Decrease In Succinate Dehydrogenase Activity Research Articles

Effect of ethanol on rat fetal hepatocytes: Studies on cell replication, lipid peroxidation and glutathione

Studies have shown that ethanol at moderate concentrations inhibits epidermal growth factor–dependent replication of fetal rat hepatocytes in culture. This may account for the growth/development impairment associated with fetal alcohol syndrome and decreased liver regeneration in alcoholic liver disease. In this study, we further define the mechanism(s) of the negative impact of ethanol on fetal rat hepatocytes and provide evidence that ethanol-induced injury to these cells is associated with membrane damage caused by lipid peroxidation, altered cell glutathione homeostasis and deranged mitochondrial structure and function. Exposure of fetal rat hepatocyte replication to ethanol (2 mg/ml) promptly resulted in blockade of replication, as indicated by a 40% reduction in DNA synthesis (p < 0.05). Assessment of cell injury on the basis of lactate dehydrogenase and ALT leakage indicated a statistically significant but not appreciable effect, whereas 51Cr leakage was more substantially increased (p < 0.05). Within 6 hr of ethanol exposure, superoxide radical levels increased more than twofold (p < 0.05). We noted a 56% increase in levels of diene conjugates, a 131% increase in malonaldehyde concentration and a 66% increase in fluorescent products of lipid peroxidation (all p < 0.05). Glutathione levels were decreased to 47% below control values (p < 0.05). Electron microscopic studies illustrated a slight disruption of mitochondrial structure (enlargement of mitochondria and dilation of cristae). This disruption was accompanied by mitochondrial swelling (increased permeability), altered mitochondrial membrane potential (a 16% decrease in rhodamine uptake), a 28% decrease in succinate dehydrogenase activity and a 30% decrease in cellular ATP level (p < 0.05). Pretreatment of fetal rat hepatocytes with 0.1 mmol/L N-acetylcysteine or S-adenosylmethionine for 24 hr prevented the ethanol-induced reduction of ATP and glutathione levels, essentially restored cell replication, ameliorated 51Cr leakage and decreased malonal-dehyde and diene conjugate levels to 41% to 65% and 25% above control values, respectively. Pretreatment with 0.1 mmol/L vitamin E fully normalized malonal-dehyde and diene conjugate levels and 51Cr leakage but failed to improve ATP levels or to increase significantly cell replication and glutathione levels. Concomitant administration of glutathione precursors with ethanol, rather than pretreatment, did not alter the impaired cell replication. Thus our data underscore the importance of cellular glutathione and ATP in preventing ethanol-induced decreases in fetal cell replication and suggest that alleviation of cellular lipid peroxidation alone is not sufficient to prevent this abnormality in fetal rat hepatocyte function. (HEPATOLOGY 1993;18:648–659.)

Effect of ethanol on rat fetal hepatocytes: Studies on cell replication, lipid peroxidation and glutathione

