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Effect of hexavalent chromium on testicular maturation in the rat
ReproductiveToxicology,Vol. 4, pp. 223-228, 1990
0890-6238/90 $3.00 + .00 Copyright© 1990PergamonPresspie
Printed in the U.S.A.
• Short Communication
EFFECT OF HEXAVALENT CHROMIUM ON TESTICULAR MATURATION IN THE RAT DAYA K. SAXENA, RAMESH C. MURTHY, BACHCHU LAL, RATAN S. SRIVASTAVA a n d SATYA V. CHANDRA Neurotoxicology Division, Industrial Toxicology Research Centre, Lucknow, India Abstract -- Daily intraperitoneal administration of hexavalent chromium (Cr6÷; 1, 2, and 3 mg/kg tntraperitoneaily as potassium dichromate) in weaned rats for an entire duration of 55 and 90 days of age produced doseand duration-dependent enzymatic and pathologic alterations. At 55 days, the pathologic changes were not seen in testes of C r 6+ treated rats, hut the activities of sorbitol dehydrogenase, lactic dehydrogenase, ~/-glutamyi transpeptidase, and glucose-6-phosphate dehydrogenase were significantly altered. When the treatment was prolonged to sexual maturity, that is, 90 days of age, the alterations in enzyme activities were greater, and there were dose-dependent pathologic changes in the testes of Cr6÷-treated rats. These alterations suggest a risk to growing testes if rats are exposed to Cr 6+ during the prepubertal stage of development, which, in turn, may disturb normal testicular physiology at adulthood. Key Words: potassium dichromate; testicular maturation; rat.
of Cr6+ in the growing rat from weaning to 55 days of age, a stage around which the rate of stage VII and VIII spermatogenesis approaches that of adult rats (5), and until the stage of sexual maturity, that is, 90 days of age (6), to ascertain the effect of this metal on the testis attaining sexual maturity.
INTRODUCTION Chrome ore mining and chrome-based industries like tannery, chemical processing, and pigment and paint manufacture release chromium (Cr) into the surrounding air, water, and soil. Exposure to hexavalent chromium (Cr6+) is known to cause pulmonary carcinoma, dermatitis, hepatotoxicity, nephrotoxicity, and gastrotoxicity in humans and laboratory animals (1). The affinity of Cr for the testis and incorporation into sperm has also been suggested (2). Behari et al. (3) reported biochemical alterations in the testes of rabbits exposed to potassium dichromate. However, there is hardly any literature dealing with the effect of Cr6+ on the developing testis. This is significant in view of the fact that young, growing animals are more susceptible to the toxicity of metals than adults (4). Furthermore, there may be a risk of Cr6+ exposure during the prepubertal stage of sexual maturity leading to permanent damage to the gonads. We have, therefore, investigated the effect of various doses
MATERIALS AND METHODS Sixty weaned male albino rats (40 to 50 g) from Industrial Toxicology Research Centre (Lucknow) breeding stock were divided into four groups of 15 rats each. Rats of group 1 were administered saline intraperitoneally while rats of groups 2, 3, and 4 were treated with 1, 2, and 3 mg/kg Cr6+ (as potassium dichromate, Aldrich Chemical Company, Milwaukee, WI, 99%) intraperitoneally daily up to 90 days of age. The animals were housed in stainless steel cages in an air-conditioned room where regular alternate cycles of 12 h light and darkness were maintained. The animals had free access to pelleted diet (Hindustan Lever Laboratory Animal Feed, Chandigarh, India) and tap water. Their body weight was recorded on alternate days. Six animals each were killed at 55 and 90 days of age by cervical dislocation and blood was collected in heparinized tubes for Cr estimation. The testes were dissected free, weighed, and kept for histopathology, biochemistry, and metal
Address correspondence to Dr. Satya V. Chandra, Deputy Director, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, India. Received 1 July 1989; Revised 28 September 1989; Accepted 30 September 1989. 223
224
Reproductive Toxicology
Volume 4, Number 3, 1990
estimation. In 90-day-old rats, the epididymis was dissected free, weighed, and the caudal end was teased in saline and processed for sperm motility, counts, and morphology.
200"
180"
Histopathologic studies The testes were fixed in 10% neutral buffered formalin, embedded in paraffin, cut at 5 ixm, and stained with hematoxylin-eosin and periodic acid Schiff's hematoxylin for routine pathology and VIIth stage germ cell counts (7) respectively. The reason for choosing this stage is that in addition to spermatogonia, two generations of spermatids and spennatocytes are present at this stage, so that the effect of treatment on cell yields during the different phases of spermatogenesis can be assessed. For each germ cell type, a ratio was calculated comparing the number of germ cell nuclei to the number of Sertoli cell nuclei. The analysis was performed as described earlier (8) in order to determine if cell death had occurred at any specific stage of spermatocyte maturation (9). Tubular and Leydig cell nuclei diameter were measured with an ocular micrometer (10). The sperm counts, motility, and morphology were evaluated by teasing the weighed cauda epididymis into a known volume of normal saline at 37 °C over a slide by a method described earlier (8).
