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Manganese induced testicular changes in monkeys
Exp. Path. 18, 240-244 (1980) Industrial Toxicology Research Centre, Lucknow, India
Manganese induced testicular changes in monkeys By R. C. MURTHY, R. S. SRIVASTAVA, S. K. GUPTA, and S. V. CHANDRA With 5 figures (Received July 26, 1979)
Address for correspondence: Dr. S. V. CHANDRA, Scientist, Industrial Toxicology Research Centre, Post Box No. 80, Lucknow - 226001, India Key words: manganese chloride; seminiferous tubules; testis, metal induced damage; monkey
Summary Oral administration of manganese chloride (25 mg/kg b. w. daily) to monkeys for a period of 18 months produced congestion and marked increase in weight of testis. Histopathologic examination revealed interstitial oedema and degeneration of seminiferous tubules. Activities of succinic dehydrogenase, glucose-6-phosphate dehydrogenase and acid phosphatase were significantly inhibited whereas NADH-diaphorase and alkaline phosphatase activities showed only slight inhibition in seminiferous tubules of treated monkeys. It was concluded that chronic exposure to manganese does not produce severe degenerative changes in the testis earlier than metal induced encephalopathy in primates.
Manganese has a wide variety of industrial applications such as manufacture of ferromanganese alloy, dry cell batteries, calico printing, in the manufacture of wood preservatives and fertilizers (TOLONEN 1972). The workers engaged in these industries are liable to suffer from neurological disorders, the symptoms of which resemble parkinsonism (RODIER 1955). Manganese also produces biochemical and morphological changes in the brain of experimental animals (MUSTAFA and CHANDRA 1971; CHANDRA and SHUKLA 1978). Besides, this metal is injurious to testicular tissue resulting in infertility in animals (CHANDRA 1971; CHANDRA et al. 1973). Testicular injury occurs much earlier than the symptoms of encephalopathy in rodents. It is, therefore, of significance to study manganese induced testicular changes in higher mammals so that the derangement in the function of male reproductive organs may be predicted in manganese exposed humans.
Material and Methods Treatment: Eight male adult rhesus monkeys (Macaca mulatta) weighing on an average 5 kg were procured from the local animal supplier and divided into 2 equal groups. They were housed in individual cages and provided with monkeys pellets (Hind Lever Ltd.), 2 bananas each per day and water ad libitum. 4 monkeys of group I were given orally 25 mg/kg MnOI 2 • 4H 2 0 daily dissolved in 1 ml of physiological saline for a period of 18 months and 4 monkeys of group II received 1 ml of physiological saline in an identical manner for the same period. After 18 months the animals were sacrificed after pentobarbitone anaesthesia, their testis removed, weighed and processed for detailed histological and histochemical studies. Small pieces of unfixed testis were mounted on microtome block holders at -25°C and sections of 6 - 8,um were cut in cryostat, mounted on slides and air dried at room temperature. The activity of succinic dehydrogenase (NACHLAS et al. 1957) (SDH, EC 1.3.99.1), Glucose-6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49) and NADH-diaphorase (NADHD, EC 1.6.4.3) were determined (PEARSE 1972). Controls for each enzyme were run simultaneously by incubating sections in solutions without respective substrates. Small pieces of tissues were fixed in 1 % cold calcium for mol for 16 hours at 4°C. Cryostat sections were cut at 6-8,um and incubated for acid phosphatase (GOMORI 1939) (AP, EO 3.1.3.2) and alkaline phosphatase (GOMORI 1941) (ALP, EO 3.1.3.1). The remaining pieces of testis were fixed in 10 % neutral buffered formalin, embedded in paraffin, cut at 6,um and stained with hematoxylin eosin.
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Fig. 1. Testis of a control monkey showing normal structure of seminiferous tubules and interstitial tissue. HE, x 205.
