cyclcdiur group is cxluwivcly urul irq 

UI in..;ricide ntdnly in ~g~iculm and in some cpunlricr 

ii~ public 114d1['~. Ar a ruulr of ik wide s p d luc, it 

rial lr a p~sntid cnvimtd unlill~u~uif ud may 

mmlu: a pUic lu.dlh l*wrd('l. hKbPulf;m 11s b t l 

----- 

Cura&lrr to I*. P. P. hlYlur 

c.nml: ~plulluOydrn.mnn 

'1~1: +91.41:4.

~lLllc mu ~LYIC~L~ II*: ~[IL'IIII WIIIS ill UC u~UJ..II cpi. 

didyt~lis :eul d,c izi~nacs~icul;~r slrniuilid rw11u, ilrur;i. 

.ILI iviilr uii ck~rrIio~i tii UIC xlivili~ of sl~xific IG\I~CU. 

rru~gc~ri~J using Ralli R~llu-Uvd!jcni Inx~n~gcnircr. 

I0 5, I ~ul~t~wlC Wuv prclmi~d ill IUWIEII s:ilittt: ~1 11 

si~p,~l;runi ru.ul lw Iiicrlu~rio~l &'d:1y. 'llr cxrr*cii, 

1;v ~ii;utcr CII~IIW. s so~lrilol dcl~yhwga~w, IWLIC u~rl ~klmniwtion of M A mlJ RNA m wnie~ 

~kliydmgcir:~~!, ~.glur.u:~yl muqrpl~d:~~ 

uul gluc.cru C. !ull~,wi~rg Ur l

enbw1h1.W mu IUY Wcab unpiliml~t at &ticactivity 

ilrw with llic dcvclojr~~~e~ti of sr~ntr;,lo 

Ub. piluilalY. Cf ~1yp~IPliY1lic lcwl, 'nu ~pididyi~ii~, 

C~ICK~"'~. Acliviliu; ot truc ly~u,~~ul UIL~IU !wvr iull 

:cini~ud wick wid vc~~utll INWUI~: an: tl11 ul&rwl. dwWl\ In nu, dm enlicuhr slcruicbgc~uiis il; ill. 

~&lI orpimr, dying On ICstoslM~ ,fur kir ucuu~I'*'~. A &acmc in UIC %id ohosDhahr ill lrer 

grwnh nnd fUnclion[lol. A rcducljon in (heir wcidlk stlr ww~d bus rcthl d d lesicu~pr smido8em- 

11uy r c b u ~ Jcuwred bianvi~ilability wd pralunion of sis in Ur W - m u ad UI my ba cmdnkd will1 1110 

aI~enr("'. 

ducal rasrerion of g ~ p h i A ~ deacare . in tk 

nl!dinc phosphaurw ocuvity in cndosulfan.mlcd n$ in- 

J i W lhnl emlobulf~~~ unLrw111 prduccd s sue of deurarcd 

stMidogwcris whuc Ihc inrcr- and invrcllular 

Wnrporr Was &ccd as rhc melabolic d m lo channnlize 

the MU~Y~ input8 for slemidogurais slowed 

do*n("l. 

In conclusion, 'he own1 studics indicate hat en- 

Pmeid IO..%tz.r) 13.12+1.lrF 

IINA' ll.l:l*l.:L~~ ?,i9*(1,#~> 

WIA' ;1.1')tU.W 0.93t0.2ib 

AX.5.W acid' 2.5+0.81 I.M+O.IP 

LYw' 4.5.08+7. 32.64+Y.L? 

M-2 31.nts.m 1r.utz.w 

3 POH.uarxd 3.WtI.W l.e8*0.31" 

JC~>*IWJ 

Aca -y' 10L.07+25.1 79.%~10,&5~ 

Wim phy.p"ouel W.50r.X.42 55.61*15.2UL 

' III~L wet wcidll 01 1 ~~1~s 

ln~~~V~og 11tu1ein 

' ruwl of NAD conuuled lo NADIUmidnrp pmlcin 

'l,nwl rt.~~~~~~~l~lw~~~l 

IIIC~;~I~I~:I~ T/::lI III~II/IIXI (21; 

I8,Ulflll 

PPM sc*norvlcgcs the rscipl of financial support 

horn Uc Popllation Council, New York. USA. 

References 

1 WllO kpn. Inlau!hm! yropmmr m c l a d olsly. 

In, Lv(mwW IWL Cri~iu-W. Gx!dlu~ L WllD. 

Gus"") 1w: I. 

2 Siwl, SK, lbrky US 'rvricilg or ctuknilr.lll ax! LLlirg ,,I 

llule NY LI .IYIIUI le chg nrohiiul~s c~u)%sa ;yul ltucm. 

rarrrl lipicl pwrilluim. llwlisll J Lp llid I'm): 27: 725 - 

:I l\>l.b" VII, l\$d

Effect of lindane on testicular antioxidant system and 

steroidogenic enzymes in adult rats 

K. Sujeh. K.C. ChiVd, C. L utchoumyct, P.P. Mahur 

*hd MLp 5clcnw, Pndrclvny Un,uniIy, Pmdichny 6% ill4 . Ida 

Abreact 

&: To fird wl hc efiozl of lib 

on Ic~iiculw arlioxihl system nnd lesiiculer rlcmidupmsis in dull male 

r.B. Methods: Adult male rats wus orally dminislued wilh iindan at a dm of 5.0 rng/kg body wighl per day 

fiu RO duyr. Twenty-[our hours nRCr LhC 13Y Mllmenl Ulc mLs w a kilkd ~ using n d i c &r. Testes, cpididymis. 

wnlinttl v~%iclc% ed wtml pmtake wcrr mwd nnd wigM. A 10% (alicuk lrgenute wa prcpDlsd ud mn- 

1lirug4 nl ,IT. Ihc wpcmacun~ w 4 tor biwlmn~wl eslimntions. Results: 'Ihe buly wiyhl MII he 

weights of ~sler, qdddymis, reminal &la end vend p w ware red& in limhw-Mlllcd mls. Then was a 

rignikm klinc in rhc Daivilia of Mlioxhnt cmyms mpuoxidc dismutasc (SOD). nnd glubthiar Rduc- 

L= dlik cut it- in hfi- pcmxide (YO,) gencmlion wa* churned. 7he spwiiic activilie% or Lerlicular 

urnihqar av.ria:+hrJRUwd ddlyil~~$uwp nnd llp-hydmxyslemid dehydquwsc wn 4324. Tk 

kuslr o( DNA. RNA anl pmdn wcrr alsn dsnased in lindmr-vmtcd ram. Conclusion: Lindme indum oxi&- 

liw.mxrMddoourstaPntiO*dPIIf~~~~in&ltmalcm~. (kiv1JAnlml2WI: 3: 135-138) 

V+ 

on rqxodunive functions in wildlife ad hunw~"~. Lin- 

1 InmJdvction 

dam has ban pviwly shown to muse loxic cfiozls on 

The orpwchlolinc insmid& lih ( 7-hu- tcrres of m. In fanulc rats li& exem anlialm- 

Irhlorocyc~) is widcly 4 m a pacicidc in genic sctivity by diwping ulmus cycle with a reduction 

m y muwria. In India it b widcly used in agriculhln in rhc ulaine vhi$~[~). Lindsm hss been rcponod to al- 

Md puhlic MUI 7. 

Lindm WUWx lcci rhc dcvelymeru d MLW crnbv in vifm in e 

lip*bilk: ClPMQ MJ enm Inlo Ur food chin mull- dosbdcpndcnl rmnd'). 

ing in bbmmdmim in Iho kdy Iixue6, Mood rind L i h when given orally lo mia during wrly, 

brrwtmiikofhuwrMdrvi~~ilc"~. L i h d o k mid Md lalo ppmm& hna been repfled lo muse IMal 

nlxac of imlnnmtion aim, locnl rcfiaption of fHusC.. 

d low binh'weighc of plp. wilh M i-v in number 

n*'adoaitm &n ad my cut dvslst 

of dad faw, rrspclinly[", Thc m1m arc highly 

nnaplib* D lindm r It the blaod-ds bsrricr 

Cl%-al a. P.P. k(l.h.. SkddWa%m=. ud with s n d c Rduction . 

IWdq WW. RwwImYaw. ha. 

Td, + P14~2lZ Rr, +9lW912 

Ed. ~ O p . p a . d c . h o ~ O ~ . u a \ 

YIWW SILI-17 

rntllwl

cbniuis 11~11 b"nci:~lc ~wlive oxygen 

01 5 .(I mvkg buJy wdght/JPy ra 3) drys. The control 

IROS)(~~. 

animals &ved a dmilor volw or lk whiele h. 

ROS nto M impumnt p;m or hu & r e m- ~wcnty-r~ hours slkr ~hc lat -I ~hc 

nisn iyninq inrmum. bt excnive gwwlion ol rm u+hd and killed ming mahdc akr. T*, opi. 

Oxygm dials #my ILunogwfc lisw. ROS arc fwmal in dkJymis, anid rrsickx and vcmd wee n. 

b h phys'rdogiul d pahlogicol mndilhs in mm- mod. c!mdofIhc.dhaingriaus.nlusi@. A 

mlim ti-. ROS inclula supmxidc mion. hydmryl 10% Igcia*rhmagar*.gnawinmmulsalin 

r;rlicll, hydncm pmai(k and r,aypcn ion, nll or which wing IWW hampim ml [he hmmwge~le WT 

;I= u~w~ahk i(ld rnllly wwliw It1 9111~2 LJIv1m b Ik ~t111ri1ubpI 01 HIXI x 8 fir X) min at .I?. The .wp 

- 

huly mlting ill WOS mwmh lo subk sun* end IIuMl wa5 lgcd lor vnria. biachcmi mays. Raein 

~10luuks~'~. When hu balm bawepl ROs and m- war eslimted by the mthd of lawyI1". Euimstions 

tioxibnt .spyrtar is losl ' oxidalive m' multsiq). Re- of auprmidc dLmdd8l. ~.ld'~). 

gluliuhim m. 

wliw ox&! wies has tun dawn to be inn44 in d~xnd"~ end hhydmBan -ids mian rcpylzlJ 

inlatilily due lo drreuive f ~ ~ l ' n and ~ " in ~ wac daa. 'he aivicies d -ic mzymcs 3p. 

ClyFlorchiistn a upm upmum! lo toxic &mi- hydmxyW 

rd 17phydmaysbroid 

Culs"','~J. I1 lu7 ha\n shown Ih hu (hc m dchdlqmm vsc eaimld irmrding 10 &gmyeozymrr 

01 hu Semi&&' pathway ux mdccular oxy- dP1, The cxaCan md d.caminnciar or DNA end 

gen Jnd ekama mfu fmm NADR1 lo hydmxylnre RNA nwe CdWW M fdkwing lk lshique d 

the mh~nld"'. In chis pmxxs, rupmxide mion or ~chnidd"' 

Mher oxygen fm dials wre pmmad a e rcrult ci 

elecuon leakage in normal wlim or hr lo inlcracfion 

o f s t a v i d ~ ~ o r a with h hum- a ~ thelhcdmpmmprrnavdntts. Stalirialmlyzymt 

1%) &r pviuur vmk ha% dawn rhol cndaulrvl sit wn &amd wing Stu(a('a ' I' test ~nsdomr["l. 

oltus tcui~ular furujons of pubcror mtst"' Md Signi~mnccordiff~uwse~~ pc0.05. 

mhxychla i- restive oxym ud &- 

u r n g,idiily~~ul 

~ ~ m*i~,xkliull eri~yma in dull IWIO rab 

~ ~ ~ i r W d a L 1 ) . T l r ~ 1 ~ ~ 1 ~ r o 

evalusreIhecllslofLindYronlk~~idru lh bqdy wigha or !inbae-lwcd rarq did rm 

spm and it? inn- on geroidoprsis in Ihc helaun. h a y n@fi dugesung cflk 

mamen. Ho*crrr. lhs bady &gku uar algniWy 

2 Mamink and m e W 

~.rlkendo~lkuamanammpndtoIhc 

~ i n g ~ d m o(Ftgurr v I). d the ~ 

mi*ofdIbc,enb,,pllinl~)and 

vaunl pawc werr M dpifdy han thmc 

ofhumnPd~mupdninuh(T.MeI). 

Lildac (7-huphlaocyslohaae) was b giR fmn 

layakridm Rslicidg, Sah. Tmil Ndu, India. Andmsmmc 

3, 17 dim, p-niuophcnyl -. p-niv 

o p h e n d M d d e l l 7 w - 

fmnSigmaUankaI6. (Sl.Louis, Mo.. USA). All 

thc drmblr ud wn. d mlylial gdc obW [nwn 

kwl m m b l aurEs. 

2.2 ~Lrandrdrrrmmmr 

Wirvrmkmu(10-12wr+ktdgc)nweobwizd 

Cmn Ihc CaurJl Anid Fsiiily d Um lsWdllPl 

llrrll~lc or PBcgndujc WiI Eduoslm nd bmdl 

(JIPMER). Wichary. I*. Ths m .me mdnwlwd 

undn a wll-roguQtal li@ uJ darL (1%: IZh) 

ululukrZ4tR't~~~~~lr.aa~M 

crsiuy dm* and mp Wa. Lindrme M dial4 in 

o~~vc oil iu a amx~mtmirni or 5.0 464. ml Ike tau 

~nwp W" giw> by crd iauhuritnl u( S i u a hap

ILXlll hnl, .I 121; 1.0 -/.a 

. Ill 

CinvrJ 

. - -.- -- ...- 

uuly wigla (8) nl * 16 1at9 

Tcxw 

(111g) llXP t 50 &%$id 

I b .W5t10 SI~~,@ 

t$*I,"i~ 

(#I+) .ULltttB :2u + ah 

0 I b . w l WJtW %*& 

Sonin*l &kd 

< 11%) (YL3*70 575 * Sb 

- 

hlal 

Wwwim Cuvol Twcd 

Slrpau.* Jlxnuslc' 23.41 t5.68 11.2ltZ.W 

ChILJ;lr' e.05+0.~5 1.n*0.ul1 

Oldur~ n&w Ill .let 3.P U~.Iulf 2.7dk 

HPhiP w L5.16+0.05 ZI.GStZ.@ 

d 

- 

n my' 

Rucin' &5.25+0.3 38,16tO.& 

DNA' Z.PiO.11 O.B7oO.Lt? 