EFFECT OF C~ 6 EXPOSURE ON BODY WEIGHT OF RATS : c. CONTROL e----o lmg/k 9 e.- • -1 2mg/kg I~-- • - - o 3 m g / k g
160" s i
140-
--.= I,-
"1"
I&l
• lOO>,,
o
2 ao-
/,O"
20"
Biochemical assays A portion of testis was homogenized in 0.32 M sucrose and centrifuged at 1200 × g at 4°C for 20 min to obtain a clear post-mitochondrial supernatant in which the activities of glucose-6-phosphate dehydrogenase (G6PD), lactic dehydrogenase (LDH), and sorbitol dehydrogenase (SDH) were measured according to Lrhr and Waller (11), Komberg (12) and Gerlach (13), respectively. For ~/-glutamyl transpeptidase (GGT) estimation, the tissue was homogenized in chilled 0.5 M Tris buffer (pH 7.0) and enzyme activity assayed in the homogenate according to the method of Roomi and Goldberg (14). Chromium estimation The measured volume of blood and weighed testes of six animals from each group were digested in a mixture of HNO3:HC104 (6:1) on a mantle heater. The residue was dissolved in 0.1 N HC1, and the total Cr in these samples was measured using a DC Plasma Emission Spectrophotometer (Beckman Spectraspan V, Geneva) and spiked samples were also processed and analyzed simultaneously. Statistical analysis Data were analyzed using one way analysis of
0
b AGE
IN
lb
f2 f3
WEEKS
Fig. 1. Body weight change in the rats treated with hexavalent chromium.
variance. The technique was applied after ascertaining the homogeneity of variance and normality assumptions of the data. If the overall F value was significant, the significance of change between the two groups was determined by least significant difference method. Differences at P < 0.05 were considered significant. RESULTS The animals of groups 1 and 2 appeared normal during the entire period of the experiment, while 2 and 3 rats died in groups 3 and 4, respectively. The cause of death could not be ascertained during autopsy.
Body and testicular weight Body weight gain registered a significant reduction from one week onward in groups 3 and 4 as compared to
Chromium-induced testicular degeneration • D. K. SAXENAET AL.
225
Table 1. Effect of Cr 6+ exposure on testicular weight, seminiferous tubule and Leydig cell nucleus diameter, sperm counts, and motility in rats
Groups 1
Testes wt. (g) (R+L) 2 55d 90d
Body/testicular weight ratio × 100 55d 90d
Seminiferous tubule diameter (nm)t
Leydig cell Sperm nucleus diameter counts (nm)t (million/mL)t
Sperm motility (%)t
0.62 --- 0.11
1.19 ± 0.03
0.6 - 0.05
0.6 ~- 0.04
268.60 ___ 13.03
6.71 - 0.15
45.78 ___ 3.3
73.49 ± 2.46
0.53 --- 0.06
1.07 ± 0.05
0.6 ___ 0.04
0.6 ± 0.10
259.30 ± 5.9 (-3.5)
6.28 _ 0.09 (-6.4)
34.23 ~ 4.27
66.03 ± 5.49
0.37 ± 0.04 (ab*)
0.82 ± 0.07 (ab*)
0.5 ± 0.03
0.7 ± 0.05
238
± 6.87 ( - 11.4)
6.07 ± 0.10 ( - 9.5) (a*)
24.01 ± 3.82 (a*)
13.19 +- 3.72 (ab*)
0.36 ± 0.02 (ab*)
0.53 ± 0.10 (abc*)
0.5 ± 0.03
0.7 _+ 0.05
181.9 ± 5.16 ( - 32.3) (abc*)
5.35 +-- 0.09 ( - 20.14) (abc*)
16.52 ± 1.61 (abc*)
7.07 ± 1.48 (abc*)
(control) 2 (1 nag Cr6÷/kg) 3 (2 nag Cr6+/kg) 4 (3 mg Cr6+/kg)
Values represent the mean ± SE of 6 animals per group. Statistical significance was calculated by one way ANOVA. The values represented in parentheses designate percent change and level of significance. a = versus group 1; b = versus group 2; c = versus group 3. *P < 0.05. t = parameters determined at 90 days only.
g r o u p s 1 a n d 2 (Figure 1). W h e n c o m p a r e d to o n e a n o t h e r , rats o f g r o u p 3 a n d g r o u p 4 did n o t s h o w a n y s i g n i f i c a n t d i f f e r e n c e in testicular w e i g h t at 55 days, b u t a s i g n i f i c a n t d i f f e r e n c e w a s s e e n at 90 days. A s i m i l a r p a t t e r n w a s also o b s e r v e d w h e n the r e l a t i v e b o d y / testicular w e i g h t w a s c a l c u l a t e d a m o n g the g r o u p s (Table 1).
Tubular and Leydig cell nuclei diameter T h e a n i m a l s in g r o u p 4 s h o w e d a s i g n i f i c a n t r e d u c tion in s e m i n i f e r o u s t u b u l e a n d L e y d i g cell n u c l e u s
Fig. 2. Control rat testis showing normal structure. HE x 105.
d i a m e t e r c o m p a r e d to all o t h e r g r o u p s , w h e r e a s g r o u p 3 a n i m a l s h a d s i g n i f i c a n t l y l o w e r L e y d i g cell n u c l e u s d i a m e t e r c o m p a r e d to g r o u p 1.
Histopathologic changes A t 55 a n d 90 d a y s o f age, the testes o f c o n t r o l rats ( g r o u p 1) r e v e a l e d a n o r d e r l y a r r a n g e m e n t o f g e r m cells, n o r m a l interstitial tissue, a n d L e y d i g cells (Figure 2). T h e testes o f rats o f g r o u p 2, 3, a n d 4 d i d n o t s h o w a n y p a t h o l o g i c c h a n g e s at 55 days. All testes s h o w e d a n o r m a l a p p e a r a n c e o f t u b u l a r a n d interstitial tissue.
Fig. 3. Testis of rat treated with 1 mg/kg Cr 6+ from weaning till 90 days of age showing a few damaged seminiferous tubules surrounded by normal tubules. HE x 105.