Results None of the animals died during the experiment. On gross examination testes of experimen tal monkeys were congested and swollen. Weight of testes from control monkeys was 11-13 g whereas those from the treated monkeys weighed 19-21 g showing marked increase in the weight of testis in experimental animals. Examination of sections from normal animals revealed an orderly arrangement of germ cells in seminiferous tubules and sparsely cellular strands of interstitial Leydig cells (fig. 1). Testis of monkeys after 18 months of manganese treatment revealed marked interstitial oedema (fig. 2a) and degenerative changes in the groups of seminiferous tubules (fig. 2b). At places these tubules were lined only by a single layer of cells consisting of Sertoli cells and spermatogonia. Degeneration was observed in about 40 % of the tubules. Leydin cells did not show any abnormality. Histochemistry Succinic dehydrogenase: Moderate SDH activity was distributed throughout the seminiferous tubules and interstitial cells in normal monkeys (fig. 3a). Testes of treated monkeys showed marked loss of enzyme activity in groups of seminiferous tubules (fig. 3 b). Glucose 6-phosphate dehydrogenase : G-6-PD activity was demonstrated in tubular epithelium and interstitial cells of normal monkeys (fig. 4a). Marked loss of G-6-PD activity was observed in the interstitial tissue and groups of seminiferous tubules (fig. 4 b). NADH-diaphorase: The activity of this enzyme was mainly distributed in seminiferous tubules and some activity was seen in interstitial tissue in normal animals. Slight loss was observed in manganese treated animals. 241
Fig. 2a, b. Testis of a monkey treated with MnCI 2 • 4H 2 0 (25 mg/kg) for 18 months. (a) showing interstitial oedema. (b) showing degenerating seminiferous tubules. HE, x 145. Fig. 3 a. Testis of control monkey showing distribution of SDH activity. x 145.3 b. Testis of manganese treated monkey showing marked reduction in the SDH activity from the tubules. x 145.
Fig.4a. Testis of control monkey showing distribution of G-6-PD activity. x 145. 4b. Testis of manganese treated monkey showing marked reduction in the G-6-PD activity from the interstitial tissue and tubules. x 145. Fig. 5 a. Testis of control monkey showing distribution of acid phosphatase activity. x 145. 5 b. Testis of manganese treated monkey showing appreciable loss of acid phosphatase activity from the tubules. x 145.
Acid phosphatase: AP activity was demonstrated in granular form in epithelial cells of seminiferous tubules in normal monkeys (fig.5a). In treated animals, marked loss of enzyme activity was visible in groups of seminiferous tubules (fig. 5 b). Alkaline phosphatase: ALP activity was uniformly distributed in the cytoplasm and basement membrane in normal animals whereas in treated monkeys the activity was slightly reduced.
Discussion A single intratracheal injection of Mn0 2 to rabbits produced marked destruction and calcification in the testicular tissue at 8 months post inoculation (CHANDRA et al. 1973). Chronic manganese administration to the monkeys for a period of 18 months, in the present study have produced relatively mild changes in the seminiferous epithelium of some of the tubules while more than half of the testicular tissue remained unaffected. Interstitial oedema appears to be responsible for the increase in the weight of the testis in treated monkeys, however, such a change has not been noticed in our previous studies with manganese (IMAN and CHANDRA 1975). It may be possible that in higher mammals manganese produces oedema in the interstitial tissue by a similar mechanism as in cadmium induced testicular oedema (JOHNSON et al. 1970). Loss in the enzyme activity in the groups of seminiferous tubules of manganese treated monkeys appears to be due to the degenerative changes in the cells of seminiferous epithelium. Normal activity of most of the enzymes is noticed in the tubules which have not undergone necrotic changes. The manganese treated monkeys, in our investigation, had developed symptoms of neurological syndrome at the time of sacrifice (unpublished data) indicating that the testicular dysfunction resulting in sterility may not occur earlier than manganese induced encephalopathy in higher mammals. It may be possible that further exposure to manganese may also produce infertility in monkeys. Acknowledgement The authors gratefully acknowledge the technical assistance of Mr. G. Husain and Mr. Bachchu Lal.