WAS 4.Wt0.01 3.46+0.l@ 

W W W ~ &).J)t8.(U 61.%3*2.YP 

M~y*lrln.l~ur* 

17l~hyruwnul s,.a+d.a U.W*~.~JF 

',,ml( mwnl~ wmaWmin pa nu pusin d 32F 

5nd ycb, m-~.rvnlin p.r s. w#&n 6 IT. 

bs.4 NAWW ~xaa.V#9rn pr a&! ~ ld X?t. n 

Crrol~~,awna~n~,ponlspadn~~~. 

*hy( a &$I dm. 

'brtml NAD 011tnnal 10 NMmin pa F a n d 32%. 

1111;l1ion (WHO Repon) under lhc wagoly or whnchnlcal 

pmluCls a% being moJMlely hplnrdous Md it is still 

uscd arnpslicidcinmywwnuiw.. Adoseof5rngof 

liWkg body weight ha? been consi- by WHO LV 

No Chscrved Elk4 Level (NOEL). Bul in the present 

study the testis might8 of lindane-treated rat8 were significanlly 

dsueascd. Thc tcrlis hat been shown to be 

highly susceptible to lin& a8 it C- bid-testis 

h e r ilnd deprrsse ynmwlogemis["l. The d~cnose in 

the lcaticulor weight ul lindam-lrenled me my be due lo 

d u d Iubule sizc, spmatogenic m$t and inhib~lion 

01 steroid biosynbis af Lcydig 

I l l g h . . Tiftt.fl 4W+& 

The mighe of cpididymis. wninnl vaicles md 

VLI*~~ Inuiuc. 

venml pwle ill lindnne-1mLed me wen decreased. 

Oll6) lUU*Pl IzutIP 

(m~ttllyb.~.) Ill taO 9)oP 

Sevual stdi have sham chat !he epididymis and ac- 

=\wry KX orgnns quire n wntinuws Yld~genic slimuintion 

fw vwntion ol their noml smcturnl and 

7hc qxcific oelivilics of supemxi& dimuIn%. functid int~~ril$~'. l%us Lhe slight rtduction in the 

uwlw sul gluulhicne dubwc domd while the 

levels or hydmgm peroxide was found lo be elcvald in 

mlcd rats when mpand to the corrapcnding grwp of 

mlrol animals. 7hC wific oetivities of ~Midocenic 

weight ol che cpididymis and ncctasoly sex orgm in be 

vcated rats may be due 10 low bioavdlabilicy of andm- 

The mtioxidnnt sysm plays an errective role in 

arg~~xrr. :I&hyJruxywuid Jchydmgcw nnd I7khy. pmwcting Lcrm onl Mher biologicul tissu*; below a cricdruxyslwid 

kl~ydm~m~~w uue deumd signiliwrlly id lhrrxhvld or wdve oxygen specie? thus prrventilrg 

(P cO.a5). 'Iherr ms u sipiftam dswEpsc in the lev. 

Icrticulor dysl~nctionl''~. Anlioxidmt ciuymc$ constiUte 

cis of lcviculv DNA. RNA md win in mtal goup 

a rnudly suppoRive ltam or defence ngninsl Mctive 

W~II urn@ lo Ilr mud mim11I6 (ToMe 2). 

oxygen npcicr (RChS). In ~hc present study IhC acdviticq 

T.h*Z. 

of antioxidant wym, wpemride di.unulpsc, malase 

E i T a d l i n l v n m ~ ~ i n W e ~ o l 

~nuknu, ,,=!a. A*S. bP< 1l.a vrlhccmrulp~up. 

iuvl glutnlhiw redwin.. were (!ecreuced in limlune.wl. 

ed me. Thc levels of hydmgen pmxide gencmlion were 

loud (o be elevaed which ma ar a h e r of in. 

crud ROS. Thus in he p m l study m increaw In 

hydrogen peroxide wilh a duction in snrioxidnnt cnzym 

idiw LhC.oxi&live Ues. induced hy lindime. 

Ihc dsreDsnl aivities of slemidogenic enrymer 3&hydmrysWuiJ 

dchydmpa~ and ll~hydmxystemid dehydrogenas 

were indicative of the duced Isticulx 

slemidopunsis, Lidme hns been shown to inhibit 

h lo thc puthqu'wis or li&r inducal rrpmlwtive 

dysfundionl". 7he Wiun 01 supruxide dint~ulw 

suggestis that it is involved in antioxidant defmce and it 

h;r$ ken Nwm~ to WI ns an aitcwa ngulalury swikb in 

lnticulilr nwidogelursidZ']. Ilr cyld~nulc PC41 alzymcs 

of Lhe sluuicbgenic plhway M known to produce 

rtte radiEals. Thesc free mdids M pmluad s s resull 

of elecm kSP.p due to Ik inlem~iun ol steroid pmJ- 

WL$ or & pwwbubxlmlc? wilh thc cnayllrcq. lhc inability 

of LhC ~ ~ lo be orygwtd l phmola e 

the relmse or wtive oxygen spie~(~"I. In lildururuld 

1x1s IhC (!ecm in IhC teSli~vlx NnIelllS of

DNA, RNA ;uxl Wcin wcrr abarval, whkh show 

Ihac wlive oxygcn rpccies WI atmk vllnl mnparnt~ 

or the all-like 11w1cic uci& and pieim. 

In oMlflusicm, che pant s!wh indiik bl )ind;ur 

sllrrs lwiUUlilr fuwtiam PhTiMy by i&ig rroctin: 

~u).yl~ qxim md &ansing LI\C a~tiw.ii tnxyilcs 

lhuchy disrupting tntk rrpNJuEtion. 

Ilw: ;w~lxm hi~t cb SUIT o( Binbmwicn Centre, 

Wickny Univmicy, Rmdichary lor help. PPM 

&nnvlalw (he rtipl or iimisl rmn he I'M8 4: 2m - 6. 

Itl(ul;~~iis Cinl~il. New Y$11,. USA (CSUII Nth. WJJ. 17 LWIY OH, k- NJ. Ih AL. -1 K). !+*in 

M7P-I)/ lCMC onl t3YJ.W KM;). 

maurcmerr~U1PoBrr~reaa.IBblorm 

IR1; 198: %-15. 

I8 hlskhxi S. hbkhd 0. hwdVemn d -a nim 

References 

nbdh.liolldrbndm.dlm*ibn*ml 

I WH0lkp.m. l-prgrracm~rd~. 

lawcYnur. %I&mtem1974:.yU-l4 

In: WIh ad Sufny M- 5( Unic (W.-) 

19 CUbm A. C*.lr mivky. Ln: Onan*lld R. edlw 

IVJI: p 14, 

GKlmtu&dnrhoc(lfammrrknlmrch. Bax 

2 aAnm7. n.nsdR.Su"Au.yurard RM. M; CRC Mi IW. pan-81. 

mlrrinc-diming ~ 4 ~ in ~ viMrlc 1 % rrl hmn. Enw- XI ~I.MnnsvU81.W~dcmEahnsnd 

nn HwXh IWJi 101: 378-8(. 

hRvmz,m~-fmrmIher. JBi 

:I Caxq.~ Kt.. Clsch&+ RW. RJrhab OL. GrUm IM. aa Irn, 261: M74-a,. 

Ik.111 KC. Ihn Jl,. M by. t!X~%i 41(l*cn hmn- El lk'tB,kbiY.Syuu&~nrrll~l.A,jmJaivlhy 

UI ~xlnll

Effect of lindane on antioxidant enzymes in 

epididymis and epididymal sperm of adult rats 

K.C. Chitre, R. Sujah, C. Latchoumycandanc, P.P. Malhur 

M#(lk-, Pm&myUn(wniy. W " y M Z O l 4 , Idk 

-: 1-1 Y -I epididymir: Mcioxidulai mUvc oxygen species 

Ab.tnct 

d lbr- W. h: h llerhllbd mis, hum we& ignitka& duaiona inin& qklkiy' 

nrlwdqhc.rpieQrml~~~~oDuadmocUiryrrmpndwlBth?~. SigniTmcdaMlerinchewpmjd. 

(80D),-, #atidam nhrPr d @uUthiare pm*duc dvitia ud signitmt inmaws in thc 

~plarbodtlpld~~r*o~inUlscpididymirudepididyrmltprmoflindan~ucPted 

a. Lip*D dPElOIDl the bYOL of mdoddmf CN- in lk epididymia and epididyd ~gcrm of 

dor~WS~lndushp&wM. ( ~ ~ ~ ~ J A I ~ ~ ~ ~ W I S ~ ~ I S : M S - ~ I % O B ) 

ud d v e oxygen spkkr (ROS) thereby inducing ox- 

Mvc we&". ROS have bkn shorn a damage 81- 

most all chs rmcmmksvlcs of che dl incluing mm- 

LXUIO~O~~~~~~IWRWI f.lty~c/ds (WFA). CBUSiw 

hpnimm or plldor fudons[" ). Thc cpididyznis 

ir highly rW in RlFA aal lhusfoa suPcqxible IO (la 

&vs MO. &pcsm lo wvimnmenlal conlominpnl 

lindur bu tea Ibom lo enhsna oxidalive urrrr in liver 

and d"". Ihe -1 6lUdy WM wdcnnken I0 C- 

vducdc PL U T d ~ linJMs on che pnciaxht cmym 

d cpididymL ad epLWytrlnl yrnn ie &I1 NU. 

Iiadna (99%) w a gin fmn Jayhrishn. &tic&, 

Srbm. %nil Nadu. India. All Mhcr drmicnls

Mt~~m(10-lZsu&)vacob- 

Wfmn Ihehml ~ n i m l H a a o l h ~ ~ & ~ 

InUilUc d Rnlpdue Medi 6Judion nd Rkirch 

(JIPMER). hdiehey, Ida. Mmb *as mintnincd 

in pMic cya u*la 8 dl-qiwd S i d 

drlr(lZh:lZh)~k*(#t5)~.Sadrd 

m m r r d a l ~ d n w a d l Q ~ * c n r ~ e d 

l k . Undnc (5.0 m&hL, h o h dl) wm @vcn 

dlyMa~of~.Om&!xdyrdgpr&y14 

ad. fhcmmmllnimhnoind8rbnibrrdvrrol 

chevehick. BodywighQmt~oncnyIlcanale 

dny mil IC ad d Ihe qahml. 'Ibo mhmb 

ux killal by Uhcr 24 h llla OD Y -. Bpld 

i vim mval, dcnsd dl Ihe ldahy ligucr 

md wished. Thc epidi qona a- ad d h y 

weir. dm bmrd*cty.nd mddq qam vac 

cdkc4alMduscdfn~m)n.

WnCr, inyminn~nt of spmri motility, nducsd futiliza- dicnlg (hsl lLdan inducts oxidative s w in epididymis 

licm ability, lbnamal ~pum in mcn and and cpididymal sprm. ?hs significant incresrc in Lhs 

wildlifd"' . Our @OUS Uudia on onc of be avimn- lipid puoxidation of cpididymal spem in linjarr.tmled 

maurl mnminanta adosulfan 

- 

have shaMl to esusc im- rnts has dm brm ascciaLed with the dsnsscd sprm 

primant of tentkulur furrcionr Pnd k t the Bndrogmic- mocilily. 'Ihc gcnascion of ROS has bm mrnlated with 

ityinrsul". AQr&kanlinIhcpcsmtuudy (5.0 f&alogicaI cprm ~ o r as i oligosprmis ~ tanned as 

~oflindac/~~bodywdght)h~becnrmridacdas defeaivc spmns' with Rduced cprm motility, futility 

No Obaaved meu Led (~05)['). Lindsrr treat- Md span-mcyfc 

In the prrrcnt atudy, tke he. 

muH haa dmvn a tignirmnt in thc body wight pididymd spam mts and spm motility wn &- 

ud thc 4ghU of mtia. vcnlnl pmsfate and 6aninal crraccd in IindPnc-treaud rats as cwparod to the mnml 

~eoickr('~. Ln thc -I .udicr Ibc epididw wight anhla ilxlbiq thst epididymia was undsrgoing oxidaof 

lindnne-W IUIO was deacsrd eignifu.nntly and liw rmss. 

Illis my be due to lk drrravd biavaihbility and produccim 

of albgas(~~. 

Ro-oxidon1 onl ~ hiQIidPI1( bdum k viml r~ nornvllbidoplcPlrunaioningorlk~. 

lrsnyofthc 

can&, ampmu arh rc cllvirmnccltll motsminonta 

nff~Urhl.nacanpmvukeuecssiwpoduc(ion 

of ROS that is effaivcly wawgcd by ~~ antioxidant 

defapt apbn"). In 7, sewrnl an- 

(w paein) 

(mg mu) 

SuprmkCdim?Smb 

(ma padn) 

(mg DNA) 

Glu*hions-c 

(ma W) 

(ma DNA) 

aunhDncpm*duce 

(nlg ~m(cin) 

(w DNA) 

&or w -9 

(ms Imuin) 

(mg mu) 

wpaojdsaon' 

(W &) 5.28tO.!a 5.61tO.M' 

(mo DNA) 4.Pr0.37 5.74ro.39' 

PC0.05 W m a d u a m 

liOxi&"l *lg P6 glutahhe pauxidasI"', supmai* 

ciismu~py[al catadm) u. kmwn to w. 