Studies have shown that ethanol at moderate concentrations inhibits epidermal growth factor-dependent replication of fetal rat hepatocytes in culture. This may account for the growth/development impairment associated with fetal alcohol syndrome and decreased liver regeneration in alcoholic liver disease. In this study, we further define the mechanism(s) of the negative impact of ethanol on fetal rat hepatocytes and provide evidence that ethanol-induced injury to these cells is associated with membrane damage caused by lipid peroxidation, altered cell glutathione homeostasis and deranged mitochondrial structure and function. Exposure of fetal rat hepatocyte replication to ethanol (2 mg/ml) promptly resulted in blockade of replication, as indicated by a 40% reduction in DNA synthesis (p < 0.05). Assessment of cell injury on the basis of lactate dehydrogenase and ALT leakage indicated a statistically significant but not appreciable effect, whereas 51Cr leakage was more substantially increased (p < 0.05). Within 6 hr of ethanol exposure, superoxide radical levels increased more than twofold (p < 0.05). We noted a 56% increase in levels of diene conjugates, a 131% increase in malonaldehyde concentration and a 66% increase in fluorescent products of lipid peroxidation (all p < 0.05). Glutathione levels were decreased to 47% below control values (p < 0.05). Electron microscopic studies illustrated a slight disruption of mitochondrial structure (enlargement of mitochondria and dilation of cristae). This disruption was accompanied by mitochondrial swelling (increased permeability), altered mitochondrial membrane potential (a 16% decrease in rhodamine uptake), a 28% decrease in succinate dehydrogenase activity and a 30% decrease in cellular ATP level (p < 0.05). Pretreatment of fetal rat hepatocytes with 0.1 mmol/L N-acetylcysteine or S-adenosylmethionine for 24 hr prevented the ethanol-induced reduction of ATP and glutathione levels, essentially restored cell replication, ameliorated 51Cr leakage and decreased malonaldehyde and diene conjugate levels to 41% to 65% and 25% above control values, respectively. Pretreatment with 0.1 mmol/L vitamin E fully normalized malonaldehyde and diene conjugate levels and 51Cr leakage but failed to improve ATP levels or to increase significantly cell replication and glutathione levels. Concomitant administration of glutathione precursors with ethanol, rather than pretreatment, did not alter the impaired cell replication. Thus our data underscore the importance of cellular glutathione and ATP in preventing ethanol-induced decreases in fetal cell replication and suggest that alleviation of cellular lipid peroxidation alone is not sufficient to prevent this abnormality in fetal rat hepatocyte function.

Use of the succinate dehydrogenase inhibition test in assessing the heat sensitivity of tumor cells.

The in vitro succinate dehydrogenase (SD) inhibition (SDI) test was used to determine the heat sensitivity of tumor cell lines and human tumor cells. The tumor cells were exposed to heat in vitro and the decrease in SD activity was assayed using a colorimetric assay, the SDI test. With respect to survival curves of HeLa cells, the SDI test correlated well with the clonogenic assay and the dye exclusion assay. Decrease in the SD activity of HeLa cells after the heat treatment (41-44 degrees C, 10 min to 5 h) depended on both the temperature and the duration of heat exposure. S- and G2/M-phase-rich HeLa cells were more sensitive to heat than were the G1-phase-rich cells. The SDI test exhibited a wide variation in the heat sensitivity among four cell lines (HeLa, B-16, V-79, and a human GT-1 squamous cell carcinoma). Variation in heat sensitivity was also detected among individual tumor tissues obtained from clinical specimens of gastric, esophageal, and colorectal cancers. Gastric cancer tissues were more sensitive to heat than were esophageal and colorectal tissues. We recommended that if the SDI test is used to assess the heat sensitivity of clinical tumor tissues in vitro, appropriate therapy for individual patients can be designed.

In vitro succinate dehydrogenase chemosensitivity of gastric carcinoma?relationship to DNA content

Relationships between in vitro chemosensitivity and cell nuclear DNA content were investigated in malignant cells from 41 patients exhibiting advanced gastric carcinoma. The chemosensitivity was evaluated by measuring the succinate dehydrogenase (SD) activity in drug-exposed cancer cells and the DNA content was microspectrophotometrically determined. Following exposure of malignant tissue to carboquone (CQ) and cisplatin (DDP), the mean SD activity in cells displaying a relatively regular DNA distribution (type II) was significantly higher than that in those exhibiting a widely scattered DNA distribution (type IV; P less than 0.01 in CQ, P less than 0.05 in DDP). A similar tendency was recognized in cells that were treated with aclacinomycin A (ACR), Adriamycin (ADM), and mitomycin C (MMC). Such a decrease in SD activity in cells exhibiting a type IV pattern was remarkable, especially in cases undifferentiated adenocarcinoma. Mitotic counting analysis revealed a significantly higher value for DNA pattern type IV as compared with the findings for type II (P less than 0.01). These results demonstrate that gastric carcinoma displaying a high malignant potentially shows a better response to antitumor drugs. Adjuvant chemotherapy prescribed following drug-sensitivity testing should be effective against such tumors.

