- Email: [email protected]
Protective role of vitamin A in the male reproductive toxicity of Hexachlorocyclohexane (HCH) in the rat
ReproductiveToxicology,Vol. 4, pp. 325-330, 1990 Printed in the U.S.A.
0890-6238/90 $3.00 + .00 Copyright© 1990PergamonPresspie
P R O T E C T I V E R O L E O F V I T A M I N A IN T H E M A L E R E P R O D U C T I V E TOXICITY OF HEXACHLOROCYCLOHEXANE (HCH) IN T H E R A T J. PIus, T. SHtVANANDAPPA, and M. K. KRISHNAKUMARI Toxicology Unit, InfestationControl and Protectants Discipline, Central Food TechnologicalResearch Institute, Mysore-570 013, India Abstract -- The reproductive toxicity of the organochlorine insecticide, hexachlorocyclohexane (HCH), was investigated in male albino rats fed a diet free of vitamin A or containing vitamin A at 2000 or 100,000 IU/kg diet. Diets containing 1000 ppm HCH for 7 weeks did not cause testicular toxicity in the vitamin-A-deficient and supplemented rats. However, reproductive toxicity was clearly manifested 2 weeks after withdrawing HCH from the diets and was more pronounced in the vitamin A deficient rats compared to their vitamin A supplemented counterparts. Reduction in the testicular weights was accompanied by atrophy of epididymides and seminal vesicles in the vitamin A deficient rats alone. Inhibition of spermatogenesis was further confirmed by decreased sperm count in the epididymis. Biochemically, the activities of the steroidogenic enzymes were drastically reduced. Supplementation of vitamin A after withdrawal of HCH accelerated the recovery and restored spermatogenesis and enzyme activities in the deficient rats. These results demonstrate the greater susceptibility of the male reproductive system to HCH toxicity during vitamin A deficiency and also the protective effect of vitamin A supplementation. Key Words: hexachlorocyclohexane; vitamin A; male rats; testes; epididymides; seminal vesicles; steroidogenesis.
INTRODUCTION
countries (11), several organochlorine insecticides, including technical grade hexachlorocyclohexane (HCH), can cause secondary vitamin A deficiency in animals (12-14). In spite of such close relationships, the role of vitamin A in HCH-induced testicular toxicity in animals is not known. Therefore, in the present study, the male reproductive toxicity of HCH has been investigated in rats fed vitamin A free or supplemented diets.
The complex process of reproduction in mammals is influenced by various physical, chemical, and nutritional factors (1-3). In male animals, testes and accessory reproductive organs are the targets for many xenobiotics, including industrial chemicals, heavy metals, and pesticides. For example, the industrial chemical tri-o-cresyl phosphate (TOCP) and the heavy metal cadmium resulted in various reproductive abnormalities such as testicular atrophy, reduced epididymal sperm count, and inhibition of several testicular enzymes in rats (4,5). Similarly, many organochlorine and organophosphorus insecticides are toxic to the male reproductive system in animals (6-8). Adequate nutrition is essential for the normal functioning of the reproductive system (3). Testicular dysfunction, as judged by failure of spermatogenesis and atrophy of secondary sex organs, has been reported in deficiencies of thiamine, riboflavin, pyridoxine, calcium pantothenate, and vitamin E (3,9). Vitamin A is also essential for normal reproduction (10). In addition to the wide prevalance of vitamin A deficiency in developing
MATERIALS AND METHODS Animals and diets Six-week-old male albino rats, Rattus norvegicus, of CFT-Wistar strain, maintained at the animal house of Central Food Technological Research Institute (Mysore) were randomly allocated into six diet groups. They were individually caged under standard laboratory conditions with free access to the respective synthetic diets and tap water. The six diet groups were: 1. Vitamin A-free (Vo); 2. Vitamin A-free diet + 1000 ppm HCH (Vo+HCH); 3. Vitamin A supplemented at the recommended dietary level, that is, 2000 IU/kg diet (V2ooo); 4. Vitamin A supplemented at 2000 IU/kg diet + 1000 ppm HCH (Vzooo+HCH); 5. Vitamin A supplemented at 100,000 IU/kg diet (Vloo,ooo); and 6. Vitamin A supplemented at 100,000 IU/kg diet + 1000 ppm HCH (V~oo,ooo+HCH). To prepare the vitamin A free diet, fat-free casein
Address correspondence to: Dr. M. K. Krishnakumari, Senior Scientist, Toxicology Unit, Infestation Control and Protectants Discipline, Central Food Technological Research Institute, Mysore-570 013, India. Received: 1 June 1989; Revision rece&ed: 12 November 1989; Accepted: 22 December 1989. 325
326
ReproductiveToxicology
was mixed well with cornstarch, and all essential minerals and vitamins with the exception of vitamin A. Fat-free casein was prepared by refluxing casein with ethyl alcohol, washing in diethyl ether, and drying at 60°C for 12 h. For vitamin A supplemented diets, vitamin A acetate (HiMedia Laboratories Pvt. Ltd., Bombay, India) was dissolved in unrefined groundnut oil (vitamin A free) (15) and mixed well with the respective diets. Technical grade HCH obtained from Hindustan Insecticides Ltd. (Udyogamandal, India) was suspended in groundnut oil and mixed well with the respective diets.
Experimental design At the end of the 7 weeks of feeding, 4 rats from each diet group were killed by ether anesthesia for various studies on testes, epididymides, and seminal vesicles. Simultaneously, HCH was withdrawn from the diets of the remaining rats, which were then fed for 4 weeks the respective control diets. Four rats from each group were killed at intervals of 2 weeks to assess the toxicity of HCH on the testes and accessory reproductive organs. Two weeks after the withdrawal of HCH from the diet, some of the vitamin A deficient rats (previously HCH fed and control) were supplemented with vitamin A at 100,000 IU/kg diet for 2 weeks and autopsied. The postautopsy studies included: a. Relative weights of reproductive organs Testes, epididymides and seminal vesicles were dissected and weighed. Relative weights (g/100 g body weight) were calculated from the absolute weights. b. Sperm count The sperm count was done in the cauda epididymis according to the procedure of Amann (16). c, Histopathology One testis and epididymis from each rat was fixed in Bouin's fluid; paraffin sections (6 ixm thick) were stained with hematoxylin and eosin and observed for histologic changes. d. Testis biochemistry One testis from each rat was washed in ice-cold saline and a 10% w/v homogenate prepared in 0.25 M chilled sucrose using a motor driven Potte-Elvehjem tissue homogenizer. The homogenate was centrifuged at 1250 x g for 20 rain (4 °C), and the following enzymes were assayed in the supernatant. All enzyme assays were conducted under conditions giving activities that were linear with respect to protein concentrations and incubation times. 1713-Hydroxysteroid dehydrogenase,
Volume 4, Number4, 1990 (1713-HSDH, 17 13-Hydroxysteroid: NAD (P)+ oxidoreductase, E.C. 1.1.1.51) and 2x5 3-[3-Hydroxysteroid dehydrogenase (3[3-HSDH, 3[3-Hydroxy-2xS-steroid: NAD + 3-oxidoreductase, E.C. 1.1.1.145) were assayed by the method of Talalay (17) using dehydroepiandrosterone and testosterone as the substrates, respectively. The method described by Lohr and Waller (18) was followed for glucose-6-phosphate dehydrogenase (G6-PDH, D-glucose-6-phosphate: NADP + 1-oxidoreductase, E.C. 1.1,1.49) and that of Whitaker (19) for lactate dehydrogenase (LDH, S-Lactate: NAD + oxidoreductase, E.C. 1.1.1.27). Acid phosphatase (ACP, Orthophosphoric-monoester phosphorylase, acid optimum E.C. 3.1.3.2) and alkaline phosphatase (ALP, Orthophosphoric-monoester phosphorylase, alkaline optimum E.C. 3.1.3,1) were assayed by the method of Walter and Shutt (20). Phenolphthalein-[3-D-glucuronide was used as the substrate for the assay of 13-glucuronidase ([3-GLR, [3-D-Glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) by the method of Fishman (21) while nonspecific esterase (NSE, carboxylic-ester hydrolase, E,C. 3.1.1.1) activity was assayed by the method of Murphy et al, (22) using e~-naphthyl acetate as the substrate. Total protein in the testis homogenate was estimated by Lowry's method (23).
