Blockade of PKCdelta proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death.

The use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 microM MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase Cdelta (PKCdelta), resulting in persistently increased kinase activity. The proteolytic activation of PKCdelta was suppressed by treatment with 100 microM Z-VAD-FMK and 100 microM Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKCdelta. Pretreatment with 100 microM Z-DEVD-FMK and 5 microM rottlerin (a PKCdelta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKCdelta(K376R) mutant or the caspase cleavage resistant PKCdelta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKCdelta in MMT-induced dopaminergic degeneration and suggest that PKCdelta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.

2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induces oxidative stress in the epididymis and epididymal sperm of adult rats.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is one of the most potent environmental contaminants, which has been shown to induce oxidative stress in testis and epididymal sperm of rats. However, the nature and mechanism of action of TCDD on the epididymis is not clear. The aim of the present study was to investigate whether induction of oxidative stress in epididymal sperm was direct effect of TCDD on epididymis. In the present studies, TCDD (0.1, 1.0 and 10 micro g/kg body weight per day) was administered orally to rats for 4 days. Twenty-four hours after the last treatment the animals were killed using anesthetic ether. Both epididymides were dissected out and epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F-12 medium at 35 degrees C. The epididymal sperm and caput, corpus and cauda epididymides were homogenized and used for biochemical studies. Epididymal sperm counts did not decrease in the rats treated with TCDD. Administration of TCDD increased the production of reactive oxygen species such as hydrogen peroxide while the activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were found to be decreased in the epididymal sperm as well as in cauda epididymides. Lipid peroxidation also increased in the epididymal sperm and in the various regions of the epididymides after exposure to TCDD. The results indicated that TCDD induces oxidative stress in the epididymis and epididymal sperm by decreasing the antioxidant enzymes through induction of reactive oxygen species. Thus, the adverse effects of TCDD on the epididymal sperm were due to direct effect of TCDD on epididymis.

Induction of oxidative stress by bisphenol A in the epididymal sperm of rats.

Bisphenol A has been shown to affect the reproduction of male rats and mice. However, the mechanism of action of bisphenol A on the epididymal sperm is not elucidated. The present study was undertaken to evaluate the effect of bisphenol A on the antioxidant system of rat epididymal sperm. Bisphenol A was administered orally to male rats at the dose levels of 0.2, 2 and 20 microg/Kg body weight per day for 45 days. After 24 h of the last treatment, rats were weighed and killed using anesthetic ether. The body weight of treated rats did not show significant change as compared with the corresponding control groups. In bisphenol A-treated rats there was a significant decrease in the weight of the testis and epididymis; the weight of ventral prostate increased significantly whereas there was no significant change in the weight of seminal vesicles as compared with the corresponding group of control animals. Sperm collected from the epididymis were used for sperm count and biochemical estimations. Administration of bisphenol A caused a reduction in the epididymal sperm motility and sperm count in a dose-dependent manner. The activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were decreased while the levels of H(2)O(2) and lipid peroxidation increased significantly in the treated rats as compared with the corresponding group of control animals. The results suggested that graded doses of bisphenol A elicit depletion of antioxidant defence system and induce oxidative stress in epididymal sperm of rats. In conclusion, the adverse effect of bisphenol A on male reproduction may be due to induction of oxidative stress in sperm.

Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor.

Methoxychlor is one of the environmental contaminants that has been shown to induce reproductive abnormalities in male rats. The mechanism of action of methoxychlor on the male reproductive system remains unclear. In the present study we have sought to investigate whether short-term administration of methoxychlor induces oxidative stress in the testis of adult rats. Methoxychlor (50, 100, or 200 mg/kg body weight per day) was administered orally for 1, 4, or 7 days. The animals were killed using anesthetic ether on the day following the last dosing. The weights of epididymides, seminal vesicles, and ventral prostate decreased after 50, 100, or 200 mg/kg per day for 7 days but remained unchanged after 1 and 4 days of treatment. The production of superoxide anion and hydrogen peroxide increased in the animals that received methoxychlor for 4 and 7 days. The activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased, while the level of lipid peroxidation increased in the testis after 4 or 7 days of treatment. The results indicated that short-term exposure to methoxychlor induces oxidative stress in the testis by decreasing antioxidant enzymes and increasing lipid peroxidation, possibly by inducing reactive oxygen species. In conclusion, the adverse effect of methoxychlor on the male reproduction could be due to induction of oxidative stress in testis.