Literature CHANDRA, S. V., Cellular changes induced by manganese in the rat testis - preliminary studies. Acta, Pharmacol. Toxico!. 29, 75-80 (1971). - R. ARA, N. NAGAR and P. K. SETH, Sterility in experimental manganese toxicity. Acta bioI. med. germ. 30,857 -862 (1973). - and G. S. SHUKLA, Manganese encephalopathy in growing rats. Environ. Res. 10, 28-37 (1978). GOMORI, G., Microtechnical demonstration of alkaline phosphatase in tissue sections. Proc. Soc. Expt. Bio!. Med. 42,23-28 (1939). - Distribution of acid phosphatase in tissues under normal and pathological conditions. Arch. Pathol. 32, 189-199 (1941). IMAM, Z., and S. V. CHANDRA, Histochemical alterations in rabbit testis produced by manganese chloride. Toxicol. Appl. Pharmacol. 32, 534-544 (1975). JOHNSON, A. D., W. R. GOMES and N. 1. VANDEMARK, The Testis, vol. III. Academic Press, New York and London 1970. MUSTAFA, S. J., and S. V. CHANDRA, Levels of 5-hydroxy-tryptamine dopamine and norepinephrine in whole brain of rabbits in chronic manganese toxicity, J. Neurochem. 18, 931-933 (1971). NACHLAS, M. M., K. C. Tsou, E. DE SOUZA, C. S. CHENG and A. M. SELIGMAN, Cytochemical demonstration of succinic dehydrogenase by the use of new p-nitrophenyl substituted diazole. J. Histochem. Cytochem. 0, 420-436 (1957). PEARSE, A. G. E., Histochemistry, Theoretical and Applied, Vol. II, 3rd ed. Churchill Ltd., London 1972. RODIER, J., Manganese poisoning in Moroccan miners,.Brit. J. Ind. Med. 12, 21-35 (1955). TOLONEN, M., Industrial toxicology of manganese, Work. Environ. Health 9, 53-60 (1972).
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Manganese induced testicular changes in monkeys By R. C. MURTHY, R. S. SRIVASTAVA, S. K. GUPTA, and S. V. CHANDRA With 5 figures (Received July 26, 1979)
Address for correspondence: Dr. S. V. CHANDRA, Scientist, Industrial Toxicology Research Centre, Post Box No. 80, Lucknow - 226001, India Key words: manganese chloride; seminiferous tubules; testis, metal induced damage; monkey
Summary Oral administration of manganese chloride (25 mg/kg b. w. daily) to monkeys for a period of 18 months produced congestion and marked increase in weight of testis. Histopathologic examination revealed interstitial oedema and degeneration of seminiferous tubules. Activities of succinic dehydrogenase, glucose-6-phosphate dehydrogenase and acid phosphatase were significantly inhibited whereas NADH-diaphorase and alkaline phosphatase activities showed only slight inhibition in seminiferous tubules of treated monkeys. It was concluded that chronic exposure to manganese does not produce severe degenerative changes in the testis earlier than metal induced encephalopathy in primates.
Manganese has a wide variety of industrial applications such as manufacture of ferromanganese alloy, dry cell batteries, calico printing, in the manufacture of wood preservatives and fertilizers (TOLONEN 1972). The workers engaged in these industries are liable to suffer from neurological disorders, the symptoms of which resemble parkinsonism (RODIER 1955). Manganese also produces biochemical and morphological changes in the brain of experimental animals (MUSTAFA and CHANDRA 1971; CHANDRA and SHUKLA 1978). Besides, this metal is injurious to testicular tissue resulting in infertility in animals (CHANDRA 1971; CHANDRA et al. 1973). Testicular injury occurs much earlier than the symptoms of encephalopathy in rodents. It is, therefore, of significance to study manganese induced testicular changes in higher mammals so that the derangement in the function of male reproductive organs may be predicted in manganese exposed humans.