Cytqbn of spmnmm is uuanly liud in volume In mnclusicm. fhe pent studies mflcct that linand 

WWi, a the polpmauwd fmty acids that dune indm oliiduiw smas in rat epididym* Md epibound 

in Ihe spurn p l w mmbnrr are wy sumbk 

lo RQS d~Y'. 

els or an~ioxidmt sraym~l with a pdbie raductiMl in e- 

didw spm, by inacasing ROS snd dsming levkubxidut 

auym rmrrliurce a mutually support- pididymal sprm mnD Md qididyd sperm molility. 

in ICM of defence against ROS. In Ua pocnt sudy 

thc rpoific rtivitic. of atnlar, rupaoxidc dimtase, 

#lulnthnrr tuk@nsE onl gluwi pcrmk WM 

lurd lo hc k& whik nn inumff in thc hydmgen 

m ilulhas h1k UT SW~T of Bioinfmtics CenpaoxWcpaxmcionMdlipid~wuc~ 

UP., Pmlichcrry University. P~dichury lw pviding 

in~Md@did~rpnnoflklindPno~- -a. fdtia. KCC vluawicdges My Tata MMOed 

n(r. Tlnm in Ua pwuu thc incrasc in lipid rhl Twt. W, India fw the Junior Sehol~6hip. 

-Mi with a rrduction in .n(iori atym in- a Pcxmwkdgu Mian Cauril of Medical Ra+arrh,

New Delhi, llrliil Ib SCI~KK Rcm& Miavship. PPM 

nckmwkdgm Ihe receipt or fi~ndnl auppn fmn Ropl. 

inlim Council. New Yak, USA (Grant Nm. 699. 

(H7P79/ HMC md B93.M W IQrlC). 

Ref- 

: RmW Kt%. Mia. P. *a* P. Ddta KK, D**rilh 75. 

Wcal rwn~~tk 

~ilwy I* I k n B r in rar. J ljrvinn 

m h 11111; r,: 4ln -Ln. 

2 Oudvid; RW, Cmpa RL, 0-q J. Rdnbsl a. M9- 

myW. Rmjbk-r(iviyd1irdncinfanJe 

ma. I Wichcln Tm'd I'AU; 3: 147-XI. 

- 

n m. w e. 

4 Imh D. OIyp midy ad aygn 

~nhnp 

W* I. ~sts HI. M I. 

RqnI*ni~ luxbly a] ti- mrarmbs d iingr in 

irWl nmk mt. Hun Toriml 1%: 15: U6- 10. 

-ips 

inmmmmls, liaRrlBidMad1989: 7r 67-1OB. 

pl4. 

mi mioxib aNm in implnn*kn R1 m: m- 

A h H, T- U. Tam H. ldvarc d mpc&m+rn 

qrra m III~&UI*: .-. Tol- 15%: 50: 2. 

~ o n ~ d r m a c m b r p r * . i l m Rqmd . 

6 -A, ti-&4~cdFlrJhna- 

T& 1%: 10: al-s. 

~ r ~ s c i d . ( m ~ ) : ~ ~ ~ a d pmmKC. o l i-C. 

- 

MhrPP. --el- 

Mcsmmprr-. FmuBiacWr 5: 1-15. 

~mdm+mlfmmtk~~ffvllirmdn. kimJ 

7 Vukh IA, hna Am. Iem, AP. K d OR. JWn Amhd IWt I: m-6. 

VW. I*+& Haubrbiuly .taws Ur arly 

mWrcswm~inbra(inUrhiu~rUeIm. 

dncimkam. Toxd MLtam 115: 45-51. 

ti r*lpul R. a"- KC. ~ilcmun)cn*rr C. ~ d u 

w. 

U i e d l i n L u c m C I i c v l r ~ ~ d ~ MaebMRF, kn R. RnprCYl d*+rmamJ npmrgarmzpminorhhm~.AdmJAnW~l;3: 

- 

135- i a d . M n d ~ d ~ d ~ k 

R 

m.ca+dl46dpid&hb,ema. BiahrmJ 

9 tie RE, be& I.?. MFmy WK. Marrmcn d cpi. l5Uq 1919 289-97. 

~slqmnmilrlys.mnvtCinUrU. BJIPnvi. Jdin C, .%z% IC. Weka P, Mmln DL. C.l**rr R. 

mCm;lmTmml LW; ?&: 317-24. 

uJ+ah*ykhmm~ndadnlC*m 

lo adxme A. mi*iy In: 0,eawdd R,aiIa. OmsrRa15u';I, Is-%. 

CXC Hanhxt oTmIhxh fa m m &-. 

Rm- MlienRS.WD.WD. UedaIl.)irrm- 

'h. UuHncn: a\CRa: l'B. pm-4. 

~kndd&.yanmimwehcFpaol* 

II M ~ S . -0. Imdmmnd-aim 

a7wdrrrh*awenprdsanh."mprmrrm. J 

n a b t ~ ~ o l a m d ~ n d * m r m a a m y 

lusmmz'&dnm*k. WJBiDdan19741 47: 4W- 

5086-91. 

w ; R . K a b a i Y . ~ r i o n d Y 0 , ~ 4 . 

chsnbllydkklprW--inbadbrmhi. 

p* -*ldi. Qll tmnml lulli Wl XI1 - Iu. 

-H.ONNN, YadK.kay(aUpldpcd&h 

ln~auabymkbrbl&~rcrtim.~Bioda 

l!691%: ll-8. 

Lo*rl ai. lldnQl@ NJ, b7 AL. Prim a. Rein 

~VimhDfolinphaDlrrqpr. JBidOan 

IS11 1% 2s-75. 

BuwK. AO.*dUrmnetand-dtk 

~~tukcdabrra*ahrdmd&- 

oxyWm&k dd. Biahcrn J lP561.52: 316-a). 

m c g ~ l c a n a i a l a s m r m ~ l l aIn: 

y . 

HmK, d SJay OuiQLin*nr. Oarn: WHO; IW1. 

uHer lo, sis.q BT. Rde d $udim pxwnm? in 

pmse&ynnnrf*rlanrua*hanladmtilly 

dbyqarmaalipidrumbnbl. OmeCRa198); 

23, n-50. 

Rz+dRnillPPJi Wr441-I. 

-T.WT. M H . Sdlill WB. RadlUmYp+dmbllarrberrmm-hrmaia~llvlnhnn-. 

InJAnWIP)9;P:S7-4.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin 011 the 

antioxidant system in mitochondrial and microsomal 


C. Lalchoumycandane, K.C. Chitra, P.P. Mathur * 

Scllwl oJ UJr Srmtrrr. Porsbrlirrry U#zri.rr.rrly, l'un,rllerrj, - WS 014 hrao 

Rc-cc~vcJ 21 Auyun ZMlI: rutivcd in rvvircd lirrtll I S Scplclnhr IKII: acceplctl i Norcmhr !lull 

The abilitv 01 2.3.7.8~tetrachlorodiben~~~~n-diorio (TCDD) to inducc oxidntive alrerr in heoutic and some 

extmhepatic iirrua of animals hu ban reporied. The precise nature and mhanism of action ~ ~TCDD on the male 

rc~roductive rwsm is not dcar. In the orucnl sludv, we hnve invcstiaaled the induction of oaidalivc slrcss in tllc 

leilic of ral dncr expoaurr to low do& 01 TCDD. TCDD (I. 10,-and 100 nukg body weipht pcr dey) wus 

udministercd orally lo the rat lor 45 dam. Aner 24 h of the lust treatment the ruts were killed using uncslhctic e~hcr. 

The weight8 ofthc -ti&, cpididymu, remind verrlo and renlral prostate dccrekvd uh~le the bod; uclghl rcmalneJ 

uncksn@ tn the mu adm~n~stercd wllh TCDD. M~lochondr~ul and micrmmal lrsct~ons of the lesl!s %ere oblnlncd 

by thc niclhd or dilTcrcnlivl antrilugulion. Thc ~lclivicy of u~rtioxidulrr cnzymcs such ax snperaxi~tc d~smnv~~sc. 

C I~IM, glululhionc rcducluse, und gluluthio~~c puroaid;~se dc%redml sig~~ilic~~~lly in llic LII~~II~IIIS trci~lc~l wilh TCIIU 

in u doae4cw11dcnt tliunncr in the milocho~idriirl UIJ ~I~C~OWIIIUI 

huelions 01 rut tc6lis. Tlic 1cvcI1 of ihydr(1~C11 

peroxde pencntlon (HIOI) and llpd prox~datton Increased In m~~ochondr~al, and m~crosomrl Iracl~onr of 111e lest s 

Thc ruolrr su9~crttd that the bw dovr of TCDD cllc~l dcp~el~on of ant~oatdant enzymes and concomltdnl tncrerw 

in the lcvclr O~H,O, and lipid peroxidation difkrentially in mitochondrial and microsomsl fraclions of rat testis. In 

conclusion the adverse erst oTTCDD on mslc reproduction could bc due to induction oloxidslive arcs. 0 202 

Elrvlcr Scicllcc Ireland Ltd. All rights rcrrvd. 

Kqmrw~lr: 2.3.7.B.Te1mchiomlibc1~o.pdw1in: Tutir Anliuxkdnn~ mzymn: Lipid prroxidstion: Mi~ocllondriul Irucltons: MIcroro. 

mu1 fnniollr Rul 

Rececltly there has been increased awareness or 

tlic possible clTects of environmental contanli- 

'Cormrm~3ina uulhor. Tel.: +91413455211; far: +91 

nRnts on ma,c ~eproduction (Shrrpe, 19g3: Chis, 

4136>3211. 

E.,~WII .ilu*h.~~~: p p ~ t h ~ ~ ~ ppmrthur~hol. 

~ . ~ ~ . ~ i ZOOU). ~ . i We ~ have reporlcd the effect ofenvironate~imuiI.com 

(P.P. Mulhurl. 

lal co~ilarninnnls endosulrun ultd li~idil~te 011 tile 

Il.r(URJXnI26 . n: Iron1 nmtler 0 M2 Elrvier Scicmr lrclilnd LJ. All rich11 mrvd 

PII: S0300-4I3XfOI 100563-1

tiii~lc rcprmluction or rut (Cllitru ct HI., 1999: 

Suj:ltl181 ct ill.. 2(K)I: Cliilnl Ct el.. 2001). Onc of 

lllc mtist ptcnt cnvironmcnlnl contaminants is 

2..1.7.R-tctr;1chlo1~dibenzo-p-tlioxi11 (TCDD). 

wl~iclis Ihrnicd ;IS ii by.ptoducl in the manuracrllrc 

ol'chlorini~lcd Iiydrmrbons. This compound 

is :!lso Fcirnicd in the incineration p m of niu. 

~~iciliill ri3i~slc, pilpcr and pulp hlcilching, cmissio~~ 

Ijt)lll \Iccl li~~~~iilrics i~nd lnotor vcliiclcs (Skc~ic ct 

ill.. 1'189). Tlie lipphilicily and low rate or 

rncli~h~ilism of TCDD leads to its accumuhtion 

;lncI ~~rsistc~icc in udipor tissues (Enan et HI.. 

I0'18) It II;IS lrcl~ rcptirtd 1Iii11 niillc rills cxpml 

to TCI)D displ;~y rcditccd fcrtility, dcliiyed pebcrly 

;lnJ altcrcd rcprductivc organ weights 

(Cri~y ct ;~l.. 1997). Millc MIS cxpoxd to TCDD 

sllt~wcil dwrc;~snl spcrln cou~irs (Gray ct ill.. 

1997) ~IIIJ IIII ~I~L~ICCJ numbcr or ubnonnsl 

svrlli (I::~qi ct ill.. 1997). Scvec~l niwhi~nis~ns 

llilvc Iwcn prop~rcd to cxplilin llic toxicity or 

TCI)I) i111c1 irs coIigcncn (SIR. 1990). Thc most 

co~isis~cnt biwhcmic;~l cnht or TCDD hi~r been 

sliown lo be the induction of cytcchrome P450 

iasax~tll~tl nnlonnoxygcnas ilnd rcletcd protcin 

(Pal:~~~il i~nil Kimbrough. 1984). Reantly nryl 

I~ydrwi~rbon (Ah) rwplor mdinled xnnrliinc ox. 

idi~x and xunthine dchydrogcnasc system has 

been rclated lo oxidative stma in vivo in the 

TCDD.tra11cd rats (Sugiharn et nl.. 2001). Acule 

highdore exposure to TCDD results in oxidative 

strcss in mulriplc tissua and species (Mohammnd. 

pur CI al.. 1988). TCDD has ken nporled to 

indacc suproxidc anion, lipid peroaidution und 

DNA damage in the hepai~ and brain lhua of 

rat (Hassoun et al.. 2001). Mitochondrial and 

niicros

I kl 

wciglit (b.wt.) per day for 45 days. For administration 

of TCDD, the pipclte tip was aently 

placed just inside the mouth and the dosing solution 

was then slowly explkd from UK tip al. 

lowing the animal to lick the compound. 

Corresponding groups of control animals were 

niai~ltni~ied und admi~~istered with vehicle nlone. 