Statistical analysis of data Data were assessed for the significance of differences between the respective control and experimental groups by the Student's t test (24). RESULTS
Relative weights of reproductive organs At the end of the 7th week, relative weights of the testes, epididymides, and seminal vesicles of all the HCH-fed rats were not significantly different from the respective controls (data not presented). However, at the 9th week, significant reductions in relative weights of the reproductive structures were observed in the Vo+HCH rats alone (Table 1). Testes of these rats showed 21% atrophy while 65% and 68% atrophy were noticed for epididymides and seminal vesicles, respectively. Four weeks after the withdrawal of HCH from the diets, the reproductive organs exhibited considerable recovery in their relative weights (Table 2). Testis weight was comparable to the control, showing complete recovery, while epididymides and seminal vesicles were atrophied considerably. However, vitamin A supplementation at 100,000 IU/kg diet for 2 weeks from the second week after withdrawal of HCH from the diet resulted in complete recovery in the relative weights of
Male reproductive toxicity • J. Pros ET AL.
327
Table 1. Epididymal sperm count and relative weights of reproductive organs of vitamin A deficient and supplemented male albino rats at the second week after withdrawal of HCH (1000 ppm) from the diets (values are mean _ SEM of 4 rats in each group) Relative wei[ht Diet group Vo Vo+HCH V2o~o V2ooo+HCH Vmo.ooo Vmo.ooo+HCH
Sperm count (Million/Cauda) 113.33 81.25 138.04 110.42 120.83 91.88
± 5.03 ± 3,52** ± 6.79 ± 9,71 --+ 9.73 ± 6.55*
Epididymides
Testes 1.17 0.93 1.11 1.13 0.97 1.03
± ± -+ ± -+ ±
(~/100 ~ b.w.)
0.03 0.04** 0.10 0.04 0.04 0.02
0.38 0,13 0,35 0,34 0.30 0.29
± ± ± ± -+ ±
0.01 0.03*** 0.02 0.02 0.01 0.01
Seminal Vesicles 0.25 0.08 0.23 0.30 0.26 0.30
± 0.03 _.+ 0.01"* _+ 0.04 ± 0.05 ± 0.02 ± 0.02
P values: *P < 0.05; **P < 0.01; ***P < 0.001. Comparisons are between HCH and same diet without HCH.
the reproductive structures in these rats (data not presented). None of the reproductive structures exhibited significant atrophy in the V2ooo+HCH and Vloo,ooo+HCH rats at any stage of autopsy.
diet resulted in significant recovery (79% of control) of sperm count (data not presented).
Histology of testis and epididymis Very mild histologic changes were noticed in the testes and epididymides of the Vo+HCH rats alone at the end of the 2nd and 4th week after the withdrawal of HCH from the diet. At the 2nd week, the testes of these rats contained occasionally degenerating seminiferous tubules with slight reduction in tubular diameter. However, spermatogenesis was normal. Epididymides of these rats showed a slight reduction in tubular diameter. At the 4th week after withdrawal of HCH, histologic alterations similar to those observed at the previous stage of autopsy were noticed in the testes and epididymides. In addition, mild spermatogenic arrest was also observed in the testes of these rats at this stage. However, vitamin A supplementation at 100,000 IU/kg diet for 2 weeks to the deficient rats during the postexposure period resulted in almost normal histology of the testes and epididymides. Rats of the V2ooo+HCH and Vloo,ooo+HCH groups exhibited normal histology of
Sperm count Epididymal sperm count was normal in all the rats at the end of the 7th week (data not presented). However, significant reductions in the sperm count of most of the previously HCH-fed rats were observed at the end of the 9th week and beyond. At the second week after withdrawal of HCH from the diet, rats of the Vo+HCH group exhibited nearly 30% reduction in sperm count, while in the V2ooo+HCH and Vmo,ooo+HCH rats, the reductions were relatively less compared to their controis (Table 1). At the 4th week after withdrawal of HCH from the diet, the sperm counts of the Vo+HCH and V2ooo+HCH rats were further reduced (53% and 37% respectively), while rats of the Vmo,ooo+HCH group had normal sperm counts (Table 2). Vitamin A supplementation at 100,000 IU/kg diet for 2 weeks to the Vo+HCH rats after withdrawal of HCH from the
Table 2. Epididymal sperm count and relative weights of reproductive organs of vitamin A deficient and supplemented male albino rats at the fourth week after withdrawal of HCH (1000 ppm) from the diets (values are mean --- SEM of 4 rats in each group) Relative weight (g/100 g b.w.) Diet group Vo Vo+HCH
V2ooo V2ooo+HCH Vmo,ooo VIoo,~o+HCH
Sperm count (Million/Cauda) 127.29 60.21 132.29 82.90 118.96 110.42
± 15.68 ___ 12.81"** _+ 11.96 ___ 10.98" ± 25.09 _.+ 14.87
Testes 1.09 1.28 1.12 1.23 1.15 1.09
_ ± _ ± ± ±
0.08 0.08 0.05 0.05 0.05 0.07
*P < 0.05; **P < 0.01; ***P < 0.001. Comparisons are between HCH and same diet without HCH.
Epididymides
Seminal vesicles
0.34 0.25 0.34 0.33 0.35 0.36
0.28 0.18 0.28 0.30 0.29 0.30
_ 0.01 ± 0.04 ___ 0.01 ± 0.01 ± 0.01 -+ 0.01
--± ± ± --_
0.01 0.02** 0,04 0.03 0.01 0.02
328
Reproductive Toxicology
Volume 4, Number 4, 1990
Table 3. Testis enzymes of vitamin A deficient and supplemented male albino rats at the second week after withdrawal of HCH (1000 ppm) from the diets (values are mean _-_ SEM of 4 rats in each group)
Diet group
Vo Vo+HCH I/2ooo V2ooo+HCH Vloo,ooo Vtoo,ooo+HCH
17[3-HSDH (SEM)
3 [3-HSDN (SEM)
G-6-PDH (SEM)
LDH (SEM)
[3-GLR (SEM)
NSE (SEM)
ALP (SEM)
ACP (SEM)
Protein (mg/g) (SEM)
171.08 (12.31) 81.00"* (14.30) 161.02 (13.01) 142.15 (9.30) 164.35 (13.18) 176.15 (13.60)
122,35 (6.17) 51.32"** (6.95) 123.61 (14.60) 109.01 (11.01) 141.08 (5.50) 142.35 (14.31)
18.01 (5.80) 10.11 (4.80) 18.70 (5.89) 14.81 (7.20) 19.20 (6.41) 16.30 (5.41)
121.02 (1.20) 82.08*** (3.51) 141.31 (5.20) 125.08 (4.12) 132.00 (6.70) 121.35 (4.18)
18.61 (1.40) 26.02** (0.77) 16.87 (1.83) 19.71 (0.89) 23.51 (3.9) 20.61 (5.1)
200.07 (4.91) 149.31"** (2.60) 158.03 (6.01) 159.35 (2.8) 233.00 (7.90) 241.00 (2.61)
5386.21 (85.01) 3502.00*** (67.80) 4240.15 (184.00) 3994.00 (132.00) 5060.01 (819.00) 4925.00 (249.00)
4536.17 (101.45) 4417.08 (131.15) 3941.87 (102.35) 3745.18 (104.31) 3380.18 (109.61) 3492.01 (152.01)
26.02 (0.75) 29.00* (0.66) 27.00 (1.32) 31.33" (1.01) 28.50 (0.43) 28.01 (0.87)
*P < 0.05; **P < 0.01; ***P < 0.001. Enzyme activities are expressed as nmoles/mg protein/h. Comparisons are between HCH and same diet without HCH.
