Kayo Adachi

Suppression of the formation of megamitochondria by scavengers for free radicals.

In the present study we have attempted to suppress the formation of megamitochondria by scavengers for free radicals since conditions for the formation of megamitochondria are often intimately related to the generation of free radicals. We employed three different experimental conditions to induce megamitochondria in the liver: ethanol, hydrazine and chloramphenicol (CP). Scavengers for free radicals tested were: alpha-tocopherol, coenzyme Q10(CoQ10) and 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO). Allopurinol (AP), a xanthine oxidase inhibitor, was also tested. Results obtained were as follows. (1) Changes observed in the liver of animals treated with ethanol, hydrazine or CP were: formation of megamitochondria; decreases in the body weight and the weight of the liver; remarkable increases in the level of lipid peroxidation; increases in the activity of xanthine oxidase. (2) 4-OH-TEMPO was most effective in improving these changes. A mechanism of the formation of megamitochondria is proposed stressing the role of free radicals in the mechanism.

Changes in physicochemical properties of mitochondrial membranes during the formation process of megamitochondria induced by hydrazine

Changes in some biochemical and physico-chemical properties of rat liver mitochondrial membranes during the formation process of megamitochondria induced by hydrazine were analyzed. Hepatic mitochondria obtained from rats placed on a 1% hydrazine diet for 3 days became slightly enlarged and sometimes elongated, while they became gigantic after 7 days of hydrazine intoxication. Changes were observed in mitochondria from rats treated with hydrazine for 3 days. Total amounts of phospholipids extracted from mitochondria and submitochondrial fractions were increased. Among phospholipid species, relative amounts of acidic phospholipids were increased. Contents of Ca2+ in mitochondria were increased. Differential scanning calorimetric analysis of mitochondria, especially that of the outer membrane fraction, showed that the thermotropic lipid phase transition temperatures were elevated accompanying the broadening of thermograms and the increase in transition enthalpy. Contents of water in mitochondria were increased significantly with the ratio of freezable water to unfreezable water unchanged. Among the changes observed was that the total amount of phospholipids (except for that of the outer membrane fraction) and the contents of water and Ca2+ nearly returned to normal in megamitochondria after 7 days of hydrazine intoxication. Relative amounts of phospholipids and thermotropic lipid phase transition temperatures of megamitochondria did not return to normal levels and yet changes were smaller than those obtained from 3 days of hydrazine intoxication. The fluidity of mitochondrial membranes was not affected by hydrazine treatment. These data would suggest that hydrazine-induced megamitochondrial formation is not due simply to the swelling of mitochondria, but might be due to the fusion of adjacent mitochondria by Ca2+-acidic phospholipid interactions, and once megamitochondria are formed the mitochondrial membranes are stabilized.

Effects of Alkyl Alcohols and Related Chemicals on Rat Liver Structure and Function: III. Physicochemical Properties of Ethanol-, Propanol- and Butanol-treated Rat Liver Mitochondrial Membranes

The physicochemical properties of mitochondria in liver tissue obtained from rats given 32% ethanol, 32% propanol or 6.9% butanol in drinking water for up to 3 months were investigated using differential scanning calo‐rimetry and fluorescence polarization measurements. The results obtained were as follows: 1) Phospholipids extracted from mitochondria showed increases in the relative amounts of phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and a decrease in the relative amount of phosphatidylethanolamine. An increase in the unsatu‐rated/saturated fatty acid ratio of phospholipids was also observed. 2) Elevation of the thermotropic lipid phase transition temperature with a decrease in the enthalpy value (δH) was revealed by differential scanning calo‐rimetry. 3) The elevation of the lipid phase transition temperature was detected also by fluorescence polarization measurements using 1,6 diphenyl 1, 3, 5 hexatriene (DPH) as a probe. Elevation of mitochondrial membrane fluidity was found in some of the experimental animals, but most showed no changes in comparison with the control. A possible role of membrane fusion in the mechanism of formation of ethanol‐, propanol‐ and butanol induced hepatic megamitochondria is discussed on the basis of these results. Acta Pathol Jpn 42: 549–557, 1992.

Suppression of the hydrazine-induced formation of megamitochondria in the rat liver by coenzyme Q10

The effects of coenzyme Q10 (CoQ10) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ10. Results obtained were as follows: (a) treatment of animals with CoQ10 prevented the hydrazine-induced formation of megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ10 to hydrazine-treated animals suppressed enhanced lipid peroxidation and improved lowered adenosine diphosphate/O ratios of mitochondria. The present data suggest that CoQ10 suppresses the hydrazine-induced formation of megamitochondria by scavenging free radicals generated from hydrazine and its metabolites.

Studies on urea synthesis in the liver of rats treated chronically with ethanol using perfused livers, isolated hepatocytes, and mitochondria

Changes in urea synthesis in the liver of rats treated with 32% ethanol in the drinking water for up to 6 months were studied using perfused livers, isolated hepatocytes, and mitochondria. Results obtained from ethanol-treated rats are summarized as follows: (1) the mitochondria of the hepatocytes of rats treated with ethanol for 2 months or longer became enlarged to various degrees, (2) the levels of ammonia in the serum remained within a normal range, while those in liver tissue were elevated compared with the control, (3) urea synthesis from ammonia in perfused livers was decreased markedly, while that from citrulline remained in the normal range, (4) the activities of carbamyl phosphate synthetase (CPS; EC 2.7.2.5) and ornithine transcarbamylase (OTC; EC 2.1.3.3) in mitochondria were unchanged compared with those of the control, and (5) the levels of ATP in liver tissue and the ability of mitochondria to synthesize ATP were decreased markedly compared with the control. Both the level of ATP in the hepatocytes and the synthesis of urea from ammonia by perfused livers of rats treated with ethanol were resistant to externally added ethanol, while those of control animals were severely affected. These results suggest that the intracellular level of ATP is intimately related to urea synthesis in both control and ethanol-treated animals, and lowered levels of ATP may be a key factor in the suppression of urea synthesis in ethanol-treated animals.