Histologic differentiation and chemosensitivity of human head and neck squamous cell carcinomas

The chemosensitivities of 42 human head and neck squamous cell carcinomas were examined using the in vitro succinate dehydrogenase inhibition (SDI) test. The tumor tissues obtained at surgery or biopsy were exposed to five different antitumor drugs: adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil (5-FU), and 1-hexylcarbamoyl-5-fluorouracil (HCFU). The results were analyzed according to the histopathologic degree of differentiation of well, moderately, and poorly differentiated squamous cell carcinoma. The average decrease in succinate dehydrogenase activity was 43.2 +/- 24.9 for ADM, 29.0 +/- 14.2 for CDDP, 32.9 +/- 17.6 for CQ, 64.2 +/- 20.6 for 5-FU, and 26.8 +/- 16.9 for HCFU. There was a statistically significant difference in the decrease of succinate dehydrogenase activity between well and poorly differentiated squamous cell carcinomas. These data suggest that, for patients with a poorly differentiated squamous cell carcinoma, the response to anti-cancer drugs may be more satisfactory than in those with a well-differentiated squamous cell carcinoma.

Estrogen-Receptor-Negative Breast Cancer Tissue Is Chemosensitive in vitro Compared with Estrogen-Receptor-Positive Tissue

The chemosensitivities of 18 estrogen-receptor-positive (ER+) tissues were compared with that of 38 estrogen-receptor-negative (ER-) tissues, using the in vitro succinate dehydrogenase inhibition test. These human breast tissue were exposed to six antitumor drugs: carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin and 5-fluorouracil. Decrease in succinate dehydrogenase activity was noted in ER- compared to ER+ tissues, exposed to six antitumor drugs, in particular to adriamycin (p less than 0.001) and aclacinomycin A (p less than 0.05). The sensitive rates were higher in ER- than in ER+ tissues, against all six antitumor drugs. The resistance rates to all drugs tested were 25% in ER- and 45% in ER+ tissues. A higher chemosensitivity is associated with the absence of ER. It appeared that the ER status in case of breast cancer is an important predictor of the response to chemotherapy.

Azo-dye-induced primary hepatoma and a gradual increase in thermosensitivity.

To determine whether or not the cytotoxic effects of hyperthermia are directed primarily to malignant cells, we examined changes in thermosensitivity during hepatocarcinogenesis in rats, as induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). The findings were compared with those in livers of rats fed a commercial diet. The cell viability was determined using the succinate dehydrogenase inhibition (SDI) test. The succinate dehydrogenase (SD) activity of liver cells, when exposed to heat (43 degrees C) for 2, 5, or 10 hr, decreased in a time-dependent manner, in each tissue. The decrease in SD activity was evident in 3'-Me-DAB liver for 5 hr of heat treatment on day 57, compared with findings in the normal liver. Significant differences were present for 2, 5, and 10 hr on days 93 and 136. Thus a chemically induced hepatoma is more sensitive to heat than are the normal cells. As this thermosensitivity gradually increased during the hepatocarcinogenesis, the malignant cells are particularly vulnerable to hyperthermia.

Pharmacological correction of experimental alcohol embryopathy

In the experiments on the progeny of ethanol-exposed rats it was shown that consumption of 15% alcohol instead of water during pregnancy resulted in the worsening of shuttlebox avoidance learning and decrease in succinate dehydrogenase activity in the visual and sensorimotor cortex. These data are indicative of cerebral hypoxia during embryogenesis. The injection of synthetic opioid peptide dalargin during critical periods of development (at the end of embryogenesis and early ontogeny) prevented partially the disturbances of higher nervous activity and tissue breathing which were induced by alcohol intoxication. Possible mechanisms of positive dalargin effect are discussed.

Excised human neoplastic tissues are more sensitive to heat than the adjacent normal tissues.