testes and epididymides at all stages of autopsies.
Testis biochemistry The pattern of results of the testicular enzyme assays were similar to those of the other parameters. Activities of the various testicular enzymes were comparable with those of the respective controls in all the HCH-fed rats at the end of the 7th week (data not presented). At the end of the 2nd week, after withdrawal of HCH from the diets, rats of the Vo+HCH group showed significant alterations in the activities of the enzymes (Table 3). Dehydrogenases were the most affected among all the enzymes. In these rats, A 5 3[3-HSDH, 17[3-HSDH, and G-6-PDH showed reduc-
tions of activities as high as 57%, 53%, and 45%, respectively, while that of [3-GLR was increased by 39%. Activities of NSE, ALP, and LDH were also significantly reduced. In the V2ooo+HCH group, some of the enzyme activities exhibited significant alterations. On the other hand, A 5 313-HSDH, 17[3-HSDH, NSE, and [3-GLR activities in the Vloo,ooo+HCH rats were comparable to those of their controls, though marginal alterations were noticed in the activities of the other enzymes. At the 4th week after withdrawal of HCH from the diets, rats of all the groups showed considerable recovery in the activities of various enzymes (Table 4). In the Vo+HCH rats, all the enzymes with the exception of
Table 4. Testis enzymes of vitamin A deficient and supplemented male albino rats at the 4th week after withdrawal of HCH (1000 ppm) from the diets (values are mean --- SEM of 4 rats in each group)
Diet group
Vo Vo+HCH 112oo0 V2ooo+HCH Vloo.ooo Vloo,ooo+HCH
17 [3-HSDH (SEM)
3 I3-HSDH (SEM)
G-6-PDH (SEM)
LDH (SEM)
I3-GLR (SEM)
NSE (SEM)
ALP (SEM)
ACP (SEM)
Protein (rag/g) (SEM)
143.31 (14.01) 117.08 (8.28) 124.08 (4.04) 116.08 (6.80) 172,31 (13,10) 168.18 (8.70)
105.18 (15.01) 84.00 (11.04) 134.35 (4.86) 133.09 (5.00) 136.18 (6.10) 134.17 (3.50)
15.80 (5.12) 10.28 (6.21) 17.21 (1.27) 18.60 (0.63) 19.50 (1.57) 20.12 (0.98)
121.00 (6.10) 92.01" (7.10) 141.01 (4.70) 142.91 (5.72) 140.09 (6.10) 136.85 (5.70)
16.08 (1.10) 18.51 (1.20) 19.90 (0.78) 20.70 (1.20) 19.71 (1.70) 20.20 (2.10)
158.02 (7.50) 150.13 (3.20) 194.08 (5.91) 208.37 (15.32) 215.06 (11.07) 207.85 (25.08)
3223.18 (243.01) 2600.00 (177.18) 3731.07 (330.18) 3653.93 (380.57) 3452.37 (310.31) 3417.97 (295.39)
4053.08 (60.08) 4500.00*** (44.31) 6025.18 (260,71) 590,73 (270,38) 5987.21 (320.30) 6013.31 (410.97)
33.00 (0.71) 34.17 (1.13) 30.00 (1.00) 31.00 (0.90) 30.72 (0.98) 29.07 (1.08)
*P < 0.05; ***P < 0.001. Enzyme activities are expressed as nmoles/mg protein/h. Comparisons are between HCH and same diet without HCH.
Male reproductivetoxicity • J. PIus ETAL. LDH showed considerable but not complete recovery. In the VEOoo+HCH rats, all the enzyme activities were close to the controls, while complete recovery of the enzyme activities was noticed in the Vto0,oo+HCH rats. Supplementation of vitamin A at 100,000 IU/kg diet to the vitamin A deficient rats previously exposed to HCH resulted in complete recovery in activities of all the enzymes (data not presented). DISCUSSION Vitamin A is required for the normal processes of reproduction, and deficiency results in atrophy of testes, degeneration of seminiferous tubules, and spermatogenic arrest in male animals (10). Similar effects on the reproductive system have also been attributed to HCH. Shivanandappa and Krishnakumari (8) have reported that feeding rats HCH at 1500 ppm for 90 days produced marked effects on spermatogenesis and steroidogenesis. It was found in the present study that dietary HCH at 1000 ppm was apparently without any toxic effect on the male reproductive system at the end of the feeding period of 49 days. During the postexposure period, manifestation of the toxic effects of HCH on the male reproductive system was more pronounced in the vitamin A deficient rats as evidenced by atrophy of the testes and accessory reproductive organs, spermatogenic arrest, reduced sperm count, histologic changes, and alterations in various testicular enzymes. These results demonstrate the latent effects of HCH at some stage prior to 7 weeks and that the male reproductive system is highly vulnerable to the toxic effects of HCH during vitamin A deficiency. Similar atrophy of the reproductive structures associated with spermatogenic arrest has been reported in animals as a result of exposure to pesticides and other chemicals (25, 26). Histologic alterations found in the testes and epididymides are supportive of the findings of reduced sperm count and relative organ weights. Atrophied testis with shrunken seminiferous tubules and spermatogenic arrest has been reported in rats fed 1500 ppm HCH for 90 days (8). Similar observations have been reported by Dikshith and Datta (25). Also, alterations in the activities of testicular enzymes such as ALP, ACP, NSE, LDH, and 13-GLR have been reported in conditions of chemical toxicity (4, 27, 28). Therefore, histologic changes, marked reduction in sperm count, and the greater alterations of the enzymes in the vitamin A deficient rats confirm the enhanced testicular toxicity of HCH during vitamin A deficiency. The lack of such changes or only mild alterations found in the vitamin A supplemented rats are suggestive of the protective role of vitamin A, particularly in excess, but not at a hypervitaminotic level that is 100,000 IU/kg diet. A5 313-HSDH and 17 f3-HSDH are key enzymes in
329
the biosynthesis of steroid hormones. G-6-PDH is the potential generator of NADPH, which is required for hydroxylation reactions in steroid biosynthesis (29). The Leydig cells and Sertoli cells of the seminiferous tubules are known to be the principal sites of steroid biosynthesis as they possess these important steroidogenic enzymes (30). Inhibition of steroidogenic enzymes in the testis and adrenals of rats following HCH administration has been shown histochemically by earlier workers (8, 31). Therefore, the reduced activities of the important enzymes of steroidogenesis observed in the present study imply that the toxic action of HCH was exerted by impaired steroidogenesis in the Leydig cells and Sertoli cells, accentuated by deficiency and protected by supplementation of vitamin A. This toxicity was reversible, as withdrawal of HCH from the diets resulted in recovery of the activities of the steroidogenic enzymes, which was further improved by vitamin A supplementation. Reproduction in animals is controlled by several hormones (32). The testicular toxicity of xenobiotics can be due either to their direct action on the reproductive structures or to indirect action through the endocrine system. Thus, methoxychlor, an organochlorine insecticide, results in an elevation in prolactin concentration and release, which in turn influences the hypothalamic gonadotropin-releasing hormone, ultimately resulting in testicular dysfunction (33). On the other hand, TOCP, an industrial chemical, brings about testicular toxicity without any alterations in the circulating hormones, showing that the toxicity is due to its direct action on the reproductive structures (4, 27). Our results indicate that HCH effects testicular toxicity indirectly by impairment of steroidogenesis, which is further accentuated by vitamin A deficiency. Further, supplementation of vitamin A, particularly in excess (not hypervitaminotic) of the normal requirement, could protect the steroidogenic inhibition of HCH and thereby reduce its toxicity. authors wish to thank Dr. B.L. Amla and Mr. S. K. Majumder for their keen interest in this investigation. The assistance of Mr. K. Narasimhamurthy and Mr. S. Viswanatha is highly appreciated. J. Pius was a recipient of CSIR (India) research fellowship.