Material and Methods Treatment: Eight male adult rhesus monkeys (Macaca mulatta) weighing on an average 5 kg were procured from the local animal supplier and divided into 2 equal groups. They were housed in individual cages and provided with monkeys pellets (Hind Lever Ltd.), 2 bananas each per day and water ad libitum. 4 monkeys of group I were given orally 25 mg/kg MnOI 2 • 4H 2 0 daily dissolved in 1 ml of physiological saline for a period of 18 months and 4 monkeys of group II received 1 ml of physiological saline in an identical manner for the same period. After 18 months the animals were sacrificed after pentobarbitone anaesthesia, their testis removed, weighed and processed for detailed histological and histochemical studies. Small pieces of unfixed testis were mounted on microtome block holders at -25°C and sections of 6 - 8,um were cut in cryostat, mounted on slides and air dried at room temperature. The activity of succinic dehydrogenase (NACHLAS et al. 1957) (SDH, EC 1.3.99.1), Glucose-6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49) and NADH-diaphorase (NADHD, EC 1.6.4.3) were determined (PEARSE 1972). Controls for each enzyme were run simultaneously by incubating sections in solutions without respective substrates. Small pieces of tissues were fixed in 1 % cold calcium for mol for 16 hours at 4°C. Cryostat sections were cut at 6-8,um and incubated for acid phosphatase (GOMORI 1939) (AP, EO 3.1.3.2) and alkaline phosphatase (GOMORI 1941) (ALP, EO 3.1.3.1). The remaining pieces of testis were fixed in 10 % neutral buffered formalin, embedded in paraffin, cut at 6,um and stained with hematoxylin eosin.
240
Fig. 1. Testis of a control monkey showing normal structure of seminiferous tubules and interstitial tissue. HE, x 205.
Results None of the animals died during the experiment. On gross examination testes of experimen tal monkeys were congested and swollen. Weight of testes from control monkeys was 11-13 g whereas those from the treated monkeys weighed 19-21 g showing marked increase in the weight of testis in experimental animals. Examination of sections from normal animals revealed an orderly arrangement of germ cells in seminiferous tubules and sparsely cellular strands of interstitial Leydig cells (fig. 1). Testis of monkeys after 18 months of manganese treatment revealed marked interstitial oedema (fig. 2a) and degenerative changes in the groups of seminiferous tubules (fig. 2b). At places these tubules were lined only by a single layer of cells consisting of Sertoli cells and spermatogonia. Degeneration was observed in about 40 % of the tubules. Leydin cells did not show any abnormality. Histochemistry Succinic dehydrogenase: Moderate SDH activity was distributed throughout the seminiferous tubules and interstitial cells in normal monkeys (fig. 3a). Testes of treated monkeys showed marked loss of enzyme activity in groups of seminiferous tubules (fig. 3 b). Glucose 6-phosphate dehydrogenase : G-6-PD activity was demonstrated in tubular epithelium and interstitial cells of normal monkeys (fig. 4a). Marked loss of G-6-PD activity was observed in the interstitial tissue and groups of seminiferous tubules (fig. 4 b). NADH-diaphorase: The activity of this enzyme was mainly distributed in seminiferous tubules and some activity was seen in interstitial tissue in normal animals. Slight loss was observed in manganese treated animals. 241
Fig. 2a, b. Testis of a monkey treated with MnCI 2 • 4H 2 0 (25 mg/kg) for 18 months. (a) showing interstitial oedema. (b) showing degenerating seminiferous tubules. HE, x 145. Fig. 3 a. Testis of control monkey showing distribution of SDH activity. x 145.3 b. Testis of manganese treated monkey showing marked reduction in the SDH activity from the tubules. x 145.
Fig.4a. Testis of control monkey showing distribution of G-6-PD activity. x 145. 4b. Testis of manganese treated monkey showing marked reduction in the G-6-PD activity from the interstitial tissue and tubules. x 145. Fig. 5 a. Testis of control monkey showing distribution of acid phosphatase activity. x 145. 5 b. Testis of manganese treated monkey showing appreciable loss of acid phosphatase activity from the tubules. x 145.