226
Reproductive Toxicology
Volume 4, Number 3, 1990 congested blood vessels were also evident. The Leydig cells were atrophied in places (Figure 5). This indicates duration and dose-dependent Cr6+-induced damage in the testicular tissue of rats.
Germ cell damage The degree of decrease in late-stage spermatids and germ cell numbers at stage VII was dose dependent, the higher doses revealing a marked decrease when compared with lower doses of Cr 6+. The percent decrease in the number of germ cells in preleptotene stage (13, 39, and 70), pachytene stage (5, 23, and 82) and spermatids (8, 48, and 71) in groups 2, 3, and 4, respectively, was evident in the testes of Cr6+-treated rats compared to controls. Fig. 4. Testes of Cr6+-treated rat (2 mg/kg from weaning until 90 days of age) showing disturbed spermatogenesis in the form of multinucleated giant cells and pyknotic nuclei. A few tubules are also devoid of sperm in their lumen.
Testes of rats treated with 1 mg/kg Cr 6+ until 90 days of age revealed damaged germ cells in a few tubules surrounded by normal tubules (Figure 3). Chromium-(2 mg/kg)-treated rat testes showed many of the tubules with disturbed spermatogenesis in the form of multinucleated giant cells and pyknotic nuclei. Interstitial edema was also evident in places (Figure 4). Testes of rats treated with 3 mg/kg Cr 6+ showed a larger number of severely damaged tubules. The tubules were shrunken and were devoid of sperm in their lumens. A few tubules were lined by only a single layer of Sertoli cells and spermatogonia. Focal interstitial edema and markedly
Biochemical changes Figure 6 shows the activity of enzymes associated with various doses of Cr 6+ in rat testes at 55 and 90 days of age. Sorbitol dehydrogenase and G6PD activities showed no significant change at 1 mg/kg Cr 6+ at 55 days of age. However, activity of these enzymes decreased in a dose-dependent manner in the rest of the Cr6+-treated rat testes at both 55 and 90 days of age. GGT and LDH activities also showed a similar pattern, but enzyme activity increased compared to controls.
Sperm counts, motility, and morphology In the rats treated up to 90 days of age, the percent reduction in sperm counts and sperm motility was dose dependent; the lowest dose did not reveal any significant reduction compared to controls, while the higher doses showed correspondingly higher incidences of reduced sperm counts and sperm motility (Table 1). No increase in sperm malformation was seen in any of the treated groups; their incidences corresponded with the controls.
Chromium contents There was a significant accumulation of total chromium in the blood and testes of Cr6+-exposed rats which was dose and duration dependent (Table 2). DISCUSSION
Fig. 5. Chromium-(3 mg/kg)-treated rat testes with most of the tubules showing severely damaged spermatogenesis. Tubules are lined by only a single layer of cells. Blood vessels are congested and dilated in places. HE x 160.
It is well established that at the age of 21 days in rats (weanling stage) only spermatogonia and spermatocytes are present, and at 55 days of age, stage VII of spermatogenesis, similar to adults, is reached (5). Thereafter, at 90 days of age, the testis reaches full maturity (6). With this background the exposure duration for Cr 6+ was chosen to investigate its effect on the maturation of male gonads. The results show that Cr 6+ administration in graded daily doses intraperitoneally in weaned rats from weaning until 55 and 90 days of age produced
Chromium-induced testicular degeneration
•
D. K. SAXEr~AET gL.
.D 0
160'
SDH
40-
O~g
0
FI C*rS-3mg/kg
80
rl O
U o's 0
C~'fi--ling/kg [ ] C*r6--2mg/ kg
120
(+)
227
G6PDH
9o,i
55d
LSSd
l
90d
55d
I
LDH 40'
0""
(-) 80'
U n 0
U 0
120 Fig. 6. Effect of Cr6÷ administration (up to 55 and 90 days of age) on certain testicular enzymes in rats. Values expressed as percent changes compared with controls --- SE.* Changes are significant compared with (a) controls, (b) 1 mg/kg Cr6+, and (c) 2 mg/kg Cr6÷ . SDH = sorbitol dehydrogenase, G6PDH = glucose-6-phosphate dehydrogenase, GGT = ~/-glutamyltranspeptidase, and LDH = lactic dehydrogenase.
dose-dependent enzymatic and pathologic alterations. The metal content in the testis also increased in a similar manner. Maximum degrees of enzymatic alteration together with marked pathologic changes at 90 days indicated the involvement of mature spermatogenetic cells, which consequently resulting in reduced testicular weight, sperm counts, and motility. Changes in the activity of SDH, LDH, GGT, and G6PD have been reported to be early biochemical indicators of testicular injury. SDH and LDH enzymes reflect germ cell damage (15, 16). G6PD is needed for steroid biosynthesis (17). Similarly, GGT enzyme activity is reported to parallel the pattern of Sertoli cell
replication and maturation (18, 19), which has been shown to be affected by various xenobiotics like di(2-ethylhexyl) phthalate (20), gossypol (21), sodium selenite (16), and manganese (22). It may take a longer duration of exposure with this dose regimen to produce pathologic changes that are dose dependent at 90 days. Since the extent of enzymatic alteration, together with the dose- and duration-dependent pathologic changes, correspond with the increasing Cr accumulation in the blood and testes of treated rats, the possibility that the metal accumulated in the testicular tissue produces enzymatic and pathologic changes cannot be ruled out. The observed decrease in body weight gain in the
Table 2. Chromium level in different tissues of rats Group 1
55 days Group 2
Group 3
Group 4
Group 1
90 days Group 2 Group 3
Group 4
Blood (ixg/mL)
0.039 --- 0.007
0.74 --- 0.02 (a*)
1.09 + 0.02 (ab*)
1.82 ___ 0.19 (abc*)
0.04 --- 0.004
1.46 ___ 0.13 (a*)
2.95 --- 0.13 (ab*)
4.19 ___ 0.47 (abc*)
Testes (p.g/g f.w.)