'I'Ac inrinrals wcn? I'asicd overnight, wcighcd 

and killed by using anesthetic ether on the day 

following the lasl dosing. Testis. cpididymir, 

seminal vesicles and vend prostate wre rc- 

~iiovcd und clcnred from the adherin8 tissues. 

The weights of the tissues wrr recorded in mg 

as well as mgj100 g b.wt. Tatis wu used for 

subccllulur fratlion~lion and bialiemiw~l nud. 

ics. 

Mitocl~ondri&l and microsomal Iructions of 

Ihc tutis wen obtained by the dilTutntial an. 

trifugation method as duaibd by Chainy et al. 

(1997) afler rtandardizution. Briefly. n 2P/. (wlv) 

homopnutc was preprcd in &sold 0.25 M sucrose 

wlulion with the help of r motor-driven 

glass leflon homogcnh. Tbc hmogcnatc was 

antrifugcd at lOW x 8 for \O min nt J *C to 

obtain the nuclear pllet. Mitochondrial pllet 

was obtained by anvifuging tbe port-nuclear 

ruprnnlnnt at 10000 x 1 for 10 min at 4 *C. 

The microsomnl plkt wu prepad by the calcium 

chloride (CaCIJ Pedimcnlrtion method of 

Kamath and Nmym (1972). BMy. the plmilochondriel 

superrutant vu diluted with icecold 

CnCI, (I M) w that the final caranIralion 

of CaCI, was 0.8 M. 11 w bblted at 4 T 

for I0 min with occasional stirria& Tbm th 

snmpk wan cmlrifu@d at 10000 x 8 br. 10 min 

at 4 *C and UIC microtorml pellet wu obtnined. 

All the frnctiom wn wuhed UIk with iced 

d 1.1 5% polouium chloride solution md dirrolvnl 

in tlu 0.25 M sucnuc wlution (I mg 

prolcin/O.l ml). Thc mitochondrial, and microwmal 

fraclioru wn uud for biochuniarl dUdis. 

Superoxide ditmulu~ (EC 1.15.1.1) was 

sryed by the method of Morklu~ld , 

Mnrklund (1974). Briefly. the uswy ~nixtuve c 

tained 2.4 ml of 5U mM Tris-HCI buffer 

wining I mM EDTA (pH 7.6). 300 p1 of 

mM pyrogallol und 300 111 elizylnc source. 

ilwrcux in clbaorh~nce was mci~sund irli~n 

rctely ut 420 nm ugtlinst blllnk co~ilnining HII 

cwmponenb cxctpt tl~c enzyme and pyrogullo 

I0 s intervals for 3 min on a Hitschi U-2 

Spsctrophotometer. The activity of enzyme 

expcard in pmol pyrogslbl oxidizcd/min 

mg protein. 

Catnlaw (EC 1.11.1.6) was arrayed by ! 

method of Clviborne (1985). BrieRy. the ass 

mixture conbitled 2.40 ml of phosphate bur 

(SO mM. pH 7.0). 10 pl of 19 mM hydr 

peroxide and 50 pl enzyme souru. The dcc 

in nbwrbmcc wsr mwud immediately at 

nm against bhk conmining all the compon 

exccpt the enzyme at 10 s inarvals for 3 mil? 

a Hilachi U.2000 Spctrophotomem. The ac 

icy of enzyme WM expW in pmol of hyd 

gcn peroxide consumed/min per mg protei~,. 

The oclivity ot gluUthionc reductax 

1.6.4.2) was utlyd by the method of Carl 

and Mannnik (1975). Briefly. the 1ury 

lure conlrinal t.75 ml of phaphntc bulTcr (1 

mM, pH 7.6). 100 MI of 200 mM NADPH. 

pl of 10 mM EDTA. 50 1 of 20 mM 

lathione oxidii and M pI enzyme wurcc. 

appevrancc of NADPH ww mws 

immediutcly at 340 nm rgainrl blvnk contvinl 

all the cornponenu exapl the enzyme a1 

intcrualt for 3 min on a Hiinchi U-IOM) S 

[rophotomcter. Thc ucrivity d enzyme wus 

pmwl in nmol 01 NADPH oxidi&/min 

mg prolein.

C'. I~~I~~~~IIIII~~O~,~~~~~~ 

R6+ 4,lNl' 

'Tlic sctivi~y orcutulnr Jecreuud signifiantly in a 

Joscdepcndcnl manner in the mitochondriul and 

microrotnul Iractionc when compared to the cor- 

rapondig group of control ~nimalr (Fig. 2). The 

levels of hydrogcn pemride &nention and lipid 

~roxidntion wcrc incmscd rignificantly (P < 

(1.05) in rnitcchondrial and mimsomal fractions 

of thc tatis when cornpnred lo the wrmponding 

prnup of c~>orm\ animds (Figr. 3 and 4). 

(with and wi~hout secretions) nnd ventral prost;tte 

in a Josc-dcpcndent manner. In the mi~le rats 

TCDD exposurc during adulthood lius been 

shown to dccrcnsc the weights of the leslis, sctninal 

vesicles and ventrnl prostate us well as altcred 

1csticular und epididymal morphology Oobnson et 

al.. 1992). These eTTKts may be due to reduced 

TCDD one of the envimnmntul mnominunts. 

hut been &own to induce reproductive abnormaLilia 

in both wildlie and humma with reduction 

in futility (U-Sakuwy ct 11.. 1998). In the 

present dudy, the animals mrc administered with 

TCDD at low daa (I. 10 .ad 100 nag b.wt. 

pr day) lor 45 day& Sin@ muMIl expsurc lo 

low Jwcr ol TCDD (50 nL) haa been reported to 

rsduathcwdJIuoFthetcltirand~ryrr; 

orpm of nu (Otuako sc .I., 2001). Thc body 

weight of MI& lrutod ,with TCDD did not 

chuyo indiating that l s pml metabolic conditiona 

of the mi& wom within noml nnge. 

Administration of TCDD ad duaion in the 

wi#hu of lhc mllr, rpididymk minrl wicks 

FI. I. TCDD on lhe anivitin 01 ruprcrxide din~~ulaw. 

Ju~thionr ~~JWIIL 8rd $luelhinlx pcrnxaur ill Ik mil* 

chodrid (Mill m d mieraeml lractiont (MLI 01 1111 Icblir. 

The silvilia crpd in nmollmin per 111s prolcill al 

35 .C. Tb nlw *re upraxd u man f SD (01 six rnnltal 

and a iruporimaul mimuh pr pup. 'P

0.1 00 01 

" 0.8 I . .lo Dl* 

rcnn IW *c wl ~*rbtl 

I:#* ? lin'iw or rrDo on I& nnimty d mlah in ~hc 

~llll~el~nJri;~l and micmaml fmh nf nt lalb. Thc 

;rlirilh.i .In cxpn%ud tn pmllmin pr mp pmKin 11 35 .C. 

I Ihr V~IIIM IR. PIF%~(R( ns mn f SD Inr six mntd anl is 

cr~riltrnl~~l iu~ltn~ill. rn #mu*. 'Pz0.05 hmnridcltd lo h 

.~pmik.!nl npilm.il mnlml 

numbcr or Lcydip dl. germ cell and 

steroiilogcne enzyme activity. as well na impaired 

Lcydip ell LH mponsivencs (Mably el al., 1992: 

Joli~tw~n ct 211.. 1992). fhc weight or the epi. 

didynlo und u~crssory sex orgsns rquirc r continuous 

rlndrogcnic stimulation Tor Ihcir normal 

gro~vll~ and l'u~~u~io~~s (Kli~ulcltcr und Ha. 

I'FlUt. 7'hc wcigllts or u-ry sex organs have 

hrn uwd its b~wswy or andrugens (Milrkur and 

I lc I I IILY~ or 1'c'1)1) Im ~ kwh d hydm(m Fox& 

.... f,.,., ,,, IIIC IIIII~I.~IIY~~~~:#~ awl oranwwlu!l rt.aliutr 

,,,,,.,,, IIK ,.l~lr. rtr srl~r\w+~l ~knwl lwn I-r nbp tn8*r?8l 

.,I I* '1 .Is I ~ !\I) K ~ rlv $1, ~ conlrd ~ ~ and us crpmlrltlsl 

,1,1. ~r Fvq,p .I'rllll5 n ~~,n~nlmrl In k stfnikionl 

,1. l,,,.l 1111111111 

4.4. Elrm dTCDDon lk*wkdUpidpraiiblbnin 

milochondri~l and miaolavl tnnia d N W. The 

nlua mn apnrd in Whh w mt pmccin nl I5 T as 

rmn f SO Icr ua mnlml and Sia riprirrnUl anirmls pl 

mp. 'P < 0.05 nl ranidnal lo be sbiiknt .pin. 

mnrml. 

Chattopadhyay. 1982). TCDD has ken reported 

to reduce plasma tcatwlerom. Sadihydrotcatosterone 

(DHT) and LH (Mably el al.. 1992). The 

observed reduction in the weights of ~k amnory 

su orpm rdkts reduad bioavailability and/or 

production of andmgm in the TCDD-lrci~tcd 

MIS. 

TCDD has bccn shown to ad through Ah 

mptor. a lipnd aninled nuckar lrnnrriplion 

raclor (Safe. 1990). The nuclear Ah wptor conrplea 

is a heterodimcr conlnining the Ah receptor 

und Ah naptor nuclear lrnnsloetltor prolcinr and 

the molcculur mechanism or Ah +or sction 

has bem shown to be assodated with bindinp or 

\he Merodimcr lo dioxin mponsin ekrmts in 

rcgulr~ry regionn of Ah-mponsive gene (Sure. 

H)OI). It hu bacn repod that TCDD and its 

mngnm bind to the Ah mqlor, which mulls 

in the induction of m m I enzyme rysremr that 

may be mpomiMe Tor the production or rcaftive 

intmndnta such 1s Tree rndbh IKimbmu~h. 

1974: Neben a 01. 1981: Slohr. I WO: Slohs el ul.. 

1991). The mechanism of TCDD-mediatnl rcuctlve 

oxypn spdn production has k n p r o w 

to involve the cytmhmme 1'450 IAl and lA2 

(I)iliherlc~ CI ul.. 1997). 1'1% Ah nwptor mdiolnl 

lndwtion of runthine oridnx'nnd xnntllinc Jchydropnnr 

activity by TCDI) ~IIS ~llro bccn rc- 

~I,,CI~I 1,) k n.*p~\~ihk. lor !In' pr~uhlni~~!~ 111'

'lstrr,tb~xr 171 i?lYJ?I 127 /.IS Ill 

rcuctiw oxygen rwim in liver (Sugihilrs ct ill., 

2OUI). In the pracnl aludy adminiutrution ol' 

TCDI) dccwd tla ttctivitim of ci~t~ilitw, xc~pr. 

oxide diuilutuw, &IuIathio~ic rcducttt~, itlid 8111- 

tutliiolw proxiduw. wl~ile the lcvcls of bydrop11 

peroxide and lipid peroxidntion incrwscd in tlrc 

mitochondrial and microrom~l fnctlonr of the 

testis. Mitochondrial rapintion is the main biological 

wurm of tuproxide anion radicals undcr 

physiological conditions. The mtioxidanl enzyme 

supemaide dimutasc hps bmn rhown to aculerute 

the dimutation of the superoxide anion into 

hydrogen peroxide (Nirscn and Knyscl. 1983). 

Cntulase allows degmdation of hydrogen peroxide 

into water und oxygcn (Jeulin el al., 1989) and llic 

glutathione pcroxidue/reductue system ha^ been 

rhown to calalysc the degradation of hydrogen 

peroxide and lipid hydroperoxide by using re. 

dud glutathione (Alvam and Storey. 1989). 

The reduction in the activitin of antioxidant enzymes 

and incruuc in hydrogen peroxide and 

lipid peroxidation could reflect the adverse effect 

of TCDD on tho untioxidunt system in testis 

subcellular frnctions. Oxidative stress has been 

shown to occur primarily in the mitochondria 

fol\owed by microsomn. Orievau and Lunnou 

11097) rcprtnl that ROS such as HIOt uppca,rs 

to k u key agent in the cytotoxic effects In 

spemlowe and in udJition to their dim effect 

on cellular constituent produce oxidative stress by 

dwrc~~sil~y the c11zymatic JeRm of the testis. 

Un& high ROS mnrrntrntions pre-dumuged 

rpcnnutowu an exposed to lipid peroxidatioli by 

polyunwturuted fatty acids (Ichikawrr ct al.. 

1999). The mitochondriul and microsomal membrann 

have ken reported to undergo permurbility 

changes following enhanad Lipid peroxidation 

and Jutathione depletion (Chancc ct al.. 1979). 

One of the important upatl of anlioxidwt en. 

rymer ii their nature of synergistic functioning: a 

decnrsc in superoxide dirmulav clctivity hm been 

shown to increase the level of supcroride anion, 

which is known to inactivate catlrlrro llctivily 

(KOIO and Fridovich. 1982). Similarly. when 

wtahuc or gluuthbne psmxiduc faib to climinate 

H,O1 from the all, the accumulated H,O, 

hru been ahown to caw inactivation of suproxide 

dismuw (Sine1 md Garber. 1981). Reactive 

oxygcli spccics Iisr bceci sliown 111 d;~tiii~yc ;llliioal 

ill1 ~llscromolcculcr. viz. prolein, lipids. ccc.. i111d 

lllc cliiinpr in llic rcprtnh~ctivc orpiill wciphts 

111:ly slso Iw duc lo iIi~~l~ii$c ~IIIIIIIC~~I hy IUIICI~VC 

Uxygc11 rwiw (&l~~ultdt%rl: IDYO). 'I'llc ~.acllla 

sugycst thut the low doses or TCDV clicil dcplc. 

lion in antioxidunt defense system diflerentislly in 

mitmhondrial and microsonial rrilctions indici~ling 

TCDD-induced oxidative strcsr ill tculis suhccllular 

fractions. This may lend to disruption of 

functional integrity of cell organelles. In conclusion 

the adverse long. term effects or low doscs or 

TCDD on male reproduction of mts may be due 

lo the induction of oxidative stress in testis. 