Acknowledgments -- The
REFERENCES 1. Ellis LC. Radiation effects. In: Johnson AB, Gomes WR, VandermarkNL, eds. The testis; vol. II1.New York: AcademicPress; 1970:333-76. 2. Mann T, Lutwak-MannC. Passage of chemicalsinto human and animal semen: mechanisms and significance. CRC Crit Rev Toxicol. 1982;11:1-14. 3. LeathernJH. Nutrition. In: Johnson AD, GomesWR, Vandemark NL, eds. The testis; vol. 3. New York: Academic Press; 1970: 169-205. 4. SomkutiSG, LapadulaDM, Chapin RE, Lamb JC, Abou-Donia
330
5.
6. 7.
8. 9. 10. 11.
12.
13. 14.
15.
16.
17.
18.
19.
Reproductive Toxicology MB. Reproductive tract lesions resulting from subchronic administration (63 days) of tri-o-cresyl phosphate in male rats. Toxicol Appl Pharmacol. 1987;89:49-63. Gunn SA, Gould TC, Anderson WAD. Specificity in protection against lethality and testicular toxicity from cadmium. Proc Soc Exptl Biol Med. 1968;128:591-5. Virgo BB, Bellward GD. Effects of dietary dieldrin on reproduction in the Swiss-Vancouver (SWV) mouse. Environ Physiol Biochem. 1975;5:440-50. Krause W, Homola S. Alterations of the seminiferous epithelium and the Leydig cells of the rat testis after the application of Dichlorovos (DDVP). Bull Environ Contain Toxicol. 1974;11: 429-33. Shivanandappa T, Krishnakumari MK. Hexachlorocyclohexlaneinduced testicular dysfunction in rats. Acta Pharmacol Toxicol. 1983;52:12-17. Bunyan J, Green J, Diplock AT, Robinson D. Lysosomal enzymes and vitamin E deficiency. Br J Nutr. 1967;21:147-54. Ganguly J, Rao MRS, Murthy SK, Sarada K. Systemic mode of action of vitamin A. Vitamins and Hormones. 1980;38:1-54. McLaren DS. Global occurrence of vitamin A deficiency. In: Bauemfeind JC, ed. Vitamin A deficiency and its control. New York: Academic Press; 1986:1-18. Phillips WEJ, Hatiana G. Effect of dietary organochlorine pesticides on the liver vitamin A content of the weanling rat. Nutr Rep Inter. 1972;5:357~62. Tinsely IJ. DDT effect on rats raised on alpha-protein rations: growth and storage of vitamin A. J Nutr. 1969;98:319-24. Plus J. Role of dietary protein and vitamin A in the toxicity of hexachlorocyclohexane (HCH) to the rat. Ph.D thesis submitted to the University of Mysore, Mysore (India), 1988. Woodroof JG. Composition and nutritive value of nuts. In: Woodroof JG, ed, Peanuts -- production, processing and products, Inc: Westport, CT: The AVI Publishing Company; 1966: 57-82. Amann RP. A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl. 1982;2: 37-58. Talalay P. Hydroxysteroid dehydrogenases. In: Colowick SP, Kaplan NO, eds. Methods in enzymology; vol. 5. New York: Academic Press; 1962:512-16. Lohr GH, Waller HD. Glucose-6-Phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:636--43. Whitaker JF. A general colorimetric procedure for the estimation of enzymes which are linked to the NADPH/NAD + system. Clin Chim Acta. 1969;24:23-37.
Volume 4, Number 4, 1990 20. Walter K, Shutt C. Phosphatases. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:856-60. 21. Fishman WH. 13-glucuronidase. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:929--43. 22. Murphy SD, Cheever KL, Chow AYK, Browster M. Organophosphate insecticides potentiation by carboxylesterase inhibitors. In: Proceedings of European Society of Toxicology; vol. 17. Excerpta Medica International Conference Series No. 376; 1975; 292-303. 23. Lowry OH, Rosebrough NJ, Fan AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:265-75. 24. Snedecor GW, Cochran WG, eds. Statistical methods. Ames, Iowa: Iowa State University; 1980. 25. Dikshith TSS, Datta KK. Pathologic changes induced by pesticides in the testes and liver of rats. Exp Pathol. 1972;7:309-16. 26. Linder RE, Rehnberg GL. Evaluation of reproductive parameters in adult male Wistar rats after subchronic exposure (gavage) to benomyl. J Toxicol Environ Health. 1988; 25:285-98. 27, Somkuti SG, Lapadula DM, Chapin RE, Lamb JC, Abou-Donia MB. Time-course of the tri-o-cresyl phosphate-induced testicular lesion in F-344 rats: enzymatic, hormonal and sperm parameter studies. Toxicol Appl Pharmacol. 1987;89:64-72. 28. Parmar D, Srivastava SP, Seth PK. Effect of di (2-ethylhexyl) phthalate (DEHP) on spermatogenesis in adult rats. Toxicology. 1986;42:47-55. 29. Hall PF. Endocrinology of the testis. In: Johnson AD, Vandemark NL, Gomes WR, eds. The testis; vol. 3. New York: Academic Press; 1970:1-71. 30. Blackshaw AW. Histochemical localization of testicular enzymes. In: Johnson AD, Vandemark NL, Gomes WR, eds. The testis; vol. 3. New York: Academic Press; 1970;72-123. 31. Shivanandappa T, Krishnakumari MK, Majumder SK, Inhibition of steroidogenic enzymes in the adrenal cortex of rats fed benzene hexachloride (hexachlorocyclohexane). Experientia. 1982;32:125153. 32. Steinberger E, Steinberger A. Hormonal control of testicular function in mammals. In: Knobil K, Sawyer WH, eds. Handbook of physiology; vol. 4. Washington, D.C.: American Physiological Society; 1974:325--45. 33. Goldman JM, Cooper RL, Rehnberg GL, Hein JF, McElroy WK, Gray LE Jr. Effects of low subchronic doses of methoxychlor on the rat hypothalamic-pituitary reproductive axis. Toxicol Appl Pharmacol. 1986;86:474-83.