0.015 ___ 0.005
0.14 -- 0.008 (a*)
0.23 __ 0.01 (ab*)
0.33 -- 0.01 (abc*)
0.03 -+ 0.007
0.54 --- 0.05 (a*)
0.96 -+ 0.03 (ab*)
2.93 __ 0.04 (abc*)
All the values represent the mean __. SE of 6 animals per group. Group 1 = saline control, Groups 2, 3, and 4 were given 1, 2, and 3 mg/kg/day Cr6+ ip, respectively. Statistical significance evaluated by using one-way ANOVA. f.w. = fresh weight. a --- compared with group 1; b = compared with group 2; c = compared with group 3. *P < 0.05.
228
Reproductive Toxicology
highest dosed rats in the present study, indicating the possibility of general metal toxicity, led us to consider whether these testicular changes are the direct effect of the metal on differentiating germ cells or are mediated through disturbances in gonadotropic hormones. Our study also showed Leydig cell atrophy and reduced Leydig cell nuclear diameter besides disturbed G6PD enzyme activity, indicating the possibility of disturbed androgen biosynthesis. Several other metals, such as lead and cadmium, have also been reported to affect the testis by interfering with hormone synthesis and in various intermediate pathways (23, 24). Further studies are being conducted to ascertain the role of various hormones in Cr6+-induced testicular dystrophy. In conclusion, the present study showed a risk to the growing testes of rats exposed to Cr 6+ during the prepubertal stage of development, which may disturb normal testicular physiology at adulthood.
- - The authors are thankful to the Director, Industrial Toxicology Research Centre, Lucknow, India, for interest in the study. Thanks are also due to Miss L. Sajnani for technical assistance and to Mr. G.M. Nair for his secretarial assistance.
Acknowledgments
REFERENCES 1. WHO, Environmental Health Criteria, 61: Chromium International Programme on Chemical Safety, Geneva, 1988. 2. Mertz W. Chromium occurrence and function in biological systems. Physiol Rev. 1969;49:163-239. 3. Behari J, Chandra SV, Tandon SK. Comparative toxicity of trivalent and hexavalent chromium to rabbits. III. Biochemical and histological changes in testicular tissue. Acta Biol Med Ger. 1978;37:463-68. 4. Sj6berg P, Bondesson U, Kjellen L, Lindquist NG, Montin G, P16en L. Kinetics of di-(2-ethylhexyl) phthalate in immature and mature rats and its effects on testis. Acta Pharmacol Toxicol. 1985;56:30-7. 5. Ekwall A, Jansson A, Sjtberg P. Differentiation of the rat testes between 20 and 120 days of age. Arch Androl. 1984;13:27-36. 6. Knorr DW, Vanha-perttula T, Lipsett MB. Structure and function of rat testis through pubescence. Endocrinology. 1970;86:1298304.
Volume 4, Number 3, 1990 7. Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann Acad Sci. 1952;55: 548-73. 8. Saxena DK, Murthy RC, Singh C., Chandra SV. Zinc reduces testicular injury induced by concurrent exposure to cadmium and lead in rats. Res Comm Chem Pathol Pharmacol. [In press]. 9. Dunnick JK, Harris MW, Chapin RE, Hall LB, Lamb JC. Reproductive toxicology of methyldopa in male F 344/N rats. Toxicology. 1986;41:305-18. 10. Shivanandappa T, Krishnakumari MK. Hexachlorocyclohexaneinduced testicular dysfunction in rats. Acta Pharmacol Toxicol. 1983;52:12-17. 11. Lthr GW, Waller HD. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis. New York: Academic Press; 1965:744-51. 12. Kornberg A. Lactic dehydrogenase of muscle. In: Colowick SP, Kaplan NO, eds. Methods in enzymology, vol. 1. New York: Academic Press; 1955:441-3. 13. Gerlach U. Sorbitol dehydrogenase. In: Bergmeyer J, Grosse M, eds. Methods of enzymatic analysis, vol. 3. Florida. Basel: Verlag Chemie Weinheim; 1983:352-6. 14. Roomi MW, Goldberg DM. Comparison of glutamyl transferase induction by phenobarbital in rat, guinea pig and rabbit. Biochem. Pharmacol. 1981;30:1563-71. 15. Bishop DW. Sorbitol dehydrogenase in relation to spermatogenesis and fertility. J Reprod Fertil. 1968;17:410-11. 16. Nebbia C, Brando C, Burdino E, Rasero R, Valenza F, Arisio R, Vgazio G. Effect of the chronic administration of sodium selenite on rat testes. Res Comm Chem Pathol Pharmacol. 1987;58: 183-97. 17. McKerns K. The pentose-phosphate pathway, steroidogenesis and protein synthesis. Biochem Biophys Acta. 1965;100:612-15. 18. Hodgeu GD, Sherins RJ. Enzymes as markers of testicular growth and development in the rat. Endocrinology. 1973;93:985-9. 19. Sherins RJ, Hodgen GD. Testicular gamma-glutamyl trans-peptidase: an index of Sertoli cell function in man. J. Reprod Fertil. 1976;48:191-3. 20. Oishi S. Testicular atrophy induced by di(2-ethylhexyl) phthalate: changes in histology, cell specific enzyme activities and zinc concentrations in rat testis. Arch Toxicol. 1986;59:290-5. 21. Olgiati KL, Hoffer AP, Toscano WA Jr. Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats. Biol Reprod. 1984;31:759-70. 22. Imam Z, Chandra SV. Histochemical alterations in rabbit testis produced by manganese chloride. Toxicol Appl Pharmacol. 1975;32: 534-44. 23. Wiebe JP. Effect of lead on testicular endocrinology in the developing male. Am Zool. 1981;20:899. 24. Zylber-Haran EA, Gershman H, Rosenmann E, Spitz IM. Gonadotropin, testosterone and prolactin interrelationships in cadmiumtreated rats. J Endocrinol. 1982;92:123-30.