Acknowledgments 

Tlie aulhors ock~~owledge Dr Slcplien Safc. TX. 

USA, for the generous girt of TCDD and thc still'l' 

or the Bioinrormutia Center. Pondicherry Unlversity. 

for various incilitics. C.L. acknowledges 

the Indian Council of Medicnl Rcseilrch. New 

Delhi. for Senior Research Fellowship. K.C. ilck~iowledges 

the Lady Tstu Meniorial Trust. 

Munibai for u Junior Scholursliip ~IIIJ P.I1.M. 

ucknowledges Ihe Population Council. New Yolk 

for financial assistance (Grant Nos. B99.047P-91 

ICMC md B99.048RIlCMC). 

Ra. 23. 77-90. 

Bury. I.A.. Ausl. S.D.. 1976. Lrclo~roridlw cill;tlyml Ilpal 

proriblion of micrororn~l and ~~rlilicrrl n!nn~brnnn. 

Biochim. Biophy+ Acts 444. 192-201. 

Curlbrre. I., Mmnmik. 8.. 1975. Purifimlion und chlmtctcrl. 

ulion d Ur Ilrvanzym plulrlhiom rrJurlax from nil 

livcr. J. Bbl. M. 250. Yl4-5480. 

Chainy, O.B.N.. S.nunwrry. 6.. Salnanla. L. 1991. Tnlov 

temne-induced sbnp in taticulur nnlioxiJilnl ~~(ICIII 

Androlopiu 29, 341-MY. 

Chuncc. 0.. Sie* H.. Bonrit. A,. 1979, llydrapcro~de 

nlslubolirm in murnmulwn orylnl. PII).riol, Kcv 59. $27- 

514. 

Chw. S.E.. 2WO. Endwrinr disruploi. innd nlalr rrpruducltsc 

futction - a &an ncuka. tat. 1. Andrul. 21. 45-46.

Chilr:~. K.C.. L!olnn!myci!~dum. C.. Milhur. P.P.. IWP. Kimbroufi. R.D.. 1914. The loaklty d the p+l~Io+.~cd 

('l~io~tk cN~yl ol'e~dtaulhn un Ik Inlicukr funstions or imlmciic m d s m d chffabla. CRC Crit. 

ra A'iiiln J Adrol 1. 203-206 

Rn. Toriwl. L 443-498. 

Chtlr;~. K.C.. Sul;!lha. R.. Lulchoun~yclnJan. C.. Mathur. Klirrkksr. 0.1, Heo, I.&, 1998, To* 01 lhc male 

P 1'. XKll, EGXI rf lindme nn entinxid.nl ctuymn in uarmnt d m ad r4 *.dl. In: Kod. K.s. 

cpal*ly~nir >~tmI r~t'dym~l sprm of ntlull mu. Asinn J (Ed.). R*nodustk and R*okmul Toikdqy, pp. 

Andt,-l J. ?(I5 XIR. 

-((1. -- 0- . 1 

, 

('littlwrn~.,\.. lVX5. 11,. C~RVIIWI~!~. R. (W.1. IlmJhr,k Kono. V. FtlJa!ch. I.. IPU. Supmxidc ndbl inhibits 

Mrlln

S.tlc. S.. ?IXII. Mubuli~r bioluyy ol 1 l ,411 ~ rwqvlut iwd it* IOIC Sl1n11h.S l .Alrlt.~c~l.N.J .Sh;tr;!. M,A..Al~ll,~y:tl~./. A,. Wi!ltl?.t. 

in ath'iaop~xsis. 'Turir'ol. Lull. 120. 1-7. 

Z.Z., IWI. Bvidmwr. tor IIIC it~duclioa ol ill! ox3;clive sllcrr 

Slliarp. R.M.. 1993. Oxlining rprlll ~ounlr in mcn - i: ll>elu is rut hcp;nlcmilwh~ndria by 2.3.1.8~MIrucl~l~~rodibE1~~~~1~. 

"a cdwrinc ruur'? I, lidwrinui. 116. 317. Jbll. 

dtoxin fTCI)I)). Ad", lirp. MIYI. Ricll. 2x1, &?I R3l 

Silwl. I1.M.. tirrlrr. I'.. I1)XI. i~~wliv~~lk~~~ 

a%( 11111111111 ('11 /.!I SIIFIII.II:I. K.. Kilt~~l,l~ti~. S . Yiu~tl~d:~. I .. l )Itlit. S.. )liw1:~\liil,8, 

r~~~ur~~r~Jiaa~~luwduri~\lsr~~u~Llt~O~~~~JII~O,~ 

Arch. K., YI~su~I~, M.~ lri~,i~i.Kt~riy:,~tt;t, Y,, 21811 Aryl lhycl~twa~. 

II~x.ln~t. Ilu!pl~y. 212. 41-1 4Ib. 

Skme, S.A.. Lkwltunl. IS.. (imcnhry. M.. 1989. I'oiyel!lu!i. 

IIUIL~ dibnus.(#-dit)ai8ih atd polychbrinul~d rlikwh,. 

lur~l rvruelar IAIIW I.,t!etli;llcd l~alt!eli~n~t,l xi~~~ll~i~lc ~ull~l:)~.f 

x;~t~lllilludcl~ydru&~~!i~~-;~~livily by !.~.~.R.IcI~~~cI~~~~II~II~II 

, 

I'untn.: Ilr rill 10 bulnna hullll, A Rcv. Ilunl. 'l'ari~r,l 8. llrr). 

177 ?lIJ. 

huj;ellt;t. H..Cltllr;t. LC.. I.i~lul~~nt~~~yvi~lId:~l,c.C.. M,llll\rr, I'.I' . 

S~ohx. SJ.. IW. O~idulive atw induced by 2.3.7.8-lclrd. 

?WI. E ~ of ltndsm I on Icsicubr rnl~o~iJa,lr syrlcln und 

chloralibmw.pdwxi~~ ITCDU). Fra Rud. Biol. Mcd. 9. 

79-W. 

aerotdoscnic cnzymn in adult ruts. Astun J. Androl. 3. 

135-138.

C. hteboumyeradlos . KC. CMtn . P.P. Msibu~ 

Induction of oxidative stress in rat epididymal spenn 

alter exposure to 2,~7,8-tetmchlorodi~~ioxin 

Rmclved: 23 Auewl 2001 IArrcptd: 7 Nomnkr 2WI / hbl~rhcd online: 6 February 2002 

0 SprinptVcrhg 2002 

Ahtnct The ability of 2,3,7.&tetrachlorodiknzo-p DNA) in the epididymal sperm. The results suggest that 

dioxin (TCDD) to induce oxidative 8tW in various graded do= of TCDD elicit depletion of antioxidant 

ti^^ of animala hu been reporled. The nalure and defense system in sprm, indicating TCDD-induced 

mecbanLmofwtionof~DDonthcnntioxidsntsyslem oxidstivc stress in the epididymal sperm. In conclusion, 

of spurn has no1 bccm Uudied. In tbe pnscnt study we theadvenedfccton malereproductioninTCDD-treated 

huvc wught to inwatigatc whether TCDD induas ON- ruts may be due to the induction of oxidativc stress in 

dativc mtror in the opididymal tpcrm of rau. Subchronic sperm. 

dose6 of TCDD !I. 10. and 100 ndkn bodv wci~ht Der 

day)w& pdminik& orally to m

(I't,l:~t~cl 211x1 Kin~hronglt IVX4). It has bccn'rclwrt~d 111111 Jilulr~i vllh 95 N ot dilucnlr Tbc h i p . on the wunling 

i~cutc hiflh-do~ cxwsurc lo TCDD results in oxidative ehrmbcn d Lh. impad Ncukaw4ypt -t 4% 

surd. 

strcss inn~ultiple iissues and species (~ohammad~ou; 

A ~ ION ot rbs ~ lbomWNy ~ .M diluku Y 

sp&.nm *u vudnd to oh Omdq dunk. dthc hem* 

el 811. IVXX). TCIID h;~s hccn rcpnrlcd to indua suwr- cyIanna.TLo~x.lMW(L.ndIbr5minina 

;rxi

I. 1 Ilal 01 2 1.7 # I c I ~ ~ L I ~ i IlLl>l>l u I $,,t ~ WIR. ~ ~ 1, Ellbcl ~ ~ or TCUU ~ o un ~ tllc ~ uelivlly ~ ol ~uprorldo di\mulil in 

lls rpddynrul Ipml cww SF saunu wrc ac@rmslro ,n lhc epididymul sperm ol rum. The cnzymc uctivtly $8 exprrssed sr 

dupllate lmm ul upnmntsl and w; sonlrol nu The dau are ~nomolcr pcr rmnule end mlllinrtm or rolei in or m~lli~rsm al 

upraad am mun+SD. and PCOOJ ImIerub) u corv~drrcd 13 DNA at 35'C. The dnla ate hpd'a$ mcani~~~toi~r~a 

~ndrrlr I u)nthcsnl dlllmrur telrren opnmcnut and concrol cxpenmenlal and ci% conlrol groups. P

t11.3. Wal 01 TCDD on !he acttnly ouhe !n the cpoddymll 

.p.m, nr ,a$, llc ctv>tt>e ;oafvtly I- urn 

as mlcramola pm 

-wtttae uw twll~pc~n~ ~(PIo!c~~ ~rrnilbmmofDNA .I IrC Rr 

data sm ctpnxkd 08 mntSV for-w apnmcnul and mr 

mnlml SroLF P

Fin. L Corrclatwn bclwfcn "perm counts and DNA cootent$ or 

Fil. 7. Urea of TCDD on lied mlidrtion in the s~ididvrnal nl cpididymli rprm. A paaltivc corrclaten (r-0.9% n-24) war 

Grin or la.. 7hc m m adktyu in m~rdm.i" 

found between rprm wunl and DNA contcnl 

pr 

mtnulc and rnillignnn or mein Ot millisnm 01 DNA. n o data 

urn n W M munlSD Iw ax uprimcnUl and am control 

mum. PcO.05 lm~erldx) il lo indiate a rienificant activity (Kono and Fridovich 1982). Cataiasc or 

glutathione peroxidae rails to eliminate HZ02 from the 

cell, and the accumulated H202 has been shown to cause 

inactivation of superoxidc dismutase (Sine1 and Garber 

1981). Griveau and Lannou (1997) reported lhal ROS 

dovl of TCDD (0.15 ndkg per day on 5 days/week for 

13 weeks) wuld induce oxidative stmu in liver, spleen, 

lung and kidncy following rubchronic exposure in mice 

(Skwk ct ill. WOO). In tho orrscat rtudv administration 

of TCDD cuuml II ducll& In the sperin wunts, which 

1s wnrrclcnl wtth esrlter reporu (Fwt el al 1991. Gra) 

cl uI 1997) S~nce a wry ugn~hcrat wrnlauon (r- 0 95. 

18-24) was ohncrvfd bitwan cprm counb and DNA 

conlcnb in cpididyntnl,sperm. DNA wnlcnt wus routinely 

ucsd M an indicator of sprm wunt (Mukherja 

cl ul. 19921. Adminiatnlion of TCDD decreased the 

hydrogen peroxide and lipid peroddauon roo in the 

enididvmal soerm of mla in tams of boLb ~mlcin and 

~ N *LI~~I; A doso-response wsr not obscr;od wtl! re. 

galrd lo .any ofllr hit)~.hw~luI ptmmctcn In our rlvdx\. 

which is auito similar to (he mulb obrwed in studies of 

the ctTcct.of TCDD on the hepatic and braln tluues of 

nu ( Hmun el d. 2001). M~tochondnPl mplntlon tr 

the main biolo+icnl mum of tuamxidc anion radials 

in physiolopi&l conditions. 7% antioxidant snrymc 

superoxide dimuusc lsaknla the diimuulion of Ihc 

runeroxidc anion into hvdroncn mxidc. Cnlal;~se ilc- 

such as HIOl appear to be key agents in the cytoloxic 

elTects in spennalozoa, and in addition lo their direct 

erect on the cellular wnstimcnt, to produce oxida~ve 

stress by darcasing the enzymatic defenses. Thc rise in 

the kvels of hydrogen peroxide generation by 160-170% 

reflccls the induction bv TCDD of the oroduction of 

mctlve oxygen rpcctcs~lhat arc no1 e.ttn'~nated b\ lhr 

ant~oxtdanc enzyme, rush as catalase and g.u~ath~one 

reductase/proxidase systems, and this can induce lipid 

oeroxidation. The wssibilitv of eflects other than the 

uppnrcnt darease In unt!oa~d.lnt actl\lltcs tn caus ng 1hc 

tncw m peroude product.on also cdnnol bs r~lcd 

our. Cytoplasm of the spermatozoa is extremely limited 

in volume and localization. so that the ~olvunsaturated 

fatty aads lhsl nhound In Ihe eprm plkm'a mcmnrd.w 

urc vcry suscept~blc to ROS ultdck (Al.kcn et ul tYSJ, 

When ROS &nczntrationr are high, predamaged spcrmatozoa 

are exposed to lipid peroxidation by polyunsatunlcd 

fatty acids (Ichikawa el al. 1999). The sperm 

membranes have been teported to undergo permeabilily 

chanua followinn enhanced linid mroxidation and 

glut~htone deplc

IIR 

h r k a ~ r Ilw ~ ;njllmr~ l ~ Ilnionk Dr. Stcrhcn Sitrc. TEXIIX. 