Effects of vitamin E on reactive oxygen species-mediated 2,3,7,8-tetrachlorodi-benzo-p-dioxin toxicity in rat testis.

This study was undertaken to investigate whether treatment with vitamin E protects rat testis from oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Male rats of Wistar strain were administered TCDD at doses of 1, 10 and 100 ng kg(-1) body wt. day(-1) for 45 days. Other groups of animals were co-administered TCDD (1, 10 and 100 ng kg(-1) body wt. day(-1)) and vitamin E (20 mg kg(-1) body wt. day(-1)) for 45 days. Animals administered TCDD and those co-administered TCDD and vitamin E did not show any significant change in body weight. Administration of TCDD decreased the weights of the testis, epididymis, seminal vesicles and ventral prostate. The daily sperm production decreased in the animals administered TCDD from the control values of 22.19 +/- 2.67 to 13.10 +/- 3.16 x 10(6). There was a significant decline in the activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase with concomitant increased levels of hydrogen peroxide and lipid peroxidation. Co-administration of TCDD and vitamin E did not show any significant changes in the weights of the testis, epididymis, seminal vesicles and ventral prostate. The daily sperm production remained unchanged in the animals co-administered TCDD and vitamin E. The activities of antioxidant enzymes and the levels of hydrogen peroxide and lipid peroxidation did not change in the animals co-administered TCDD and vitamin E. The results suggested that administration of TCDD induces oxidative stress in testis, and vitamin E could impart a protective effect against TCDD-induced oxidative stress.

Effect of nonylphenol on the antioxidant system in epididymal sperm of rats.

Nonylphenol, an environmental contaminant, has been shown to induce reproductive abnormalities in male rats. The nature and mechanism of action of nonylphenol on the epididymal sperm has not been elucidated. In the present study we have sought to investigate whether administration of nonylphenol induces oxidative stress in rat epididymal sperm. Nonylphenol was administered orally to male rats at 1, 10 and 100 microg/kg body weight per day for 45 days. Twenty-four hours after the last treatment, rats were weighed and killed using anaesthetic ether. The body weight of the animals treated with nonylphenol did not show any significant change. The weights of the testes and epididymides decreased significantly whereas the weights of seminal vesicles and ventral prostate remained unchanged at all doses of nonylphenol in treated rats. Epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F-12 medium at 32 degrees C. Administration of nonylphenol decreased the epididymal sperm counts in a dose-dependent manner. The activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased significantly while the levels of H(2)O(2) generation and lipid peroxidation increased significantly in the animals treated with nonylphenol when expressed in terms of milligram protein and milligram DNA. The activity of alpha-glucosidase, a negative control against antioxidant enzymes, in the sperm of nonylphenol-treated rats did not show any significant change at any of the doses. The results suggest that graded doses of nonylphenol elicit depletion of antioxidant defence system in sperm, indicating nonylphenol-induced oxidative stress in the epididymal sperm of rats.

Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis.

Methoxychlor, an environmental contaminant, which is widely used as a pesticide in many countries, has been shown to induce reproductive abnormalities in male rats. The precise nature and mechanism of action of methoxychlor on the male reproductive system is not clear. In the present study, we have sought to investigate the induction of oxidative stress in the testis of rat after exposure to methoxychlor. Methoxychlor (1, 10, and 100 mg kg(-1) body weight per day) was administered orally to the rats for 45 days. After 24 h of the last treatment the animals were killed using anesthetic ether. The body weight of the animals administered with methoxychlor did not show any significant change. The weights of the testis, epididymis, seminal vesicles and ventral prostate decreased significantly in 100 mg dose but remained unchanged in 1 and 10 mg doses. Mitochondrial and microsome-rich fractions of the testis were obtained by the method of differential centrifugation. The activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased significantly in the animals treated with methoxychlor in a dose-dependent manner in the mitochondrial and microsome-rich fractions of rat testis. The levels of hydrogen peroxide generation (H(2)O(2)) and lipid peroxidation increased in mitochondrial and microsome-rich fractions of the testis of the rats treated with methoxychlor. The results suggested that the low to medium doses of methoxychlor elicit depletion of antioxidant enzymes and concomitant increase in the levels of H(2)O(2) and lipid peroxidation differentially in mitochondrial and microsome-rich fractions of rat testis. In conclusion, the adverse effect of methoxychlor on male reproduction could be due to the induction of oxidative stress in testis.