0890-6238/90 $3.00 + .00 Copyright© 1990PergamonPresspie
P R O T E C T I V E R O L E O F V I T A M I N A IN T H E M A L E R E P R O D U C T I V E TOXICITY OF HEXACHLOROCYCLOHEXANE (HCH) IN T H E R A T J. PIus, T. SHtVANANDAPPA, and M. K. KRISHNAKUMARI Toxicology Unit, InfestationControl and Protectants Discipline, Central Food TechnologicalResearch Institute, Mysore-570 013, India Abstract -- The reproductive toxicity of the organochlorine insecticide, hexachlorocyclohexane (HCH), was investigated in male albino rats fed a diet free of vitamin A or containing vitamin A at 2000 or 100,000 IU/kg diet. Diets containing 1000 ppm HCH for 7 weeks did not cause testicular toxicity in the vitamin-A-deficient and supplemented rats. However, reproductive toxicity was clearly manifested 2 weeks after withdrawing HCH from the diets and was more pronounced in the vitamin A deficient rats compared to their vitamin A supplemented counterparts. Reduction in the testicular weights was accompanied by atrophy of epididymides and seminal vesicles in the vitamin A deficient rats alone. Inhibition of spermatogenesis was further confirmed by decreased sperm count in the epididymis. Biochemically, the activities of the steroidogenic enzymes were drastically reduced. Supplementation of vitamin A after withdrawal of HCH accelerated the recovery and restored spermatogenesis and enzyme activities in the deficient rats. These results demonstrate the greater susceptibility of the male reproductive system to HCH toxicity during vitamin A deficiency and also the protective effect of vitamin A supplementation. Key Words: hexachlorocyclohexane; vitamin A; male rats; testes; epididymides; seminal vesicles; steroidogenesis.
INTRODUCTION
countries (11), several organochlorine insecticides, including technical grade hexachlorocyclohexane (HCH), can cause secondary vitamin A deficiency in animals (12-14). In spite of such close relationships, the role of vitamin A in HCH-induced testicular toxicity in animals is not known. Therefore, in the present study, the male reproductive toxicity of HCH has been investigated in rats fed vitamin A free or supplemented diets.
The complex process of reproduction in mammals is influenced by various physical, chemical, and nutritional factors (1-3). In male animals, testes and accessory reproductive organs are the targets for many xenobiotics, including industrial chemicals, heavy metals, and pesticides. For example, the industrial chemical tri-o-cresyl phosphate (TOCP) and the heavy metal cadmium resulted in various reproductive abnormalities such as testicular atrophy, reduced epididymal sperm count, and inhibition of several testicular enzymes in rats (4,5). Similarly, many organochlorine and organophosphorus insecticides are toxic to the male reproductive system in animals (6-8). Adequate nutrition is essential for the normal functioning of the reproductive system (3). Testicular dysfunction, as judged by failure of spermatogenesis and atrophy of secondary sex organs, has been reported in deficiencies of thiamine, riboflavin, pyridoxine, calcium pantothenate, and vitamin E (3,9). Vitamin A is also essential for normal reproduction (10). In addition to the wide prevalance of vitamin A deficiency in developing
MATERIALS AND METHODS Animals and diets Six-week-old male albino rats, Rattus norvegicus, of CFT-Wistar strain, maintained at the animal house of Central Food Technological Research Institute (Mysore) were randomly allocated into six diet groups. They were individually caged under standard laboratory conditions with free access to the respective synthetic diets and tap water. The six diet groups were: 1. Vitamin A-free (Vo); 2. Vitamin A-free diet + 1000 ppm HCH (Vo+HCH); 3. Vitamin A supplemented at the recommended dietary level, that is, 2000 IU/kg diet (V2ooo); 4. Vitamin A supplemented at 2000 IU/kg diet + 1000 ppm HCH (Vzooo+HCH); 5. Vitamin A supplemented at 100,000 IU/kg diet (Vloo,ooo); and 6. Vitamin A supplemented at 100,000 IU/kg diet + 1000 ppm HCH (V~oo,ooo+HCH). To prepare the vitamin A free diet, fat-free casein
Address correspondence to: Dr. M. K. Krishnakumari, Senior Scientist, Toxicology Unit, Infestation Control and Protectants Discipline, Central Food Technological Research Institute, Mysore-570 013, India. Received: 1 June 1989; Revision rece&ed: 12 November 1989; Accepted: 22 December 1989. 325
326
ReproductiveToxicology
was mixed well with cornstarch, and all essential minerals and vitamins with the exception of vitamin A. Fat-free casein was prepared by refluxing casein with ethyl alcohol, washing in diethyl ether, and drying at 60°C for 12 h. For vitamin A supplemented diets, vitamin A acetate (HiMedia Laboratories Pvt. Ltd., Bombay, India) was dissolved in unrefined groundnut oil (vitamin A free) (15) and mixed well with the respective diets. Technical grade HCH obtained from Hindustan Insecticides Ltd. (Udyogamandal, India) was suspended in groundnut oil and mixed well with the respective diets.
Experimental design At the end of the 7 weeks of feeding, 4 rats from each diet group were killed by ether anesthesia for various studies on testes, epididymides, and seminal vesicles. Simultaneously, HCH was withdrawn from the diets of the remaining rats, which were then fed for 4 weeks the respective control diets. Four rats from each group were killed at intervals of 2 weeks to assess the toxicity of HCH on the testes and accessory reproductive organs. Two weeks after the withdrawal of HCH from the diet, some of the vitamin A deficient rats (previously HCH fed and control) were supplemented with vitamin A at 100,000 IU/kg diet for 2 weeks and autopsied. The postautopsy studies included: a. Relative weights of reproductive organs Testes, epididymides and seminal vesicles were dissected and weighed. Relative weights (g/100 g body weight) were calculated from the absolute weights. b. Sperm count The sperm count was done in the cauda epididymis according to the procedure of Amann (16). c, Histopathology One testis and epididymis from each rat was fixed in Bouin's fluid; paraffin sections (6 ixm thick) were stained with hematoxylin and eosin and observed for histologic changes. d. Testis biochemistry One testis from each rat was washed in ice-cold saline and a 10% w/v homogenate prepared in 0.25 M chilled sucrose using a motor driven Potte-Elvehjem tissue homogenizer. The homogenate was centrifuged at 1250 x g for 20 rain (4 °C), and the following enzymes were assayed in the supernatant. All enzyme assays were conducted under conditions giving activities that were linear with respect to protein concentrations and incubation times. 1713-Hydroxysteroid dehydrogenase,
Volume 4, Number4, 1990 (1713-HSDH, 17 13-Hydroxysteroid: NAD (P)+ oxidoreductase, E.C. 1.1.1.51) and 2x5 3-[3-Hydroxysteroid dehydrogenase (3[3-HSDH, 3[3-Hydroxy-2xS-steroid: NAD + 3-oxidoreductase, E.C. 1.1.1.145) were assayed by the method of Talalay (17) using dehydroepiandrosterone and testosterone as the substrates, respectively. The method described by Lohr and Waller (18) was followed for glucose-6-phosphate dehydrogenase (G6-PDH, D-glucose-6-phosphate: NADP + 1-oxidoreductase, E.C. 1.1,1.49) and that of Whitaker (19) for lactate dehydrogenase (LDH, S-Lactate: NAD + oxidoreductase, E.C. 1.1.1.27). Acid phosphatase (ACP, Orthophosphoric-monoester phosphorylase, acid optimum E.C. 3.1.3.2) and alkaline phosphatase (ALP, Orthophosphoric-monoester phosphorylase, alkaline optimum E.C. 3.1.3,1) were assayed by the method of Walter and Shutt (20). Phenolphthalein-[3-D-glucuronide was used as the substrate for the assay of 13-glucuronidase ([3-GLR, [3-D-Glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) by the method of Fishman (21) while nonspecific esterase (NSE, carboxylic-ester hydrolase, E,C. 3.1.1.1) activity was assayed by the method of Murphy et al, (22) using e~-naphthyl acetate as the substrate. Total protein in the testis homogenate was estimated by Lowry's method (23).