USA, li,r Ihr: gctwmu\ gn uITCUU, and Ihc rlail of thc 01otnr0rm:ltin 

Ccnlcr. Pondichcrry Uncvcnily lot ~riow Iscilain. 

C L tr the Iholder oli, Setunr Rwurch Fcli~w~hip Im thc Indian 

~ ...., . ", 

('rrhg iron? tk LnJy Tala Memorial Tnul. Mumhi; w t r and bn~n ((PYOI d nu Inn eubshronic 1; 

ilnd P.P M hi83 refcivd Ilwndal ;,nmtlnn: rmm tk Popvialion m~nlumo~TCDD.ndlU n IApplTrmcol2l~2lI-219 

Tclt~ml. Nrv Yolk r$mnla nn. RW YlP.P/ICMC.nd BPP.MRRI lehok.m T . ~ o d l ~ . ~ h mWB(1999) o n ~ R- oxyen 

Il'M('1 'Ihc ia~*~llvn LILYI:IP tlnill IIK cx~ri~ncnln done Jurin~ won tnfluaa Ik o a m a raa.on hut nn r.ain anans. 

tllru. ato~lnz R)III~~Y wvlh llle cl4rrelll 181~1 or lhr co~ntry. 

J&,G&:fi

Iionshii 

nepmdvctivc mxicity ad uptm m RewrA (JlPhf6Rh Pondichay. In& llx mhnls ~nr. 

chaniuls Lhat geasltc ructive Oxyw apsic. (ROS) 191. nuinainodvndcra12h:IZh~of~.ad~md 

It has ken rtur mthoxychlw urdapm -5 pmvidad*.tad.rdsommcrd.l~Wadruer 

-rygcnrrc mdialld mivation ad the 8d libilurn Metbmychla ( l . l . l - u ! ~ 2 3 ~ ~ y - 

mulunt d v e mtrbolitar, pxsibly free ndiulr, bind P ~ Y I I C X ~ ~ ~ 95%. sima mmnw Ca) wu 

mnknUy m micmroml commnu [lo]. Antioxidanw diualvtd in mna ad olive 011 (1:19) md .dmlnlncred 

free ndiul m m and suifhydyl WnWning mm- dlyndaad50. 1 0 0 . a 2 0 0 ~ b o d y ~ d a y 

pOunds inhibit wvhl binding of mihorychlm in hurmn for 1.4. a 7 days. An Antiaul group of minub was 

liM miuaomm sumttg that the mdve inmmd(aln -ldministaed -w 600 mslLI WY VclOhv 

~m+ndiulr[11].IthasllsokenrrpMtrdr)uthuman 

day)mdritMinEZD~bdy~for7d.y~. 

cytochmm P4U) cnzymar respmsibk fm conversion of 

~&~oftkdodn~d~oll,tbepfpme~p~~ 

methoxychla im 16 nqior mmblhea, the m o w - 

pcnUy pld 

jW inaide the mmlh ad tbe doring solution 

mcthylatcd dsivPive, ixluding CWlA2 play the p+e. 

dominant mlc in this reaction [Ill. Thc ability of the 

w lhsl slowly orpolled fmrn the tip dlowing the m i d m 

cyl* 

lid 

chmm P-450 systsn lo induce the poduction of d v e 

the cnmpound. A carrrpondiw Omvp of mnml ani- 

OXyECn swim in hepatic and other tisucr has bcen re- 

MIS was admini- M equal volum of vehicle alone. 

paned 1131. 

It has bcen rhom thtt mthoxychlor rndvcc oxidative 2.Z. Necm~~ 

and eo"~lb7 oj~pidIdyn~1 rprm 

stress in the epididyd sprm of gost 1141. ROS are famcd 

on h ~ physblogr h 

pnlhologic euditlon~ in mumlw. The nnimlr vtrr fmed owmight, wcighd. 

- 

and killed 

lion tisuu: due meir high -tivity, ROS my int- with by using ancrVlctic clhcr on the &y folldnp Ihc last 

biomloeuia inducing oxiduiw [IS]. This oxidative bins. The wighu of the linr. kklney. -ti& apididymis. 

rocss i mlmd m the antioxidant defense qsem, and an ~C"ind nriCla, aad vend pmM werc romrdad. The 

ovaail babct bwkn prwxidano and mti0lid.n~ is ieR cduda epididymL wu weighed aad wed fa qrrm 

q u i d m rminhin ecllvl~ hmcmasis. Environmaral mvnu and Iprm motility. Ihe E.Ph md mud. 

wnumirunb have ken rcpard m dirnvb the pm-oxidant maions Of the right Wid- and rpididyrml rpcrm vm 

- 

and mtkridant bel- of sells 1161 and to -11 in en- vocd for bbckdd ny.. Thc @id@ rpmr 

ention of oxygen free radicals aad ROS Ilfl. A mk of ROS mllsEccd by cvuinp the wididymi, into anU w in 

m inf&ity due m Mmiw spcnn function has bca m- 

q c d 118-201. cxaruve gmaslhn of ROS has been 

shorn w caw pxidnive to the plasma mmbnrr. 

W h k.dr m irnplirrd rprm funom I2 11. Suproxide 

diMHUue dcptcdon ir Wght m be auocimd wilh 

spcrmImmotility [21l.Tofisul.rpodvEfionofhsendiulr 

and rtiv&an of the ~doxjdant dmnsc rym have ken 

rrponcd aftu expome to wxic chuniulr [I8301 and tkc 

epididymal pDdvction of hse ndifals md function of the 

anuouht dmnse synrm quire fvrtbcr sndia. Rcviova 

rodia 6um ow llbaroy have sham lindur idoxidative 

meu in the twis u well as in the cpidndymia aad 

cpididyd sprm of 8duit ram 123,241. In Ihc prrynt study. 

vs have sought to duemine the effsct of nulhoxychlor on 

the epididynul antioxidant system of adult ntr by nuw- 

~ng the antioxidant rnrymr ad lipid papridUion in epidrdyd 

rpcrm d in vshs regions ofthe cpldidymis. We 

aim erdvltcd the Mccl of mahoxychlor on cpididmf 

spmuumaadspammailityinordcrm.uarimnvlc 

qmdua(ve mrieity. 

Epididyual qam rmnt wu deOBmiacd by tbe mahod 

u dmaibcd it1 tk WHO MMlul 1261. Brkfly a S-6 

diquol ofapididyrml .pnn was dild wiLh 956 dil~t 

(50prodiumbkpboartr. IOmL35%farmll4mdO.S8 

ayplablucaar.Mod.adm&qmaw~umofl 

L with dirtlllsd e). A mw llip vu .sMd a the 

monungdumbarof~Ncuhtypc~.& 

pmatnutaly 10 I3. of the Uaovghly mirod dilulld ipscinmw~aredmsuhoftbe4np~oftk 

~ ~ , v h i c h w ~ . U o v m d m ~ f m 5 m t n i n s 

bunM hanba .w -1 wlu. Tile cab ~lmnlld 

ddn~~histimmd-rmnaddChaLilh(micmopc 

m m x. 

Roprrri*c.prm~Uy*.l.~lrhUdrbmd.rdmc(bodI27YIbyobpinhy~~tbe~ 

epididymla xi* 8 pipoac Up and diktiw a 2 mL 4th 

H.m'sP12mrdi~.135.CAa.UQmcd~1alumn

C L m r ~ n . l l ~ T a d ~ 1 6 W J O m - O C O 

a. Superoxide dismutase

o. Glutathione reductage 

mg pmWn mgDNA mg Protein mgDNA mg protein mgDNA 

1 d.). 4 d.ys 

d. GlutaUlione peroxidase

c urbmwen&n= s a( I n.pradpradpradnu lad* 

I6 rm) mo-mo 

a. Hydrogen peroxide generation 

I d.Y 

b. Lipid peroxidation 

I d.)r,

IOWAME -. Tor. #558517 PME: 9 SESS: 5 OUTPUT: Tuc Jan 29 10:00:51 2002 

hieblW-mM1-#mOlm/msr26-02. 

- 

1- in th opididYIni1 Md a dcuura in the weight of thaxychlor into Its mujar melaboiitrr and CYPlA2 hrr 

Ihc 80% Orom8 my dn lea biwv&ility bccn shown lo play a predominant role bn rhlr reaction 

Or pmdvaiw Of Mdrapsn in wtbozychlor--mi rau. IIZI. The ability of the cytochrome P450 syslcm to in- 

Tho weigh1 of tho Uvu UKI Udvy dld not ihow any duce the pmdvction of ROS has ken wcll documented 

lloniacMI in tho MbmIr lmald~wid Moxy. 1131. In the present study, admnisuatian of methaxychla. 

OnY Q II. npottcd IhQ ldmiairrruion of mc- chlor dccrurrcd the activrtics of catslare, superoxids dis- 

IhOxyshlor Q dce4 &@ng from 200 c 400 mglkdday murase. plutath~onc reduclass, and giutslh~one pcroxifar 

11 nwr& dlarod tho W y wci@ htd veiphtr of the darc, and concom3untly increased the Levels of hydrogen 

live# Md Udmy 1361. Thc ebomr & of admininn- perondc and lipid . . proridstion in eddidymnl rwrm 85 

tion in out uwiy-nuy -I fcxiho I.ek of o b ~ ~ e d wcll as in the cpididymis. Suprox~dc dismutasc ia cunetion 

on (b..o a* wciphu. 

Thc acb.niim of momol;ychla-mediated oxidative 

rtdered the fimt line of defense against deleterious cffccrr 

of oxyradicsll in Ihe cell by coulynng the d~rmuiot$on 

~irfflc~butilhu~lbowncbomsdiaudby superoxide radicals to hydrogen peromde und molecuivr 

Ihc vdvuloll of nkxumnl moaoorygenue, which is 

involved in tho WII~!QXI of mDZboxysbior iaw iu re. 

vdn mmbliccr 1101. Dudly Uhir nrtioa. mtivc 

oxygen. The reduction in thc aclivity of catalase m;ly 

reflect an inability of cpididymal sperm md the epidid. 

ymir lo eliminate hydropcn pmxide prcduced by the 

M $ V & poulbly fno ndiul:, bind covllently to activation of methoxychlor and iu mclabolilcr or lo enmi- 

mmp~wmr 1101. ~stioxid.n~fioe r.dic~.i zyme mscuvauon caused by excess ROS production In 

luvenlar lad hirulfhydiyisonuini~ compounds Inhibit 

COV&lll Mlldiq of maborycNa in hwnrn llver microcpsdidymai 

sperm and the cpididymls 1371 The nnrioxldant 

enzymes caalase and peroiidaic prolocl ruperoude 

unna, ruwdng Uul UT M~VC inlediate is I. flK dirmutssc agatnst ~nactlvation by hydrogen proxidc. Rendtul 

1101. It hu been rhown that human cylahmme 

P450 my- po rolporulblc of rhc converrion of mcctpmcally. 

1hC rvperoxide d~rmulasc protects cntalasc 

and prnxidase against inhrbitian by superoxide anlon.

- aaarslu30 ,.*mra. L~jl;lr; 

-P4-w~~w'IPldnopd.oSd)o 

'wDU----w3p 

~ r c p o ~ ~ a ~ ~ ~ ~ 

~ * p a ~ l a . q l = I ~ ~ ~ ! J l ~ 

~ ~ ~ J ~ ~ ~ P ~ . C P * P F I ~ ~ ( O O J 

u08.p(rd PW% pcn wmd polorpKq u! muul 

I p p c n - ~ ~ ~ ) O . . ~ u ~ p ~ . ~ a ~ ~ 

~ ~ ~ s n n o r n ~ I r m X p l p ! & 

Po. O(tP wwandnt almpylna 

u1 pOtllWI wm-d 

or en- aq Knu wu*. WIKZw rM J.3 nu.l.q '.nu

JOBNAME: Rqm. Tm. M5W7 PAOB: 11 SBS: 5 OUmJT Tuc an 29 IO:W:~I zm2 

MsbZla-m+m,iwasw-m 

cpididyrnis against lipid peraxidation mduced by me. 

thoxychlor by inCreasing antioxidant enzymes md de. 

crcains ROS geamtion. 1n addition, tosopbcml sup- 

PrcsEM lhe prcducdon of reactive oxidants and pmly 

impmvcl s pm motility (401. It has bsaa repartcd that 

vitamin E raises superoxide dismutasc activity in bovine 

sprm [411. 

Out results suggcst that exposure to melhoxychlor 

induces depletion in antioxidant defenrc system in the 

cpldidyrnie and opididymal sperm indisarlng methorychlor-induced 

oxidative smss in the epidrdymia. Thtr 

effect may lead to disruption in the functional integrity of 

cell orgmcller. We present study also suggests thal coadnunisvation 

of vitamin E with methorychlor prevents 

the advcne cffecfa of methorychlor on antioxidant status 

8s well ar improving sperm motility in adult rats. In 

conflvrion the adverse effect of mcthorychior on male 

reproduction of rats may be due to the induction of 

oxidative rmrr. These effects could be nilevistcd by the 

antioxidant viumin E. 