Acid phosphatase: AP activity was demonstrated in granular form in epithelial cells of seminiferous tubules in normal monkeys (fig.5a). In treated animals, marked loss of enzyme activity was visible in groups of seminiferous tubules (fig. 5 b). Alkaline phosphatase: ALP activity was uniformly distributed in the cytoplasm and basement membrane in normal animals whereas in treated monkeys the activity was slightly reduced.
Discussion A single intratracheal injection of Mn0 2 to rabbits produced marked destruction and calcification in the testicular tissue at 8 months post inoculation (CHANDRA et al. 1973). Chronic manganese administration to the monkeys for a period of 18 months, in the present study have produced relatively mild changes in the seminiferous epithelium of some of the tubules while more than half of the testicular tissue remained unaffected. Interstitial oedema appears to be responsible for the increase in the weight of the testis in treated monkeys, however, such a change has not been noticed in our previous studies with manganese (IMAN and CHANDRA 1975). It may be possible that in higher mammals manganese produces oedema in the interstitial tissue by a similar mechanism as in cadmium induced testicular oedema (JOHNSON et al. 1970). Loss in the enzyme activity in the groups of seminiferous tubules of manganese treated monkeys appears to be due to the degenerative changes in the cells of seminiferous epithelium. Normal activity of most of the enzymes is noticed in the tubules which have not undergone necrotic changes. The manganese treated monkeys, in our investigation, had developed symptoms of neurological syndrome at the time of sacrifice (unpublished data) indicating that the testicular dysfunction resulting in sterility may not occur earlier than manganese induced encephalopathy in higher mammals. It may be possible that further exposure to manganese may also produce infertility in monkeys. Acknowledgement The authors gratefully acknowledge the technical assistance of Mr. G. Husain and Mr. Bachchu Lal.
Literature CHANDRA, S. V., Cellular changes induced by manganese in the rat testis - preliminary studies. Acta, Pharmacol. Toxico!. 29, 75-80 (1971). - R. ARA, N. NAGAR and P. K. SETH, Sterility in experimental manganese toxicity. Acta bioI. med. germ. 30,857 -862 (1973). - and G. S. SHUKLA, Manganese encephalopathy in growing rats. Environ. Res. 10, 28-37 (1978). GOMORI, G., Microtechnical demonstration of alkaline phosphatase in tissue sections. Proc. Soc. Expt. Bio!. Med. 42,23-28 (1939). - Distribution of acid phosphatase in tissues under normal and pathological conditions. Arch. Pathol. 32, 189-199 (1941). IMAM, Z., and S. V. CHANDRA, Histochemical alterations in rabbit testis produced by manganese chloride. Toxicol. Appl. Pharmacol. 32, 534-544 (1975). JOHNSON, A. D., W. R. GOMES and N. 1. VANDEMARK, The Testis, vol. III. Academic Press, New York and London 1970. MUSTAFA, S. J., and S. V. CHANDRA, Levels of 5-hydroxy-tryptamine dopamine and norepinephrine in whole brain of rabbits in chronic manganese toxicity, J. Neurochem. 18, 931-933 (1971). NACHLAS, M. M., K. C. Tsou, E. DE SOUZA, C. S. CHENG and A. M. SELIGMAN, Cytochemical demonstration of succinic dehydrogenase by the use of new p-nitrophenyl substituted diazole. J. Histochem. Cytochem. 0, 420-436 (1957). PEARSE, A. G. E., Histochemistry, Theoretical and Applied, Vol. II, 3rd ed. Churchill Ltd., London 1972. RODIER, J., Manganese poisoning in Moroccan miners,.Brit. J. Ind. Med. 12, 21-35 (1955). TOLONEN, M., Industrial toxicology of manganese, Work. Environ. Health 9, 53-60 (1972).
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