In tissues from patients subjected to gastrectomy or colectomy, the heat sensitivity was determined in the case of 23 neoplastic, 15 gastric, 8 colorectal, and the adjacent normal tissues, using the in vitro succinate dehydrogenase inhibition test. The succinate dehydrogenase (SD) activity of tissue fragments was assayed, following exposure to heat at 43 degrees C (heat treatment) or 37 degrees C (control) for 5, 10, 15 or 20 h. The sensitivity to heat treatment was estimated by the percentage of SD activity of the heat-treated cells, compared to that of control cells. The decrease in SD activity varied in the tumor tissue, following exposure to heat. The SD activity decreased to a greater extent in the tumor tissue than in the adjacent normal tissue, in each case. The mean +/- standard deviation of SD activity, following exposure to heat treatment for 20 h, was 32.1 +/- 14.0% for the tumour tissues and 52.4 +/- 10.4% for the adjacent normal tissues, with a statistically significant difference (p less than 0.01). These results show that the assay of heat sensitivity is meaningful for prediciting the effectiveness of hyperthermia and that hyperthermia has a selectivity for treating a malignant lesion.

Thein vivo effects of tetrahydrocannabinol on the activity of lactate and succinate dehydrogenases in rat preovulatory follicles

The effects of delta 9-tetrahydrocannabinol on the activities of lactate and succinate dehydrogenases in the theca interna and membrana granulosa of rat preovulatory follicles have been analysed microdensitometrically using the same injection regime employed in a previous study on steroidogenic enzymes. A small but statistically significant (18%) decrease in succinate dehydrogenase activity was observed in the theca interna, but none in any region of the membrana granulosa. Lactate dehydrogenase activity was unaffected by THC administration. Thus, a dosage and regimen sufficient to cause significant decreases in the activities of steroidogenic enzymes had little effect on succinate and lactate dehydrogenases in rat preovulatory follicles.

The radial distribution of succinate dehydrogenase activity in porcine muscle fibres

A microscope photometer with a computer-controlled scanning stage was used to map the distribution of succinate dehydrogenase (SDH) activity in transverse sections of skeletal muscle fibres of pigs from 9 to 29 weeks of age. Absorbance due to SDH activity was measured in successive concentric zones that converged on the central axis of the muscle fibre. In all fibres, there was less SDH activity in the axis of the fibre than in the periphery of the fibre. In fibres with strong adenosine triphosphatase (ATPase) activity and weak overall SDH activity, the radial gradient of SDH activity remained constant as fibres grew in cross-sectional area. However, in fibres with weak ATPase and strong SDH and in fibres with strong ATPase and strong SDH, the radial gradient of SDH activity increased as fibres grew larger. Changes in radial gradients were due to both decreased SDH activity in the axis and to increased SDH activity in the periphery. In all three fibre types, the overall SDH activity per fibre increased with age.

Neurotoxicity of furfuryl alcohol vapor in prolonged inhalation exposure

Three-month-old male Wistar rats exposed intermittently (5 days weekly, 6 hr/day) to furfuryl alcohol vapor at 1 (25 ppm), 2 (50 ppm), or 4 μmol/liter (100 ppm) for 4 to 16 weeks were lighter than controls at the end of the experiment at the two higher concentration levels. Urinary furoic acid excretion was in a linear relationship to the exposure at the same time. Analysis for cerebellar creatine kinase showed increased activity in all groups throughout the experiment while succinate dehydrogenase activity decreased in a dose-dependent manner at later time points. Glial cell fractions isolated from cerebral hemispheres showed decreased succinate dehydrogenase activity while 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity increased at 4 μmol/liter for 16 weeks. This change was accompanied by decreased basic protein content of isolated myelin fractions. The results indicate that furfuryl alcohol, at fairly low vapor concentrations, may have significant mitochondrial effects in the brain which lead to glial cell degeneration and initiation of demyelination.

Myocardial protection with prostacyclin after lethal endotoxemia.