IM authors aclmowledgc Dr. Ufe Tiem. Germany for 

lhe gifl of mcthoxychlor md the sfaff of the Bioinformat>ci 

Ccnlr. Pondichcny University for various foeilltier. C. L. 

thank8 lo thc Indian Council of Medical Rwrch. Ncw 

Delhi for Senior Research Fcilowrtup and P.P.M, acknawledger 

the Populntlon Council. New Yark for finunclul it*. 

r,stunce (Gnni Nu~.DY~.~U~P-Y~CMC and II1)P,lUYI

(=I 23. w- C Olln (LC W W. Ubrth d-1. 

Mrrmiaul.sWdmd-dmke-m w.7.1. 

v A = b T o l * o i . b p r r . 

1261 W O ~ ~ ( a h r ~ d h m m s , - a d 

~--I999.UhBdlt(Pr~U~. 

-.-,---. 

1321 WLI + v- WN. sw*. oa guda* w gllimw 

- d ~ ~ p l o i ( d n I l . b Q i n 

Med 1967:701SB-69. 

P31 Fid; E K dPi Y. S-M boa ad MIL -a br 

. . 

Anr LU. 19z-ml 

1351 MSh. PP. Oatop.dgq. 5.190 d- .rr 

r- 

- 

rn n-ldrnd m- (n rr ICL 

8- ,.- 873-G ...-" 

1361 om. 5 w. I, coos. RL. r 4 lops m -k ad 

~--%w..*alm~llrt 

mdbchmarhhol*-phdt-y.)I.aLnldpDarttD 

in rmb DI TmM. M H..h4 IS, 37-47 

1371 mloar.r-.r.-~-d-ccn- 

M.-.D.-J. 

19W.01.18*1- 

d~M-mdElPUCIDni.dmbY-.nda-Wd

t 

02 

ELSEVIER 

Toricology OM) (2W1) DM-wo 

wwv.el~vicr.com/lo~~le~loii~)l 

Effect of methoxychlor on the antioxidant system in 


C. Latchoumycandane, P.P. Mathur I* 

S'hwl v/ LII Scicnrrr. Pvldtchrrry Un~urriry. Pondrdcrry 605 014, Indra 

Rccctvcd 21 N~vcmbcr ZmI, rccetvcd m rcvn~d iorm 19 h m h r 2001. a~cc~~cd 28 March 2W1 

Abstract 

Mcthoaychior. an cnvironmenlal contdmlnanl, which is widely used as a pesticide in many countries and has k n 

shown lo ~nducc rcproductivc abnormalities ~n mule rats The precise nature and mechanism ot action of methoxy 

cllior on lllc lniblc rcproducllve ryslcm is no1 clwr In tho prcxnt study, we have sought to invesligille the induction 

of o*ldat~vc stress In thc tesl~l of rat after expoure to methorychlor Melhoiychlor (I, 10, and IW mg kg-' body 

wcnghl wr Jsy) was adrninisttrsd ormlly to the rat for 45days Aitcr 24 h of the last treatment the anlmals were killed 

wing :~ne\.thct~c clhcr. The body welght of thc anlmalr adm~n~slcrcd with methorychlor d~d not show any s~gn~flcam 

chungc Thc wcaghts of the testis, cpadtdymis, seminal vn!cles and ventral prostate dscreawd significantly in 100 mg 

dox but rcma~ncd unchsngtd In I and 10 ma do~s 

Miloehondrial and microsome-rich fract~ons of the test~s were 

oblatned by the method or d~llcrcntlal centrifugation. The activitles of anttoridant enzymes such as superoxide 

dlsmutasc. cslnlarc, plutath~one rcducmre and glutnthlone prox*dasc decreued s~gnificantly ,n the animals treated 

well8 tnc~horyci~lor In a dose-dependent manner In thc m~lochondilal and micromme-rich fraetloer of rat testis The 

lcvclr of hydrogcn peroiidc pncmllon (H,OI) and lip~d peroxtdatlon increased in m~~ochondriul and m~crorome-rich 

lracl~ons of the lcstts of the rats tmtd with melhoxychlor The rcsulu suppaled that the low to medlum dosn ol 

rncthuxyci,lor clcn deplet8on of antloxtdanl enzymes and concom~lant increase In the levels or HIO* and lipid 

prortdiltian dtllcrcnl\ally in mitochondrial and micro~omc-nch fractions of rat testis In conclusion, the adverse 

cflccl 01 mcthoxychlor on malc rsproducuon could be duc to lhc lnduclion of oxidal~ve stress in lestis. Q 2002 

Publl,hd by Elrvicr Sclsncc Ireland Ltd 

lcywvrdr Mc~horychior, Tuur, Anboridanl cnqmn. L~ptd pcrondalaon. Mllochondnsl fnclionr, Mncroromcrich Irlclionl: Rat 

The exposure of humans to cnvironmcntal contaminants 

that negatlvely affccl the male reproductive 

svstcm is increasing -(Sham, . 1993: Chia, 

'Cormpondlny author Tel. +91413615.211, 81. +91. 2000,, 11;~ important to notc that many cnviron. 

413.bJJ.lll 

K.n~~a1~~d~,rcu pp~~~;~~I~ut~~!l~olm~~I,com 

(P.P Muthur) mcnta' like organochlor'ne 

' B.mwl vpnnlhurgpl pon.n~.tn, cider, accumulate in fatty tissues. Therefore,

continuous exposure of humans to increasing 

numbcn and amounts of thns cnvtronmcntal 

contaminants niay have cumulative efTects that 

lcad to reproductive disorders (Lafuentc et al., 

2000). Mctlioiychlor is onc of the cnv~ronmentnl 

contamlnonts wli~clt as w~dcly urd as a pesticide 

in many countries that was developed to replau 

dichloro-diphenyl-tnchloroethane (DDT), and its 

chemical name is 1.l.l-trichloro-2.2-his@- 

mcthoxy phenyl) cthanc. Mcthoxychlor displays 

both ntrogcnic and antrandrogcnic activities in 

vivo and In vitro (Maness el nl.. 1998). Methoxychlor 

1s wnstdcred to be a proatrogcn that 1s 

mctabolired into mono- and bs-hydroxy melabohtcs, 

wh~ch have been shown to have more estrogenic 

properties than the parental compounds 

(Bulger el al., 1978). Due to the estrogcnictty of 

mcthoxychlor metabolites. exposure of either 

neonatal or adult organlsm to high doses of 

mclhoxychlor may place the male reproductive 

system at risk. It has been reported that methoxychlor 

can affect malc rcprodvction of rats by 

Jccrcnsina spc-rm counts (Chnpin ct al.. 1997). 

Mctlioxychlor has also been shown to inducc 

ox~dat~ve strm In the ep~didymal sperm of goat 

(Gnligudhar~a ct al.. 2001). It has bcen rcportcd 

th;it mcthoxyclilor undcrgo hcpatic m~crosomal 

nmnooxygcnax mcdiatcd actwallon and the resultant 

rcactlve metabol~tes possibly free radicals 

bind wvalcnlly to microsomal components 

(Bulger et al.. 1983). Ant$ox~dants/froe radical 

scavengers, and sulfhydryl containing compounds 

inhibat covalent bindlng of methoxychlor in human 

liver microsomes, suggesting that the rcastive 

intermediate is a frec radlcal (Bulgcr and Kupfur, 

1989). It has also been reported that human cy. 

tmhrome P-450 enzymes responsible for wnversion 

of methoxychlor into its major metaboliter, 

the mono-o-dcmethylatcd dcrivatlvn and 

CYPIA2, havc bcen shown lo play predomrnant 

role tn thas rcaction (Strcrxr and Kupfcr. 1998). 

Thc abklity of cylmhrorne P-450 system to mduu 

rcacuvc oxygcn spx~es (ROS) has ban reported 

(Bondy and Naderi. 1994). ROS are formed in 

both phys~ological and patholog~cal wnditions in 

mammalran ttssun, duc to lhcir high reactivity 

thcy s,;ty tmcrilct with hiomol~zuln inducing oxidat~ve 

stress (Ochxndocrf, 1999). Mitochondr~al 

respiration is the main bioloaical source of suoeror~dc 

anion radicals und~r-~h~siolo~iul conditions. 

F m radiFalsiROS generated in tissues mnd 

subcellular wmpMmonts are cfficicntly rcavcngcd 

by the antioxidant defence system, which 

co~ts(itutcs antioxidant enzymes such as supcror. 

ide dismutaac, &lase. xlutathione rcduclnse and 

glutathlonc peroxidase. Under normal physiological 

conditions ires radiulaIROS are generated in 

subcellular compartments of testis which are subsequently 

mvcngd by the antioxidant defence 

system of the corresponding cellular compartments. 

The suixzllular membranes an more sus- 

ceptible lo l~p~d pcroxidat~ons, which are rich in 

unslaturated fatty acids and has bcen shown to 

contain low amount of antiox~dants. The subcellular 

membranes have been reported to undergo 

pcrmeab~lity changes following enhanced lip~d 

pcroxidation and glutathione depletion (Chance ct 

al.. 1979). The testicular production or free radicals 

and function of the antioxidant dcfcna systcm 

have ken reported upon exposure to toxlc 

chcm~calr (Peltola ct sl.. 1994; Sujotha ct al.. 

2WI. Latchoumvcandanc el al.. 2W2a) Prevaous 

stud~es from ou; laboratory havc shown that the 

cnvironmcntal wntamrnants lake Ilndanc. 

mcthoxychlor and 2.3.7.8-tetrachlorodibenzo.pdioxin 

indua oxidauvc suns in the epid~dymrs 

and eptdidymal sperm of rats (Chttra el al.. 2WI; 

Latchournycdndane el al., 2002b,c). In order to 

explain the mechanism of action of methoxychlor 

at suixzlluhr levels In testis, the present studies 

wen undertaken to evaluate the eNm of 

methoxychlor an the antioxidant systcm in mitochondrial 

and microsome-rich frlclions of rat 

testlS. 

2. Material and arthods 

Methoxychlor (I.l.I-lrichloro-2.2.b~~[~ 

mcthoxyphenyl] cthanc), Appror. 95% purc, 

Sigma Chemical Company, was obta~ncd as gift 

from Dr Ute Ticmann. Rescarch institute for the 

Biology of Farm An~mals. Dummcntorf, Germany. 

Th~obarbitunc acid and malondialdchyde

---- 

- 

wcre obtu~ncd from E-Monk. Germany. All other 

chcmicnls were of analytical grade and obtained 

from local commercial sources. 

Alblno male rats of Wistar strain (45 davs old) 

werc used an thc present study and oblalncd from 

the Central Anhmal House. laaaharlal lnst~lutc of 

Portgradu~le Mcdteal Wucatlon and Re~earch. 

Pond~cherry. India. The animals wcre maintained 

undcr a wcll-regulated linht and dark schedule 

(12.12 hl and tcmperatureil f 3 'C and were fed 

wllh sltlndard comn>erclal pcllcted fced and water 

ad I~b>lum Mcthoaycblor was dissolved in acelonc 

and olwe oil ([:I91 and administered orally 

at the doses of 1. 10 and 100 mg kg-' body 

wcipl~t wr Jay Tor 45-days. Corrsaponding groups 

of annrnals wcrc admintstercd with vehtcle alone 

dnd scrved us conlrol. 

The animals wcrc fasted overnight, weighed and 

k~llcd by urxng ancslhclic cthcr on the day following 

the I;tst dosing Tcst~s, cptdidymts. scmtnal 

vcs~cles and ventral prostatc were removed and 

clearcd from thc adhertng tissues. The weights of 

thc ttssucs wcrc rccordcd in mg as well as mg 

kg - ' body wctght. Both thc testa of each animal 

wcrc u\cd Tor subccllvlar irilcl~onat~on and biochcmlcal 

studtcs. 

M~tochoodr~al and mtcrosome-rlch fractions of 

the tertls wcrc obtalncd by diffcrcntlal centrtfugatlon 

mcthod as dcscrabcd prcv~ously (Lutchoumyrn~nd;knc 

cl ul.. 2002a). Briefly, a 20% (w/v) 

tcst~cular homogcnate was prepared in ~cecald 

0.25 M sucrose solulnon with the help of a motordrtvcn 

xlifss tcflon homo~enircr, The homoaenale 

wa- . re8;tnfuscd -~ at 1000 ; e for 10 rnin at 4-oc to 

~ - - ~ - 

obtain the nuclear pellet. Mitochondrial pellet 

was obtatncd by ccntr~rugtng the post-nuclear supernatant 

at 1OW x I: for 10 min at 4 -C. The 

m~croromc-rlch fractions were prepared by calclum 

chloridc (CaClJ sedimentation method of 

Kamath and Narayan (1972). Briefly, the post-ms. 

tachandr~al supernatant was diluted wzth ice-cold 

CaCI, (I M) so that the final concentration of 

CaCI, was 0 8 molar. It was incubated at 4 'C for 

10 min with occasional stirring. The sample was 

then centr~iuged at IOWO x g for 10 min at 4 "C 

and the m~crosome-rich fractions were obtained. 

All Ihe lraclions werc washed three t!mes w~th 

~ce-cold 1.15% potassium chloride solution and 

d~ssolved in 0.25 M sucrose solution (1 mg protein 

per 0.1 ml). The mitochondr~al and mlcrosomerich 

iractions were used for biochemical studies. 

Superoxlde dlsmutase (EC 1.15 1 1) was assayed 

by the method of Marklund and Marklund 

(1974) Br~efly, the assay mlxture contained 2.4 mi 

of 50 mM tris-HCI buffer conlaming I mM 

EDTA (pH 7.6). 300 PI of 0.2 mM pyrogallol and 

300 PI cnzymc source. The incrcase I" absorbance 

was rnoasurcd immediatelv at 420 nm arainst 

blank contamin8 all the components except the 

enzyme and pyrogallol at 10 s intetvals for 3 mln 

on a Systronic~ Speztrophatometer. The enzyme 

activtty was cxpresscd in nmolc pyrogallol oridtzcd 

per min mg-' prolcin. 