0890-6238/90 $3.00 + .00 Copyright© 1990PergamonPresspie
Printed in the U.S.A.
• Short Communication
EFFECT OF HEXAVALENT CHROMIUM ON TESTICULAR MATURATION IN THE RAT DAYA K. SAXENA, RAMESH C. MURTHY, BACHCHU LAL, RATAN S. SRIVASTAVA a n d SATYA V. CHANDRA Neurotoxicology Division, Industrial Toxicology Research Centre, Lucknow, India Abstract -- Daily intraperitoneal administration of hexavalent chromium (Cr6÷; 1, 2, and 3 mg/kg tntraperitoneaily as potassium dichromate) in weaned rats for an entire duration of 55 and 90 days of age produced doseand duration-dependent enzymatic and pathologic alterations. At 55 days, the pathologic changes were not seen in testes of C r 6+ treated rats, hut the activities of sorbitol dehydrogenase, lactic dehydrogenase, ~/-glutamyi transpeptidase, and glucose-6-phosphate dehydrogenase were significantly altered. When the treatment was prolonged to sexual maturity, that is, 90 days of age, the alterations in enzyme activities were greater, and there were dose-dependent pathologic changes in the testes of Cr6÷-treated rats. These alterations suggest a risk to growing testes if rats are exposed to Cr 6+ during the prepubertal stage of development, which, in turn, may disturb normal testicular physiology at adulthood. Key Words: potassium dichromate; testicular maturation; rat.
of Cr6+ in the growing rat from weaning to 55 days of age, a stage around which the rate of stage VII and VIII spermatogenesis approaches that of adult rats (5), and until the stage of sexual maturity, that is, 90 days of age (6), to ascertain the effect of this metal on the testis attaining sexual maturity.
INTRODUCTION Chrome ore mining and chrome-based industries like tannery, chemical processing, and pigment and paint manufacture release chromium (Cr) into the surrounding air, water, and soil. Exposure to hexavalent chromium (Cr6+) is known to cause pulmonary carcinoma, dermatitis, hepatotoxicity, nephrotoxicity, and gastrotoxicity in humans and laboratory animals (1). The affinity of Cr for the testis and incorporation into sperm has also been suggested (2). Behari et al. (3) reported biochemical alterations in the testes of rabbits exposed to potassium dichromate. However, there is hardly any literature dealing with the effect of Cr6+ on the developing testis. This is significant in view of the fact that young, growing animals are more susceptible to the toxicity of metals than adults (4). Furthermore, there may be a risk of Cr6+ exposure during the prepubertal stage of sexual maturity leading to permanent damage to the gonads. We have, therefore, investigated the effect of various doses
MATERIALS AND METHODS Sixty weaned male albino rats (40 to 50 g) from Industrial Toxicology Research Centre (Lucknow) breeding stock were divided into four groups of 15 rats each. Rats of group 1 were administered saline intraperitoneally while rats of groups 2, 3, and 4 were treated with 1, 2, and 3 mg/kg Cr6+ (as potassium dichromate, Aldrich Chemical Company, Milwaukee, WI, 99%) intraperitoneally daily up to 90 days of age. The animals were housed in stainless steel cages in an air-conditioned room where regular alternate cycles of 12 h light and darkness were maintained. The animals had free access to pelleted diet (Hindustan Lever Laboratory Animal Feed, Chandigarh, India) and tap water. Their body weight was recorded on alternate days. Six animals each were killed at 55 and 90 days of age by cervical dislocation and blood was collected in heparinized tubes for Cr estimation. The testes were dissected free, weighed, and kept for histopathology, biochemistry, and metal
Address correspondence to Dr. Satya V. Chandra, Deputy Director, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, India. Received 1 July 1989; Revised 28 September 1989; Accepted 30 September 1989. 223
224
Reproductive Toxicology
Volume 4, Number 3, 1990
estimation. In 90-day-old rats, the epididymis was dissected free, weighed, and the caudal end was teased in saline and processed for sperm motility, counts, and morphology.
200"
180"
Histopathologic studies The testes were fixed in 10% neutral buffered formalin, embedded in paraffin, cut at 5 ixm, and stained with hematoxylin-eosin and periodic acid Schiff's hematoxylin for routine pathology and VIIth stage germ cell counts (7) respectively. The reason for choosing this stage is that in addition to spermatogonia, two generations of spermatids and spennatocytes are present at this stage, so that the effect of treatment on cell yields during the different phases of spermatogenesis can be assessed. For each germ cell type, a ratio was calculated comparing the number of germ cell nuclei to the number of Sertoli cell nuclei. The analysis was performed as described earlier (8) in order to determine if cell death had occurred at any specific stage of spermatocyte maturation (9). Tubular and Leydig cell nuclei diameter were measured with an ocular micrometer (10). The sperm counts, motility, and morphology were evaluated by teasing the weighed cauda epididymis into a known volume of normal saline at 37 °C over a slide by a method described earlier (8).