Statistical analysis of data Data were assessed for the significance of differences between the respective control and experimental groups by the Student's t test (24). RESULTS
Relative weights of reproductive organs At the end of the 7th week, relative weights of the testes, epididymides, and seminal vesicles of all the HCH-fed rats were not significantly different from the respective controls (data not presented). However, at the 9th week, significant reductions in relative weights of the reproductive structures were observed in the Vo+HCH rats alone (Table 1). Testes of these rats showed 21% atrophy while 65% and 68% atrophy were noticed for epididymides and seminal vesicles, respectively. Four weeks after the withdrawal of HCH from the diets, the reproductive organs exhibited considerable recovery in their relative weights (Table 2). Testis weight was comparable to the control, showing complete recovery, while epididymides and seminal vesicles were atrophied considerably. However, vitamin A supplementation at 100,000 IU/kg diet for 2 weeks from the second week after withdrawal of HCH from the diet resulted in complete recovery in the relative weights of
Male reproductive toxicity • J. Pros ET AL.
327
Table 1. Epididymal sperm count and relative weights of reproductive organs of vitamin A deficient and supplemented male albino rats at the second week after withdrawal of HCH (1000 ppm) from the diets (values are mean _ SEM of 4 rats in each group) Relative wei[ht Diet group Vo Vo+HCH V2o~o V2ooo+HCH Vmo.ooo Vmo.ooo+HCH
Sperm count (Million/Cauda) 113.33 81.25 138.04 110.42 120.83 91.88
± 5.03 ± 3,52** ± 6.79 ± 9,71 --+ 9.73 ± 6.55*
Epididymides
Testes 1.17 0.93 1.11 1.13 0.97 1.03
± ± -+ ± -+ ±
(~/100 ~ b.w.)
0.03 0.04** 0.10 0.04 0.04 0.02
0.38 0,13 0,35 0,34 0.30 0.29
± ± ± ± -+ ±
0.01 0.03*** 0.02 0.02 0.01 0.01
Seminal Vesicles 0.25 0.08 0.23 0.30 0.26 0.30
± 0.03 _.+ 0.01"* _+ 0.04 ± 0.05 ± 0.02 ± 0.02
P values: *P < 0.05; **P < 0.01; ***P < 0.001. Comparisons are between HCH and same diet without HCH.
the reproductive structures in these rats (data not presented). None of the reproductive structures exhibited significant atrophy in the V2ooo+HCH and Vloo,ooo+HCH rats at any stage of autopsy.
diet resulted in significant recovery (79% of control) of sperm count (data not presented).
Histology of testis and epididymis Very mild histologic changes were noticed in the testes and epididymides of the Vo+HCH rats alone at the end of the 2nd and 4th week after the withdrawal of HCH from the diet. At the 2nd week, the testes of these rats contained occasionally degenerating seminiferous tubules with slight reduction in tubular diameter. However, spermatogenesis was normal. Epididymides of these rats showed a slight reduction in tubular diameter. At the 4th week after withdrawal of HCH, histologic alterations similar to those observed at the previous stage of autopsy were noticed in the testes and epididymides. In addition, mild spermatogenic arrest was also observed in the testes of these rats at this stage. However, vitamin A supplementation at 100,000 IU/kg diet for 2 weeks to the deficient rats during the postexposure period resulted in almost normal histology of the testes and epididymides. Rats of the V2ooo+HCH and Vloo,ooo+HCH groups exhibited normal histology of
Sperm count Epididymal sperm count was normal in all the rats at the end of the 7th week (data not presented). However, significant reductions in the sperm count of most of the previously HCH-fed rats were observed at the end of the 9th week and beyond. At the second week after withdrawal of HCH from the diet, rats of the Vo+HCH group exhibited nearly 30% reduction in sperm count, while in the V2ooo+HCH and Vmo,ooo+HCH rats, the reductions were relatively less compared to their controis (Table 1). At the 4th week after withdrawal of HCH from the diet, the sperm counts of the Vo+HCH and V2ooo+HCH rats were further reduced (53% and 37% respectively), while rats of the Vmo,ooo+HCH group had normal sperm counts (Table 2). Vitamin A supplementation at 100,000 IU/kg diet for 2 weeks to the Vo+HCH rats after withdrawal of HCH from the
Table 2. Epididymal sperm count and relative weights of reproductive organs of vitamin A deficient and supplemented male albino rats at the fourth week after withdrawal of HCH (1000 ppm) from the diets (values are mean --- SEM of 4 rats in each group) Relative weight (g/100 g b.w.) Diet group Vo Vo+HCH
V2ooo V2ooo+HCH Vmo,ooo VIoo,~o+HCH
Sperm count (Million/Cauda) 127.29 60.21 132.29 82.90 118.96 110.42
± 15.68 ___ 12.81"** _+ 11.96 ___ 10.98" ± 25.09 _.+ 14.87
Testes 1.09 1.28 1.12 1.23 1.15 1.09
_ ± _ ± ± ±
0.08 0.08 0.05 0.05 0.05 0.07
*P < 0.05; **P < 0.01; ***P < 0.001. Comparisons are between HCH and same diet without HCH.
Epididymides
Seminal vesicles
0.34 0.25 0.34 0.33 0.35 0.36
0.28 0.18 0.28 0.30 0.29 0.30
_ 0.01 ± 0.04 ___ 0.01 ± 0.01 ± 0.01 -+ 0.01
--± ± ± --_
0.01 0.02** 0,04 0.03 0.01 0.02
328
Reproductive Toxicology
Volume 4, Number 4, 1990
Table 3. Testis enzymes of vitamin A deficient and supplemented male albino rats at the second week after withdrawal of HCH (1000 ppm) from the diets (values are mean _-_ SEM of 4 rats in each group)
Diet group
Vo Vo+HCH I/2ooo V2ooo+HCH Vloo,ooo Vtoo,ooo+HCH
17[3-HSDH (SEM)
3 [3-HSDN (SEM)
G-6-PDH (SEM)
LDH (SEM)
[3-GLR (SEM)
NSE (SEM)
ALP (SEM)
ACP (SEM)
Protein (mg/g) (SEM)
171.08 (12.31) 81.00"* (14.30) 161.02 (13.01) 142.15 (9.30) 164.35 (13.18) 176.15 (13.60)
122,35 (6.17) 51.32"** (6.95) 123.61 (14.60) 109.01 (11.01) 141.08 (5.50) 142.35 (14.31)
18.01 (5.80) 10.11 (4.80) 18.70 (5.89) 14.81 (7.20) 19.20 (6.41) 16.30 (5.41)
121.02 (1.20) 82.08*** (3.51) 141.31 (5.20) 125.08 (4.12) 132.00 (6.70) 121.35 (4.18)
18.61 (1.40) 26.02** (0.77) 16.87 (1.83) 19.71 (0.89) 23.51 (3.9) 20.61 (5.1)
200.07 (4.91) 149.31"** (2.60) 158.03 (6.01) 159.35 (2.8) 233.00 (7.90) 241.00 (2.61)
5386.21 (85.01) 3502.00*** (67.80) 4240.15 (184.00) 3994.00 (132.00) 5060.01 (819.00) 4925.00 (249.00)
4536.17 (101.45) 4417.08 (131.15) 3941.87 (102.35) 3745.18 (104.31) 3380.18 (109.61) 3492.01 (152.01)
26.02 (0.75) 29.00* (0.66) 27.00 (1.32) 31.33" (1.01) 28.50 (0.43) 28.01 (0.87)
*P < 0.05; **P < 0.01; ***P < 0.001. Enzyme activities are expressed as nmoles/mg protein/h. Comparisons are between HCH and same diet without HCH.
testes and epididymides at all stages of autopsies.