Catalase (EC, 1.1 1.1.6) was assayed by the 

mcthod of Claiborne el al. (1985). Br~efly, the 

ansay rnlxture contained 240 ml of phosphate 

buffer (50 mM. pll 7.0). 10 $4 of 19 mM hydrogcn 

peroxide (H20,) and 50 gl enzyme source. 

The decrease in absorbance was measured Immediatcly 

at 240 nm against blank conlainlng all the 

components except the enzyme at 10 s intervals 

for 3 min on a Systronics Spcctrophotometcr. The 

enzyme actwily was expressed in lrmole oi H A 

consumed per mln mg-' protein. 

2.7. Glurorhione reducrose 

The activity of glutathione reductase (EC. 

1.6.4.2) was assayed by the method or Carlberg 

and Manncrv~k (1975). BneRy, the assay mixture 

contatned 1 75 rnl of phosphate buffer (100 mM.

pH 7.6). I00 pl of 200 mM NADPH, IW pI of 10 

mM EDTA. 50 p1 of 20 mM glutathione oxidized 

and 50 MI enzyme soum. Disappearance of 

NADPH was measured immcd~ately at 340 nm 

seainst blank containing all tho componcnts exccpt 

thc enzymc at 10 s mtcrvals for 3 min on a 

Systrotl!u S~trophotometer. The enzyme activ- 

~ty was cxprcsscd In nmole of NADPH oxidized 

pcr min mg-' protein. 

Ci1ut;tlhionc pcroxidasc (EC 1 .I 1.1.9) was nsr;tycJ 

by tine mclllod of l'ugl~;t and Valcntlac 

(1967). linefly. thc assay mirturc contamed 1.59 

ml of phosphatc bufTcr (100 mM. pH 7.6). 100 p1 

of 10 mM EDTA. 100 g1 of sod~um azide, 50 pl of 

glutathlonc rcductasc, 100 pl of glutathione reduced 

100 p1 of 200 mM NADPH. 10 pl of H,O, 

and 10 el cnzyme sourcc. Disappearance of 

NADPH was rncasurcd immcd~atcly at 340 nm 

ng.lmst blank containing all the components exccpt 

thc cnzymc at 10 s intcrvdls for 3 min on a 

Systron~cs Spatrophotomcter. The enzyme actlv- 

~ty was cxprcsscd in nmolc of NADPH oxidized 

pcr znln mp- ' prutcitl. 

2.9. Hydrogen pcroxrde generalton assoy 

11,0, pocr;trton was aswycd by thc mcthod or 

I'lch and Kcisr~ (1981). Drlcfly, the incubatson 

mtrriurc wntaincd 1.64 ml phosphate bukr (50 

mM. pH 7.6). 54 4 of horseradish peroxidasc (8.5 

Vlml). 30 pl of 0.28 nM phenol red. 5.5 nM of 

165 pl of dextrose and 100 pl of enzyme aource 

was done at 35 'C for 30 min. The reaction was 

tcrmlnatcd by the addition of 60 p1 of 10 N 

sodtum hydrondc. Thc absorbance was read at 

610 nM against a reagent blank on a Syrtromu 

Spcctrophotomctcr. The quantlty of H,O, produccd 

was exprcsxd as nmolc H,O, pcnentcd pr 

min per mg protcln at 35 'C. For standard curve. 

known amount or H,O, and all the above 

rcagcnb except cnzymc source wcrc incubated for 

30 mrn at 35 'C and then added 60 14 of 10 N 

saltum hydroxide and optical dsnsbty WM read at 

610 nM. 

2.10. Lipid pcrox~dadorion 

A break-down product of lipid peroxidation 

thiobarbituric actd reactive subslance (TEARS) 

was measured by the method of Bucp and Aust 

(1976). Ensfly. the stock solution wntained equal 

volumes of trichloroaoct~c actd 15% (wlv) in 0.25 

N hydrochloric acid and 2-thiobarbituric acid 

0.37% (wlv) in 0.25 N hydrmhloric add. h e 

volume of the test sample and two volumes of 

stock reagent were mixed in a screwsopped centrifuge 

tube. vortexcd and heated for IS min on a 

boiling water bath. After cooling on ia the precipilalc 

was rcmovcd by centrifugation at 1000 x 

g for I5 man and absorbanes of the supernalant 

was measured at 532 nM against blank conlaming 

all the reagents except test sample. A standard 

CUPS was Constructed extrapolating the amount 

of commercially bought product malondialdchyde 

to the rneasurcd absorbance. The value is cxpressed 

In pmole of malond~aldchydc formed pr 

mg protctn. 

Data wcrc crprcrrcd as mean *S.D, lor four 

anlmals per dose and analyrcd statistically using 

one-way analysis of varlana (ANOVA) followed 

by Tukey's 161. Probability valun Ins than 0.05 

wcrc dcternllned to be statistically rtgn!ficant 

agalnst control. 

3.1. Body weaghr and organ wctghfs 

Thc body weight of methoxychlor-lrcatsd rats 

d~d not change r~gnificantly when wmparod wlth 

the corresponding group of control animals 

(Tabk I), the weights of the tutia, cpid~dymir, 

=mind ves~lcr and ventral prmtate decreased 

significantly in the rau treated with 100 mg 

mcthoaychlor. However, the weighta remained 

unchanged in the animals treated with melhory. 

shlor at lower do= (Table I).

3.2. A,III~AILNI syrlo~t of ,nIrochondrml nnd 

,nicro~o~,te-rrch ftaertons qf Ihe terrir 

The iCvCl of H202 In mltochondrlal and micro. 

some-rlch fractions of tcalis increased slgnllicantly 

In the animals administered with methoxychlor as 

compared w~th the corresponding group of con. 

Thc actwty of suprox~de dismulase In mitochondrisl 

fractions of rat testis decrea~d s~gnlfi- fro1 animals (Table 2). 

wurtly III lhc ~~llinv;lls troatcd with mcthoxychlor at ThC level of lipid proxidation incrcascd slgnin. 

the doscs of 10 and 100 mg ss wmparcd with the 

in mitochondr~al fractlons of rcstts at 10 

correspond!ng group of control animals (Table 2). and 100 doses and all the doses in mlcraromc- 

Adm!ntstration of varsous doses or methoxychlor rich fractions in the animals administered w~th 

dccrolscd thc activtty of superoxide dirmutasc methoxychlor as compared with the correspondmlCro~on~~.ri~h 

fractg Broup of control animals (Tablc 2). 

sory sex organ weights and suppression 

of tcsllcu. 

Admtnlslralion of various doses of methoxychlor 

decreased the actrvlty of glutath~one reductase 25 mg kg-) body weight per day (Gray el al.. 

s~gnlficantly ~n mlcrosomc-nch fractions as corn. 1989) Rcduc~on in fert~l~ty has also been repdrcd 

with the corresponding group of control ported in the methoxychlor treated rats (Gray el 

an~mals (Tablc 21. Adm~nistration of mcthoxy a].. 1989). It has been reported that methoxychlor 

chlor dccrcascd the activlty ol glutathione peroa- decreased the epidtdymal spcm counts and motil. 

~dusc II, rnllochondrial and m~crosome-rich ~ty In adult rats (Latchoumycandane ct al.. 

fractbons of testis as compared w~th the corre- 2002b). Female rats gavaged with methoxychlor 

spo~ldtng group of control animals (Table 2), at the doses of 5, 50 and 150 mg kg-' body 

~ K ~ ~ ~ ~ ~ ~ $ ~ ~ ~ ~ ~ ~ ~ ~ ~ , " C ~ , , " , " ' ~ ~ 

T"h1f I 

Enm, or mcthorysh~ar on the bodl vclphc md ~.,~hu or ,he ~uia. cpldtdymlr and .-row 

wx organa or *dull mt 

-- 

Melhaxychlor pi Kg body wcighl per day 

cornre1 1 nig 10 mg IW ms 

Udy wc13n UI tl6f 366 118*513 I18* l 19 114zk116 

TsrllHmgl 972 t 15.W 968f 1145 977 i 5 91 9I53II89. 

Eptdrdymlr lmg) 412311 I7 401 3 22 60 4D4k1281 367 i 499' 

Scmrnnl rcrtcln (ml) 947i 1707 941 i I5 I5 94631608 873 4 12' 

Vcntrsl pr0rt.r (mp) 

110 i I8 1s 19934 11 1W*515 176 f 432'

ioavaiiability andlor production of testosterone 

and also thc cstrogfnic and antiandrogenic activitics 

of thc mcthoxychlor and its metabolits. 

Thc rncchiinisn~ of mclhoxychlor mcdialod 0x1- 

ililllvc strcss is not "cry clcar but ~t has bocn 

shown lo bc mcdiatcd by thc activation of microsome-nch 

monooxygenax, which a involved ~n 

the convcrsnon of methoxychior Into its reacttve 

mctabolitcs (Bulger el ai.. 1983). During this reaclloll 

tllc ~cibctlvc 1I1~l.lb0lilCs. possibly free ~di. 

cals. b~nd wvaicntly to microsome-rich 

components (Bulger et al . 1983). 11 has been 

shown that human cytochrome P450 enzymes. 

rcsponslblc for tlic conversion of mothoxychlor 

lnto 11s major mclaboitler and CYPIAZ, havc 

been shown to play e predommilnt role in'thir 

reaclloii (Slrcsscr and Kupfer, 1998). The ablilty 

of cytoclirome P-450 system lo lnduce ROS has 

been wcll documented (Bondy and Naderi. 1994) 

It has bcco reported that ROS such as H,O, 

appcdrs lo be a key agent In the cytotoxtc erects 

In alxrn?.\tozo;t iwd 111 itddltlo!, to thew dlrcot 

cffcct on ccllulnr constituent produce oxcdatlve 

stress by dccreas~ng the enzymatic defenses of the 

tcais (Grlv~au and Lannou. 1997). In the present 

nudy. ad~nlnlstration of mcthoxychlor dccrcased 

thc i~cliv~t~cs of ~ttulurc, superox~dc d~smutnrc. 

piutathionc reductax, and glulathtonc peroxidase 

and concomitant incrcaw In the levels of H?0, 

and hpld pcroxldal~on dificrentiaily in the mltochondrral 

and msrosomc-rich fractions of rat 

tcrtls Thc marlmum decllnc In the actlvltlcs of 

ontioxId.tnl cnrynlcs was obscrvcd In mlcrosomc 

rlch fracuans followcd by mltachondrtal fractions 

aftcr mcthoxychlor trcntment. Superoxlde d~smutarc 

1s conskdercd the first llne of defence against 

delcter~aus cKec(s of oxyradicais In cell by catalyz~ng 

the d~rmutat!on of superoxidc rad~cals to 

H,02 and molecular oxygen. The rcduct~on in the 

ncttvaty of catalar may rcflect ~nabilzty of tesllculilr 

m~tochondrta and mscrosomes to cl~mtnatc 

11,0, produccd by thc actwellon of mcthoxychlor 

and 11s n~elabol~ler. Th~s may also be due to 

cnzyme ~naclbvation cawed by taws ROS production 

in mttochondr~s and mtcrosomes (Pigco- 

Icl et "1.. Il99U). Thc nnt~oridant cnrymcs cnlnlusc 

and pcroxtdarc protect superoxide dismutasc 

against mactlvatlon by H,O,. Rcc~procally, the 

superoxide d~smutase protects the catalase and 

peroxidase against inhibition by superoxtdc anion. 

Thus. the balance of this enzyme system may be 

crscntlal to gct rid oTsuperoxidc anion and pcror. 

ides generated in subaeliular companmmts of the 

testis. The reduction in the activities of anttoxldant 

enzymes and increase in H,O, and lipld 

peroxidation wuld reflcct the adverse effect of 

mcthoxychior on the antioxidant system in testis 

suhccllular fruchons. The ROS cause damage to 

m~lochondr~al and other cytoplasmic organelle 

membrane structures through peroxldatlon of 

phospholipids, proteins and nucleotides. Undcr 

hlgh ROS concentrations prc-damaged spermalolo* 

arc exposed to hpid peroxldatlon by polyunsaturated 

fatty acids (ichikawa el a].. 1999) The 

milochondrial and m!crosome-rich membranes 

havc been reported to undergo permeabllily 

changes following enhanced lip~d peroxidation 

and giutath~onc dcplction (Chance et al., 1979). 

The results suggested that the low to medium 

dnscs exposure or pubcrtal rat lo mcthoxychlor 

~nducen dcpletlon in antioxidant defcncc systems 

diflerentially in milochandrial and microsome. 

rich rracllons ind~cating melhaxychlor-lnduced 

ox~daltve stress tn tcrtis rubcellular fractions. Thls 

crrcct may lead to dtsruplion In functtonal integrity 

of cell organelles in testis and thus the 

advcrsc effcct of methoxychlor on male rcproduction 

of rats may be due to the induction of 

oridanve stress in testls. 

The authors acknowledge Dr Ute Ticmann, 

Research lnslrtutc for the Biology of Farm Anl. 

rnuir. Dummerstorf, Germany for the generous 

gait of mclhoxychlor and the staff of the Biolnfor 

matlcs Center. Pondcherry University for various 

facillt~es C. Latchoumycandane acknowledgcs the 

Indian Council of Medical Research, New Delhi 

for Scnior Research Fellowship and P.P. Mathur 

acknowlcdgcs the Population Council. Ncw York 

for financial assistance (Grant Nos.B99.047P-91 

lCMC and B99.048RilCMC).

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