EFFECT OF C~ 6 EXPOSURE ON BODY WEIGHT OF RATS : c. CONTROL e----o lmg/k 9 e.- • -1 2mg/kg I~-- • - - o 3 m g / k g
160" s i
140-
--.= I,-
"1"
I&l
• lOO>,,
o
2 ao-
/,O"
20"
Biochemical assays A portion of testis was homogenized in 0.32 M sucrose and centrifuged at 1200 × g at 4°C for 20 min to obtain a clear post-mitochondrial supernatant in which the activities of glucose-6-phosphate dehydrogenase (G6PD), lactic dehydrogenase (LDH), and sorbitol dehydrogenase (SDH) were measured according to Lrhr and Waller (11), Komberg (12) and Gerlach (13), respectively. For ~/-glutamyl transpeptidase (GGT) estimation, the tissue was homogenized in chilled 0.5 M Tris buffer (pH 7.0) and enzyme activity assayed in the homogenate according to the method of Roomi and Goldberg (14). Chromium estimation The measured volume of blood and weighed testes of six animals from each group were digested in a mixture of HNO3:HC104 (6:1) on a mantle heater. The residue was dissolved in 0.1 N HC1, and the total Cr in these samples was measured using a DC Plasma Emission Spectrophotometer (Beckman Spectraspan V, Geneva) and spiked samples were also processed and analyzed simultaneously. Statistical analysis Data were analyzed using one way analysis of
0
b AGE
IN
lb
f2 f3
WEEKS
Fig. 1. Body weight change in the rats treated with hexavalent chromium.
variance. The technique was applied after ascertaining the homogeneity of variance and normality assumptions of the data. If the overall F value was significant, the significance of change between the two groups was determined by least significant difference method. Differences at P < 0.05 were considered significant. RESULTS The animals of groups 1 and 2 appeared normal during the entire period of the experiment, while 2 and 3 rats died in groups 3 and 4, respectively. The cause of death could not be ascertained during autopsy.
Body and testicular weight Body weight gain registered a significant reduction from one week onward in groups 3 and 4 as compared to
Chromium-induced testicular degeneration • D. K. SAXENAET AL.
225
Table 1. Effect of Cr 6+ exposure on testicular weight, seminiferous tubule and Leydig cell nucleus diameter, sperm counts, and motility in rats
Groups 1
Testes wt. (g) (R+L) 2 55d 90d
Body/testicular weight ratio × 100 55d 90d
Seminiferous tubule diameter (nm)t
Leydig cell Sperm nucleus diameter counts (nm)t (million/mL)t
Sperm motility (%)t
0.62 --- 0.11
1.19 ± 0.03
0.6 - 0.05
0.6 ~- 0.04
268.60 ___ 13.03
6.71 - 0.15
45.78 ___ 3.3
73.49 ± 2.46
0.53 --- 0.06
1.07 ± 0.05
0.6 ___ 0.04
0.6 ± 0.10
259.30 ± 5.9 (-3.5)
6.28 _ 0.09 (-6.4)
34.23 ~ 4.27
66.03 ± 5.49
0.37 ± 0.04 (ab*)
0.82 ± 0.07 (ab*)
0.5 ± 0.03
0.7 ± 0.05
238
± 6.87 ( - 11.4)
6.07 ± 0.10 ( - 9.5) (a*)
24.01 ± 3.82 (a*)
13.19 +- 3.72 (ab*)
0.36 ± 0.02 (ab*)
0.53 ± 0.10 (abc*)
0.5 ± 0.03
0.7 _+ 0.05
181.9 ± 5.16 ( - 32.3) (abc*)
5.35 +-- 0.09 ( - 20.14) (abc*)
16.52 ± 1.61 (abc*)
7.07 ± 1.48 (abc*)
(control) 2 (1 nag Cr6÷/kg) 3 (2 nag Cr6+/kg) 4 (3 mg Cr6+/kg)
Values represent the mean ± SE of 6 animals per group. Statistical significance was calculated by one way ANOVA. The values represented in parentheses designate percent change and level of significance. a = versus group 1; b = versus group 2; c = versus group 3. *P < 0.05. t = parameters determined at 90 days only.
g r o u p s 1 a n d 2 (Figure 1). W h e n c o m p a r e d to o n e a n o t h e r , rats o f g r o u p 3 a n d g r o u p 4 did n o t s h o w a n y s i g n i f i c a n t d i f f e r e n c e in testicular w e i g h t at 55 days, b u t a s i g n i f i c a n t d i f f e r e n c e w a s s e e n at 90 days. A s i m i l a r p a t t e r n w a s also o b s e r v e d w h e n the r e l a t i v e b o d y / testicular w e i g h t w a s c a l c u l a t e d a m o n g the g r o u p s (Table 1).
Tubular and Leydig cell nuclei diameter T h e a n i m a l s in g r o u p 4 s h o w e d a s i g n i f i c a n t r e d u c tion in s e m i n i f e r o u s t u b u l e a n d L e y d i g cell n u c l e u s
Fig. 2. Control rat testis showing normal structure. HE x 105.
d i a m e t e r c o m p a r e d to all o t h e r g r o u p s , w h e r e a s g r o u p 3 a n i m a l s h a d s i g n i f i c a n t l y l o w e r L e y d i g cell n u c l e u s d i a m e t e r c o m p a r e d to g r o u p 1.
Histopathologic changes A t 55 a n d 90 d a y s o f age, the testes o f c o n t r o l rats ( g r o u p 1) r e v e a l e d a n o r d e r l y a r r a n g e m e n t o f g e r m cells, n o r m a l interstitial tissue, a n d L e y d i g cells (Figure 2). T h e testes o f rats o f g r o u p 2, 3, a n d 4 d i d n o t s h o w a n y p a t h o l o g i c c h a n g e s at 55 days. All testes s h o w e d a n o r m a l a p p e a r a n c e o f t u b u l a r a n d interstitial tissue.