Testis biochemistry The pattern of results of the testicular enzyme assays were similar to those of the other parameters. Activities of the various testicular enzymes were comparable with those of the respective controls in all the HCH-fed rats at the end of the 7th week (data not presented). At the end of the 2nd week, after withdrawal of HCH from the diets, rats of the Vo+HCH group showed significant alterations in the activities of the enzymes (Table 3). Dehydrogenases were the most affected among all the enzymes. In these rats, A 5 3[3-HSDH, 17[3-HSDH, and G-6-PDH showed reduc-
tions of activities as high as 57%, 53%, and 45%, respectively, while that of [3-GLR was increased by 39%. Activities of NSE, ALP, and LDH were also significantly reduced. In the V2ooo+HCH group, some of the enzyme activities exhibited significant alterations. On the other hand, A 5 313-HSDH, 17[3-HSDH, NSE, and [3-GLR activities in the Vloo,ooo+HCH rats were comparable to those of their controls, though marginal alterations were noticed in the activities of the other enzymes. At the 4th week after withdrawal of HCH from the diets, rats of all the groups showed considerable recovery in the activities of various enzymes (Table 4). In the Vo+HCH rats, all the enzymes with the exception of
Table 4. Testis enzymes of vitamin A deficient and supplemented male albino rats at the 4th week after withdrawal of HCH (1000 ppm) from the diets (values are mean --- SEM of 4 rats in each group)
Diet group
Vo Vo+HCH 112oo0 V2ooo+HCH Vloo.ooo Vloo,ooo+HCH
17 [3-HSDH (SEM)
3 I3-HSDH (SEM)
G-6-PDH (SEM)
LDH (SEM)
I3-GLR (SEM)
NSE (SEM)
ALP (SEM)
ACP (SEM)
Protein (rag/g) (SEM)
143.31 (14.01) 117.08 (8.28) 124.08 (4.04) 116.08 (6.80) 172,31 (13,10) 168.18 (8.70)
105.18 (15.01) 84.00 (11.04) 134.35 (4.86) 133.09 (5.00) 136.18 (6.10) 134.17 (3.50)
15.80 (5.12) 10.28 (6.21) 17.21 (1.27) 18.60 (0.63) 19.50 (1.57) 20.12 (0.98)
121.00 (6.10) 92.01" (7.10) 141.01 (4.70) 142.91 (5.72) 140.09 (6.10) 136.85 (5.70)
16.08 (1.10) 18.51 (1.20) 19.90 (0.78) 20.70 (1.20) 19.71 (1.70) 20.20 (2.10)
158.02 (7.50) 150.13 (3.20) 194.08 (5.91) 208.37 (15.32) 215.06 (11.07) 207.85 (25.08)
3223.18 (243.01) 2600.00 (177.18) 3731.07 (330.18) 3653.93 (380.57) 3452.37 (310.31) 3417.97 (295.39)
4053.08 (60.08) 4500.00*** (44.31) 6025.18 (260,71) 590,73 (270,38) 5987.21 (320.30) 6013.31 (410.97)
33.00 (0.71) 34.17 (1.13) 30.00 (1.00) 31.00 (0.90) 30.72 (0.98) 29.07 (1.08)
*P < 0.05; ***P < 0.001. Enzyme activities are expressed as nmoles/mg protein/h. Comparisons are between HCH and same diet without HCH.
Male reproductivetoxicity • J. PIus ETAL. LDH showed considerable but not complete recovery. In the VEOoo+HCH rats, all the enzyme activities were close to the controls, while complete recovery of the enzyme activities was noticed in the Vto0,oo+HCH rats. Supplementation of vitamin A at 100,000 IU/kg diet to the vitamin A deficient rats previously exposed to HCH resulted in complete recovery in activities of all the enzymes (data not presented). DISCUSSION Vitamin A is required for the normal processes of reproduction, and deficiency results in atrophy of testes, degeneration of seminiferous tubules, and spermatogenic arrest in male animals (10). Similar effects on the reproductive system have also been attributed to HCH. Shivanandappa and Krishnakumari (8) have reported that feeding rats HCH at 1500 ppm for 90 days produced marked effects on spermatogenesis and steroidogenesis. It was found in the present study that dietary HCH at 1000 ppm was apparently without any toxic effect on the male reproductive system at the end of the feeding period of 49 days. During the postexposure period, manifestation of the toxic effects of HCH on the male reproductive system was more pronounced in the vitamin A deficient rats as evidenced by atrophy of the testes and accessory reproductive organs, spermatogenic arrest, reduced sperm count, histologic changes, and alterations in various testicular enzymes. These results demonstrate the latent effects of HCH at some stage prior to 7 weeks and that the male reproductive system is highly vulnerable to the toxic effects of HCH during vitamin A deficiency. Similar atrophy of the reproductive structures associated with spermatogenic arrest has been reported in animals as a result of exposure to pesticides and other chemicals (25, 26). Histologic alterations found in the testes and epididymides are supportive of the findings of reduced sperm count and relative organ weights. Atrophied testis with shrunken seminiferous tubules and spermatogenic arrest has been reported in rats fed 1500 ppm HCH for 90 days (8). Similar observations have been reported by Dikshith and Datta (25). Also, alterations in the activities of testicular enzymes such as ALP, ACP, NSE, LDH, and 13-GLR have been reported in conditions of chemical toxicity (4, 27, 28). Therefore, histologic changes, marked reduction in sperm count, and the greater alterations of the enzymes in the vitamin A deficient rats confirm the enhanced testicular toxicity of HCH during vitamin A deficiency. The lack of such changes or only mild alterations found in the vitamin A supplemented rats are suggestive of the protective role of vitamin A, particularly in excess, but not at a hypervitaminotic level that is 100,000 IU/kg diet. A5 313-HSDH and 17 f3-HSDH are key enzymes in
329
the biosynthesis of steroid hormones. G-6-PDH is the potential generator of NADPH, which is required for hydroxylation reactions in steroid biosynthesis (29). The Leydig cells and Sertoli cells of the seminiferous tubules are known to be the principal sites of steroid biosynthesis as they possess these important steroidogenic enzymes (30). Inhibition of steroidogenic enzymes in the testis and adrenals of rats following HCH administration has been shown histochemically by earlier workers (8, 31). Therefore, the reduced activities of the important enzymes of steroidogenesis observed in the present study imply that the toxic action of HCH was exerted by impaired steroidogenesis in the Leydig cells and Sertoli cells, accentuated by deficiency and protected by supplementation of vitamin A. This toxicity was reversible, as withdrawal of HCH from the diets resulted in recovery of the activities of the steroidogenic enzymes, which was further improved by vitamin A supplementation. Reproduction in animals is controlled by several hormones (32). The testicular toxicity of xenobiotics can be due either to their direct action on the reproductive structures or to indirect action through the endocrine system. Thus, methoxychlor, an organochlorine insecticide, results in an elevation in prolactin concentration and release, which in turn influences the hypothalamic gonadotropin-releasing hormone, ultimately resulting in testicular dysfunction (33). On the other hand, TOCP, an industrial chemical, brings about testicular toxicity without any alterations in the circulating hormones, showing that the toxicity is due to its direct action on the reproductive structures (4, 27). Our results indicate that HCH effects testicular toxicity indirectly by impairment of steroidogenesis, which is further accentuated by vitamin A deficiency. Further, supplementation of vitamin A, particularly in excess (not hypervitaminotic) of the normal requirement, could protect the steroidogenic inhibition of HCH and thereby reduce its toxicity. authors wish to thank Dr. B.L. Amla and Mr. S. K. Majumder for their keen interest in this investigation. The assistance of Mr. K. Narasimhamurthy and Mr. S. Viswanatha is highly appreciated. J. Pius was a recipient of CSIR (India) research fellowship.