The effect of methoxychlor on the epididymal antioxidant system of adult rats.

Methoxychlor is widely used as a pesticide in many countries and has been shown to induce reproductive abnormalities in male rats, causing reduced fertility. The mechanism of action of methoxychlor on the male reproductive system is not clear. In the present study we investigated whether administration of methoxychlor induces oxidative stress in the epididymis and epididymal sperm of adult rats. Methoxychlor (50, 100, or 200 mg/kg body weight/day) was administered orally for 1, 4, or 7 days. The animals were killed using anesthetic ether 24 h after of the last treatment. Epididymal sperm were collected by cutting the epididymis into small pieces in Ham's F-12 medium at 35 degrees C. The body weight and weights of the testis, liver, and kidney did not show any significant changes in the methoxychlor-treated rats. The weight of the epididymis, seminal vesicles, and ventral prostate as well as epididymal sperm counts decreased after 50, 100, or 200 mg/kg/day for 7 days but remained unchanged after shorter courses of treatment. Epididymal sperm motility was decreased in a dose-dependent manner in the animals treated with methoxychlor for 4 or 7 days. The activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase were decreased while the levels of hydrogen peroxide and lipid peroxidation were increased in the epididymal sperm as well as in the caput, corpus, and cauda epididymis after 4 or 7 days of treatment. The activities of superoxide dismutase decreased while the levels of lipid peroxidation increased in the liver but not in the kidney in all groups. Co-administration of the antioxidant vitamin E (20 mg/kg body weight/ day) to the 200 mg/kg/d methoxychlor-treated rats for 7 days prevented significant changes in the antioxidant systems in the epididymis and epididymal sperm and prevented alterations in sperm counts and motility. The results indicated that methoxychlor induces oxidative stress in the epididymis and epididymal sperm by decreasing antioxidant enzymes, possibly by inducing reactive oxygen species. In conclusion the adverse effect of methoxychlor on the male reproduction could be due to induction of oxidative stress.

Induction of oxidative stress in rat epididymal sperm after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The ability of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) to induce oxidative stress in various tissues of animals has been reported. The nature and mechanism of action of TCDD on the antioxidant system of sperm has not been studied. In the present study we have sought to investigate whether TCDD induces oxidative stress in the epididymal sperm of rats. Subchronic doses of TCDD (1, 10, and 100 ng/kg body weight per day) were administered orally to male Wistar strain rats for 45 days. After 24 h of the last treatment the rats were killed using diethyl ether. The epididymides were removed and cleared from the adhering tissues. Epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F12 medium, and counted using a hemocytometer. The epididymal sperm counts in the TCDD-treated groups decreased in a dose-dependent manner from the control value of 8.2+/-0.14 x 10(8) to 5.31+/-0.15 x 10(8). Since a positive correlation (r=0.95; n=24) was observed between sperm count and DNA content of the epididymal sperm, DNA content was routinely used as an indicator of sperm count, and the results were expressed in terms of both protein and DNA. There was a significant decline in the activities of superoxide dismutase (40+/-2.17 to 27.1+/-0.76/mg protein and 32.41 to 18.07+/-0.76/mg DNA), catalase (2.49+/-0.13 to 2.03+/-0.05/mg protein and 2.01+/-0.05 to 1.35+/-0.05/mg DNA), glutathione reductase (71.2+/-3.87 to 48+/-1.79/mg protein and 57.58+/-1.52 to 31.94/mg DNA) and glutathione peroxidase (22.4+/-1.43 to 16.9+/-1.57/mg protein and 18.08+/-0.61 to 11.38+/-1.22/mg DNA) while there were increases in the levels of hydrogen peroxide (20.8+/-1.96 to 55.3+/-0.88/ mg protein and 16.18+/-1.88 to 36.87+/-0.88/ mg DNA) and lipid peroxidation (2.17+/-0.2 to 6.08/mg protein and 1.75+/-0.12 to 4.05+/-0.12/mg DNA) in the epididymal sperm. The results suggest that graded doses of TCDD elicit depletion of antioxidant defense system in sperm, indicating TCDD-induced oxidative stress in the epididymal sperm. In conclusion, the adverse effect on male reproduction in TCDD-treated rats may be due to the induction of oxidative stress in sperm.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the antioxidant system in mitochondrial and microsomal fractions of rat testis.