Fig. 3. Testis of rat treated with 1 mg/kg Cr 6+ from weaning till 90 days of age showing a few damaged seminiferous tubules surrounded by normal tubules. HE x 105.
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Reproductive Toxicology
Volume 4, Number 3, 1990 congested blood vessels were also evident. The Leydig cells were atrophied in places (Figure 5). This indicates duration and dose-dependent Cr6+-induced damage in the testicular tissue of rats.
Germ cell damage The degree of decrease in late-stage spermatids and germ cell numbers at stage VII was dose dependent, the higher doses revealing a marked decrease when compared with lower doses of Cr 6+. The percent decrease in the number of germ cells in preleptotene stage (13, 39, and 70), pachytene stage (5, 23, and 82) and spermatids (8, 48, and 71) in groups 2, 3, and 4, respectively, was evident in the testes of Cr6+-treated rats compared to controls. Fig. 4. Testes of Cr6+-treated rat (2 mg/kg from weaning until 90 days of age) showing disturbed spermatogenesis in the form of multinucleated giant cells and pyknotic nuclei. A few tubules are also devoid of sperm in their lumen.
Testes of rats treated with 1 mg/kg Cr 6+ until 90 days of age revealed damaged germ cells in a few tubules surrounded by normal tubules (Figure 3). Chromium-(2 mg/kg)-treated rat testes showed many of the tubules with disturbed spermatogenesis in the form of multinucleated giant cells and pyknotic nuclei. Interstitial edema was also evident in places (Figure 4). Testes of rats treated with 3 mg/kg Cr 6+ showed a larger number of severely damaged tubules. The tubules were shrunken and were devoid of sperm in their lumens. A few tubules were lined by only a single layer of Sertoli cells and spermatogonia. Focal interstitial edema and markedly
Biochemical changes Figure 6 shows the activity of enzymes associated with various doses of Cr 6+ in rat testes at 55 and 90 days of age. Sorbitol dehydrogenase and G6PD activities showed no significant change at 1 mg/kg Cr 6+ at 55 days of age. However, activity of these enzymes decreased in a dose-dependent manner in the rest of the Cr6+-treated rat testes at both 55 and 90 days of age. GGT and LDH activities also showed a similar pattern, but enzyme activity increased compared to controls.
Sperm counts, motility, and morphology In the rats treated up to 90 days of age, the percent reduction in sperm counts and sperm motility was dose dependent; the lowest dose did not reveal any significant reduction compared to controls, while the higher doses showed correspondingly higher incidences of reduced sperm counts and sperm motility (Table 1). No increase in sperm malformation was seen in any of the treated groups; their incidences corresponded with the controls.
Chromium contents There was a significant accumulation of total chromium in the blood and testes of Cr6+-exposed rats which was dose and duration dependent (Table 2). DISCUSSION
Fig. 5. Chromium-(3 mg/kg)-treated rat testes with most of the tubules showing severely damaged spermatogenesis. Tubules are lined by only a single layer of cells. Blood vessels are congested and dilated in places. HE x 160.
It is well established that at the age of 21 days in rats (weanling stage) only spermatogonia and spermatocytes are present, and at 55 days of age, stage VII of spermatogenesis, similar to adults, is reached (5). Thereafter, at 90 days of age, the testis reaches full maturity (6). With this background the exposure duration for Cr 6+ was chosen to investigate its effect on the maturation of male gonads. The results show that Cr 6+ administration in graded daily doses intraperitoneally in weaned rats from weaning until 55 and 90 days of age produced
Chromium-induced testicular degeneration
•
D. K. SAXEr~AET gL.
.D 0
160'
SDH
40-
O~g
0
FI C*rS-3mg/kg
80
rl O
U o's 0
C~'fi--ling/kg [ ] C*r6--2mg/ kg
120
(+)
227
G6PDH
9o,i
55d
LSSd
l
90d
55d
I
LDH 40'
0""
(-) 80'
U n 0
U 0
120 Fig. 6. Effect of Cr6÷ administration (up to 55 and 90 days of age) on certain testicular enzymes in rats. Values expressed as percent changes compared with controls --- SE.* Changes are significant compared with (a) controls, (b) 1 mg/kg Cr6+, and (c) 2 mg/kg Cr6÷ . SDH = sorbitol dehydrogenase, G6PDH = glucose-6-phosphate dehydrogenase, GGT = ~/-glutamyltranspeptidase, and LDH = lactic dehydrogenase.
dose-dependent enzymatic and pathologic alterations. The metal content in the testis also increased in a similar manner. Maximum degrees of enzymatic alteration together with marked pathologic changes at 90 days indicated the involvement of mature spermatogenetic cells, which consequently resulting in reduced testicular weight, sperm counts, and motility. Changes in the activity of SDH, LDH, GGT, and G6PD have been reported to be early biochemical indicators of testicular injury. SDH and LDH enzymes reflect germ cell damage (15, 16). G6PD is needed for steroid biosynthesis (17). Similarly, GGT enzyme activity is reported to parallel the pattern of Sertoli cell
replication and maturation (18, 19), which has been shown to be affected by various xenobiotics like di(2-ethylhexyl) phthalate (20), gossypol (21), sodium selenite (16), and manganese (22). It may take a longer duration of exposure with this dose regimen to produce pathologic changes that are dose dependent at 90 days. Since the extent of enzymatic alteration, together with the dose- and duration-dependent pathologic changes, correspond with the increasing Cr accumulation in the blood and testes of treated rats, the possibility that the metal accumulated in the testicular tissue produces enzymatic and pathologic changes cannot be ruled out. The observed decrease in body weight gain in the
Table 2. Chromium level in different tissues of rats Group 1
55 days Group 2
Group 3
Group 4
Group 1
90 days Group 2 Group 3
Group 4
Blood (ixg/mL)
0.039 --- 0.007
0.74 --- 0.02 (a*)
1.09 + 0.02 (ab*)
1.82 ___ 0.19 (abc*)
0.04 --- 0.004
1.46 ___ 0.13 (a*)
2.95 --- 0.13 (ab*)
4.19 ___ 0.47 (abc*)
Testes (p.g/g f.w.)