A previous study of endotoxemia in dogs demonstrated that exogenous prostacyclin (PGI2), normally a product of vascular endothelium, restored the cardiac index to normal and improved survival. To account for these results, a study was undertaken to test whether PGI2 would alter isolated rat or dog cardiac mitochondrial function following incubation with plasma from endotoxemic animals. A group of five animals served as anesthetized controls. A second group of seven mongrel dogs was given 1.75 mg Escherichia coli endotoxin/kg and was observed for 5 hours without treatment. Anesthesia did not alter cardiopulmonary function; however, 30 minutes after endotoxin administration, the cardiac index decreased from 148 +/- 25 (mean +/- SD) to 111 +/- 12 ml/kg . min (P less than 0.05) and further decreased to 89 +/- 20 ml/kg . min after 4 hours. Dog plasma obtained 2 to 5 hours after endotoxin infusion, incubated with rat or dog myocardial mitochondria, decreased succinate dehydrogenase (SDH) activity (P less than 0.05) and depressed mitochondrial respiration in the presence of the substrate succinate and adenosine diphosphate (ADP) from 180 to 87 Natoms oxygen/mg protein . min (P less than 0.05). There was no change in oxygen consumption when substrate alone was present, nor did plasma alter the amount of ADP phosphorylation as a function of oxygen consumption. A third group of seven animals, 30 minutes after administration of 1.75 mg endotoxin/kg, was treated with 100 ng/kg . min PGI2 for 3 hours. PGI2 infusion in this group prevented the decrease in cardiac index. Plasma obtained during and after PGI2 infusion did not decrease mitochondrial SDH activity, which remained higher than that in controls (P less than 0.001); mitochondrial respiration was also not altered. A correlation was observed between cardiac index and SDH activity (r = 0.58, P less than 0.001) and between cardiac index and mitochondrial respiration (r = 0.61, P less than 0.001). In PGI2-treated dogs cardiac mitochondria were functionally and structurally normal in contrast to the depression and disruption produced by endotoxemia, as observed by enzymatic assay as well as electron microscopy. These results suggest that endotoxemia depresses cardiac mitochondrial respiration, an event related to the decrease in cardiac index. In contrast, cardiac function and mitochondrial respiration are maintained with PGI2 treatment.

Dose-related effects of dichloromethane on rat brain in short-term inhalation exposure

Male Wistar rats were exposed to 500, 1000 or 100 ppm as time-weighted average (t.w.a.) concentrations of dichloromethane vapour. The 1000 (t.w.a.) ppm exposure consisted of two 1-h peak concentrations (2800 ppm) on a basal exposure of 100 ppm. All exposures lasted for 6 h, 5 days weekly and for 2 weeks. The solvent burdens were analyzed in the perirenal fat samples which showed a relation to the dose with the highest values in the 1000 (t.w.a.) ppm exposures. The solvent concentrations increased in the perirenal fat between the two weeks of exposure. Blood carbon monoxide concentrations did not accurately reflect the body solvent burdens. Neurochemical effects also displayed a dose relationship, and included decreased succinate dehydrogenase activity in the cerebellum at the two higher doses and increased acid proteinase activity at 1000 ppm in the cerebrum. Withdrawal of the animals for 7 days from the 2-week exposure showed that the biochemical changes were largely abolished with the exception of decreased succinate dehydrogenase activity at 1000 ppm (t.w.a.).

Influence of starvation and clofibrate administration on oxidative phosphorylation by rat liver mitochondria.

Whole cells, homogenates and mitochondrial obtained from the livers of albino rats which were starved for 6 days or more showed a 50% decrease in oxidative activity. The decrease could be corrected by the addition of cytochrome c in vitro. The phosphorylative activity of mitochondria remained unaffected. The decrease in oxidative rate was not observed when starving animals were given the anti-hypercholesterolaemic drug clofibrate. The total cellular concentration of cytochrome c was not affected by starvation. However, the concentration of the pigment in hepatic mitochondria isolated from starving animals was less than half that in normal mitochondria. Clofibrate-treated animals did not show a decreased concentration of cytochrome c in hepatic mitochondria. Mitochondria isolated from starving animals, though deficient in cytochrome c, did not show any decrease in succinate dehydrogenase activity or in the rate of substrate-dependent reduction of potassium ferricyanide or attendant phosphorylation. In coupled mitochondria, ferricyanide may not accept electrons from the cytochrome c in the respiratory chain. Starvation decreases the concentration of high-affinity binding sites for cytochrome c on the mitochondrial membrane. The dissociation constant increases in magnitude.