Acknowledgments -- The
REFERENCES 1. Ellis LC. Radiation effects. In: Johnson AB, Gomes WR, VandermarkNL, eds. The testis; vol. II1.New York: AcademicPress; 1970:333-76. 2. Mann T, Lutwak-MannC. Passage of chemicalsinto human and animal semen: mechanisms and significance. CRC Crit Rev Toxicol. 1982;11:1-14. 3. LeathernJH. Nutrition. In: Johnson AD, GomesWR, Vandemark NL, eds. The testis; vol. 3. New York: Academic Press; 1970: 169-205. 4. SomkutiSG, LapadulaDM, Chapin RE, Lamb JC, Abou-Donia
330
5.
6. 7.
8. 9. 10. 11.
12.
13. 14.
15.
16.
17.
18.
19.
Reproductive Toxicology MB. Reproductive tract lesions resulting from subchronic administration (63 days) of tri-o-cresyl phosphate in male rats. Toxicol Appl Pharmacol. 1987;89:49-63. Gunn SA, Gould TC, Anderson WAD. Specificity in protection against lethality and testicular toxicity from cadmium. Proc Soc Exptl Biol Med. 1968;128:591-5. Virgo BB, Bellward GD. Effects of dietary dieldrin on reproduction in the Swiss-Vancouver (SWV) mouse. Environ Physiol Biochem. 1975;5:440-50. Krause W, Homola S. Alterations of the seminiferous epithelium and the Leydig cells of the rat testis after the application of Dichlorovos (DDVP). Bull Environ Contain Toxicol. 1974;11: 429-33. Shivanandappa T, Krishnakumari MK. Hexachlorocyclohexlaneinduced testicular dysfunction in rats. Acta Pharmacol Toxicol. 1983;52:12-17. Bunyan J, Green J, Diplock AT, Robinson D. Lysosomal enzymes and vitamin E deficiency. Br J Nutr. 1967;21:147-54. Ganguly J, Rao MRS, Murthy SK, Sarada K. Systemic mode of action of vitamin A. Vitamins and Hormones. 1980;38:1-54. McLaren DS. Global occurrence of vitamin A deficiency. In: Bauemfeind JC, ed. Vitamin A deficiency and its control. New York: Academic Press; 1986:1-18. Phillips WEJ, Hatiana G. Effect of dietary organochlorine pesticides on the liver vitamin A content of the weanling rat. Nutr Rep Inter. 1972;5:357~62. Tinsely IJ. DDT effect on rats raised on alpha-protein rations: growth and storage of vitamin A. J Nutr. 1969;98:319-24. Plus J. Role of dietary protein and vitamin A in the toxicity of hexachlorocyclohexane (HCH) to the rat. Ph.D thesis submitted to the University of Mysore, Mysore (India), 1988. Woodroof JG. Composition and nutritive value of nuts. In: Woodroof JG, ed, Peanuts -- production, processing and products, Inc: Westport, CT: The AVI Publishing Company; 1966: 57-82. Amann RP. A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl. 1982;2: 37-58. Talalay P. Hydroxysteroid dehydrogenases. In: Colowick SP, Kaplan NO, eds. Methods in enzymology; vol. 5. New York: Academic Press; 1962:512-16. Lohr GH, Waller HD. Glucose-6-Phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:636--43. Whitaker JF. A general colorimetric procedure for the estimation of enzymes which are linked to the NADPH/NAD + system. Clin Chim Acta. 1969;24:23-37.
Volume 4, Number 4, 1990 20. Walter K, Shutt C. Phosphatases. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:856-60. 21. Fishman WH. 13-glucuronidase. In: Bergmeyer HU, ed. Methods of enzymatic analysis; vol. 2. New York: Academic Press; 1972:929--43. 22. Murphy SD, Cheever KL, Chow AYK, Browster M. Organophosphate insecticides potentiation by carboxylesterase inhibitors. In: Proceedings of European Society of Toxicology; vol. 17. Excerpta Medica International Conference Series No. 376; 1975; 292-303. 23. Lowry OH, Rosebrough NJ, Fan AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:265-75. 24. Snedecor GW, Cochran WG, eds. Statistical methods. Ames, Iowa: Iowa State University; 1980. 25. Dikshith TSS, Datta KK. Pathologic changes induced by pesticides in the testes and liver of rats. Exp Pathol. 1972;7:309-16. 26. Linder RE, Rehnberg GL. Evaluation of reproductive parameters in adult male Wistar rats after subchronic exposure (gavage) to benomyl. J Toxicol Environ Health. 1988; 25:285-98. 27, Somkuti SG, Lapadula DM, Chapin RE, Lamb JC, Abou-Donia MB. Time-course of the tri-o-cresyl phosphate-induced testicular lesion in F-344 rats: enzymatic, hormonal and sperm parameter studies. Toxicol Appl Pharmacol. 1987;89:64-72. 28. Parmar D, Srivastava SP, Seth PK. Effect of di (2-ethylhexyl) phthalate (DEHP) on spermatogenesis in adult rats. Toxicology. 1986;42:47-55. 29. Hall PF. Endocrinology of the testis. In: Johnson AD, Vandemark NL, Gomes WR, eds. The testis; vol. 3. New York: Academic Press; 1970:1-71. 30. Blackshaw AW. Histochemical localization of testicular enzymes. In: Johnson AD, Vandemark NL, Gomes WR, eds. The testis; vol. 3. New York: Academic Press; 1970;72-123. 31. Shivanandappa T, Krishnakumari MK, Majumder SK, Inhibition of steroidogenic enzymes in the adrenal cortex of rats fed benzene hexachloride (hexachlorocyclohexane). Experientia. 1982;32:125153. 32. Steinberger E, Steinberger A. Hormonal control of testicular function in mammals. In: Knobil K, Sawyer WH, eds. Handbook of physiology; vol. 4. Washington, D.C.: American Physiological Society; 1974:325--45. 33. Goldman JM, Cooper RL, Rehnberg GL, Hein JF, McElroy WK, Gray LE Jr. Effects of low subchronic doses of methoxychlor on the rat hypothalamic-pituitary reproductive axis. Toxicol Appl Pharmacol. 1986;86:474-83.