0.015 ___ 0.005
0.14 -- 0.008 (a*)
0.23 __ 0.01 (ab*)
0.33 -- 0.01 (abc*)
0.03 -+ 0.007
0.54 --- 0.05 (a*)
0.96 -+ 0.03 (ab*)
2.93 __ 0.04 (abc*)
All the values represent the mean __. SE of 6 animals per group. Group 1 = saline control, Groups 2, 3, and 4 were given 1, 2, and 3 mg/kg/day Cr6+ ip, respectively. Statistical significance evaluated by using one-way ANOVA. f.w. = fresh weight. a --- compared with group 1; b = compared with group 2; c = compared with group 3. *P < 0.05.
228
Reproductive Toxicology
highest dosed rats in the present study, indicating the possibility of general metal toxicity, led us to consider whether these testicular changes are the direct effect of the metal on differentiating germ cells or are mediated through disturbances in gonadotropic hormones. Our study also showed Leydig cell atrophy and reduced Leydig cell nuclear diameter besides disturbed G6PD enzyme activity, indicating the possibility of disturbed androgen biosynthesis. Several other metals, such as lead and cadmium, have also been reported to affect the testis by interfering with hormone synthesis and in various intermediate pathways (23, 24). Further studies are being conducted to ascertain the role of various hormones in Cr6+-induced testicular dystrophy. In conclusion, the present study showed a risk to the growing testes of rats exposed to Cr 6+ during the prepubertal stage of development, which may disturb normal testicular physiology at adulthood.
- - The authors are thankful to the Director, Industrial Toxicology Research Centre, Lucknow, India, for interest in the study. Thanks are also due to Miss L. Sajnani for technical assistance and to Mr. G.M. Nair for his secretarial assistance.
Acknowledgments
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Volume 4, Number 3, 1990 7. Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann Acad Sci. 1952;55: 548-73. 8. Saxena DK, Murthy RC, Singh C., Chandra SV. Zinc reduces testicular injury induced by concurrent exposure to cadmium and lead in rats. Res Comm Chem Pathol Pharmacol. [In press]. 9. Dunnick JK, Harris MW, Chapin RE, Hall LB, Lamb JC. Reproductive toxicology of methyldopa in male F 344/N rats. Toxicology. 1986;41:305-18. 10. Shivanandappa T, Krishnakumari MK. Hexachlorocyclohexaneinduced testicular dysfunction in rats. Acta Pharmacol Toxicol. 1983;52:12-17. 11. Lthr GW, Waller HD. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis. New York: Academic Press; 1965:744-51. 12. Kornberg A. Lactic dehydrogenase of muscle. In: Colowick SP, Kaplan NO, eds. Methods in enzymology, vol. 1. New York: Academic Press; 1955:441-3. 13. Gerlach U. Sorbitol dehydrogenase. In: Bergmeyer J, Grosse M, eds. Methods of enzymatic analysis, vol. 3. Florida. Basel: Verlag Chemie Weinheim; 1983:352-6. 14. Roomi MW, Goldberg DM. Comparison of glutamyl transferase induction by phenobarbital in rat, guinea pig and rabbit. Biochem. Pharmacol. 1981;30:1563-71. 15. Bishop DW. Sorbitol dehydrogenase in relation to spermatogenesis and fertility. J Reprod Fertil. 1968;17:410-11. 16. Nebbia C, Brando C, Burdino E, Rasero R, Valenza F, Arisio R, Vgazio G. Effect of the chronic administration of sodium selenite on rat testes. Res Comm Chem Pathol Pharmacol. 1987;58: 183-97. 17. McKerns K. The pentose-phosphate pathway, steroidogenesis and protein synthesis. Biochem Biophys Acta. 1965;100:612-15. 18. Hodgeu GD, Sherins RJ. Enzymes as markers of testicular growth and development in the rat. Endocrinology. 1973;93:985-9. 19. Sherins RJ, Hodgen GD. Testicular gamma-glutamyl trans-peptidase: an index of Sertoli cell function in man. J. Reprod Fertil. 1976;48:191-3. 20. Oishi S. Testicular atrophy induced by di(2-ethylhexyl) phthalate: changes in histology, cell specific enzyme activities and zinc concentrations in rat testis. Arch Toxicol. 1986;59:290-5. 21. Olgiati KL, Hoffer AP, Toscano WA Jr. Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats. Biol Reprod. 1984;31:759-70. 22. Imam Z, Chandra SV. Histochemical alterations in rabbit testis produced by manganese chloride. Toxicol Appl Pharmacol. 1975;32: 534-44. 23. Wiebe JP. Effect of lead on testicular endocrinology in the developing male. Am Zool. 1981;20:899. 24. Zylber-Haran EA, Gershman H, Rosenmann E, Spitz IM. Gonadotropin, testosterone and prolactin interrelationships in cadmiumtreated rats. J Endocrinol. 1982;92:123-30.