In vitro toxicity tests of zinc phosphate cement

BHK-21 (C-13) cells were exposed to freshly mixed zinc phosphate cement for 0.5, 1 and 2 h in a simulated cavity apparatus. The effect on the cells was evaluated by quantitative measurement of acid phosphatase and succinic dehydrogenase reaction product in order to determine organelle membrane permeability. After 0.5 h cell-material contact there was no significant effect. After 1 h cell-material contact, there was slight labilization of the lysosomal membranes, and a significant decrease in succinic dehydrogenase activity. The mean overall stain density in the experimental cells was 109% and 57% of the control values for acid phosphotase and succinic dehydrogenase respectively. After 2 h cell-material contact, there was significant labilization of the lysosomal membranes, and also labilization of the mitochondrial membranes. The mean overall stain densities were 114% and 75% of the control values for acid phosphatase and succinic dehydrogenase respectively. The relevance of these findings to the in vivo situation is discussed.

Effect of Carbon Dioxide on Growth and Carbohydrate Metabolism in Sclerotium rolfsii

SUMMARY: Carbon dioxide at a concentration of 1 to 2% (v/v) in air enhanced the growth rate and inhibited sclerotium formation in the fungus Sclerotium rolfsii Sacc. A CO2 concentration of 10% inhibited growth. Similar growth patterns were observed when the fungus was grown on a medium supplemented with the fungicide carboxin, which inhibits succinate dehydrogenase. A high CO2 concentration (1 to 10%) or growth on carboxin-supplemented medium caused a decrease in succinate dehydrogenase activity and significant increases in isocitrate lyase, isocitrate dehydrogenase, malate synthase and malate dehydrogenase activities. Mycelium of S. rolfsii grown at a high CO2 concentration contained less glyoxylate, lipids and glycogen than mycelium grown in air. It is suggested that sclerotium formation in S. rolfsii requires a balanced supply of carbohydrate intermediates and energy.

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.

Histochemical patterns in early lesions and infiltrating adenocarcinomas induced in mouse duodenum by n-ethyl-n'-nitro-n-nitrosoguanidine.

Intravillous, microcrater, and macroscopic invasive lesions induced in the mouse duodenum by N-ethyl-N'-nitro-N-nitrosoguanidine were examined histochemically. The cells of these neoplastic lesions and the proliferative zones of the normal crypts showed similar staining reactions in leucine aminopeptidase, alkaline and acid phosphatases, adenosine 5'-triphosphatase, and glucose-6-phosphate dehydrogenase. However, a slight decrease in succinic dehydrogenase activity and a slight increase in lactic dehydrogenase activity were observed in the intravillous and microcrater lesions compared to the activity in the proliferative zones of the crypts. The neoplastic cells of these lesions showed no mucus secretion. We discussed the origin of the neoplastic lesions using these and other findings.

EXPERIMENTAL CALCIUM-MAGNESIUM DEFICIENCY IN RATS: CHEMICAL AND HISTOCHEMICAL STUDY

Chemical and histochemical profiles of central nervous system tissues, skeletal and cardiac muscles and visceral organs of experimental calciummagnesium deficient rats and control animals were studied. Six to eight weeks after a feeding schedule of a calcium-magnesium deficient diet, the calcium concentration in the kidney increased twenty-five fold over that of the control and there was a moderate increase in the spinal cord and skeletal muscle. Fluctuation of the magnesium concentration was insignificant.A decrease in succinic dehydrogenase activity demonstrated in the cerebral cortex, cerebellum, spinal cord, kidney and skeletal muscle corresponds to tissue morphology, for example, atrophy and a decreased SDH type II muscle fiber was noted in gastrocnemius tissue specimens.Acetylcholinesterase activity in the motor end-plate of the gastrocnemius muscle showed a slight decrease in enzyme activity and a swelling of the motor end-plate.The severe histochemical changes of SDH and acid phosphatase activities in renal tubules and hepatic parenchymal cells suggest a progression of cellular catabolism related to lysosomal enzyme activity.