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce oxidative stress in hepatic and some extrahepatic tissues of animals has been reported. The precise nature and mechanism of action of TCDD on the male reproductive system is not clear. In the present study, we have investigated the induction of oxidative stress in the testis of rat after exposure to low doses of TCDD. TCDD (1, 10, and 100 ng/kg body weight per day) was administered orally to the rat for 45 days. After 24 h of the last treatment the rats were killed using anesthetic ether. The weights of the testis, epididymis, seminal vesicles and ventral prostate decreased while the body weight remained unchanged in the rats administered with TCDD. Mitochondrial and microsomal fractions of the testis were obtained by the method of differential centrifugation. The activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase decreased significantly in the animals treated with TCDD in a dose-dependent manner in the mitochondrial and microsomal fractions of rat testis. The levels of hydrogen peroxide generation (H(2)O(2)) and lipid peroxidation increased in mitochondrial, and microsomal fractions of the testis. The results suggested that the low doses of TCDD elicit depletion of antioxidant enzymes and concomitant increase in the levels of H(2)O(2) and lipid peroxidation differentially in mitochondrial and microsomal fractions of rat testis. In conclusion the adverse effect of TCDD on male reproduction could be due to induction of oxidative stress.

Effect of lindane on antioxidant enzymes in epididymis and epididymal sperm of adult rats.

AIM: To find out the changes induced by lindane on the antioxidant enzymes in epididymis and epididymal sperm of adult rats. METHODS: Adult male rats were orally administered lindane at a dose of 5.0 mg/kg body weight per day for 30 days. At the end of the treatment, the rats were sacrificed. The epididymis was removed and weighed and sperm were collected for sperm count, motility and biochemical studies. A 1% homogenate of epididymis was prepared and used for biochemical estimations. RESULTS: In lindane-treated rats, there were significant reductions in the epididymal weight, epididymal sperm count and motility compared with the controls. Significant decreases in the superoxide dismutase (SOD), catalase, glutathione reductase and glutathione peroxidase activities and significant increases in the H2O2 generation and lipid peroxidation were also observed in the epididymis and epididymal sperm of lindane-treated rats. CONCLUSION: Lindane decreases the levels of antioxidant enzymes in the epididymis and epididymal sperm of adult rats thereby inducing oxidative stress.

Effect of lindane on testicular antioxidant system and steroidogenic enzymes in adult rats.

AIM: To find out the effect of lindane on testicular antioxidant system and testicular steroidogenesis in adult male rats. METHODS: Adult male rats were orally administered with lindane at a dose of 5.0 mg/kg body weight per day for 30 days. Twenty-four hours after the last treatment the rats were killed using anesthetic ether. Testes, epididymis, seminal vesicles and ventral prostate were removed and weighed. A 10% testicular homogenate was prepared and centrifuged at 4 degrees C. The supernatant was used for various biochemical estimations. RESULTS: The body weight and the weights of testes, epididymis, seminal vesicles and ventral prostate were reduced in lindane-treated rats. There was a significant decline in the activities of antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione reductase while an increase in hydrogen peroxide (H2O2) generation was observed. The specific activities of testicular steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase were decreased. The levels of DNA, RNA and protein were also decreased in lindane-treated rats. CONCLUSION: Lindane induces oxidative stress and decreases antioxidant enzymes in adult male rats.

Chronic effect of endosulfan on the testicular functions of rat.

AIM: To find out the toxic effect of endosulfan on the testicular function of pubertal rats. METHODS: Male rats of pubertal age were orally administered endosulfan at a dose of 1.0 mg/kg body weight for 30 days. Twenty-four hours after the last treatment, the rats were sacrificed and the testis, epididymis, seminal vesicles and ventral prostate were removed and weighed. A 10% testicular homogenate was prepared for biochemical estimations. RESULTS: In endosulfan-treated rats, there were a reduction in the body weight and the weights of testis and accessory sex organs, a decrease in the testicular lactate and pyruvate activities, and in the testicular DNA and RNA concentrations, whereas the testicular protein concentration was slightly increased; the specific activity of testicular steroidogenic enzyme, 3beta-OH-steroid dehydrogenase and the ascorbic acid level were decreased, which were correlated with a decrease in steroidogenesis. The lysosomal enzyme acid phosphatase and brush-border enzyme alkaline phosphatase activities were also decreased in the testis of treated rats. CONCLUSION: In pubertal rats, endosulfan treatment inhibits the testicular functions.