Content and redistribution of vitamin E in tissues of Wistar rats under oxidative stress induced by hydrazine.

Hydrazine toxicity is associated with generation of several kinds of free radicals and oxidative stress in cell. Experiments in vivo have demonstrated that oxidative stress could either diminish or increase concentration of vitamin E in some tissues. Thus in the present study we performed experiments to determine whether hydrazine-induced oxidative stress would change the tissue levels of the vitamin. Seven days of hydrazine intoxication led to accumulation of different amounts of vitamin E: 215% in the liver, 118% in the heart, 135% in the spleen, and 100% in the muscle over control value. There were no changes in the level of the vitamin in kidney and pancreas, despite its significant depletion in the serum. In tissue that accumulated vitamin E after hydrazine treatment, an increased of oxidative stress measured by the concentration of lipid-soluble fluorophore was observed. Significant increases of 107%, 46%, 72%, and 58% over control values were observed in the liver, heart, spleen, and muscle, respectively. Rats treated with hydrazine and pharmacological doses of alpha-tocopherol accumulated higher concentrations of vitamin E in all studied tissues compared with the alpha-tocopherol-only treated rats. However, in tissues with elevated levels of fluorophore as liver, heart, spleen, and muscle, the accumulation of vitamin E was 5.03, 4.5, 4.03, and 4.6 times higher than in alpha-tocopherol-treated rats, respectively. Vitamin E concentration was much higher than in kidney and pancreas, where the accumulation was only 2.31 and 2.6 times higher. On the other hand, 3 days of hydrazine treatment did not change either the level of lipid-soluble fluorophore or the level of vitamin E in the liver mitochondria, microsomes, and homogenate. In skeletal muscle vitamin E caused decreased lipofuscin accumulation, and in pancreas vitamin E increased lipofuscin accumulation. Our data indicate that hydrazine is able to modify significantly vitamin E status in different rat tissues.

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 alpha-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.

Cerulein-induced acute pancreatitis diminished vitamin E concentration in plasma and increased in the pancreas.

Redistribution of vitamin E in the rat body was studied during acute pancreatitis induced by two intraperitoneal doses of cerulein 40 micrograms/kg of body weight at 1-hr intervals. Hyperamylasemia (2064 +/- 521 vs 6419 +/- 129 U/dL) and pancreatic edema (pancreatic water content, 71 +/- 1.2% vs 78 +/- 2%) were observed. In this model the increased level of lipid soluble fluorophore was also observed (274 +/- 18 vs 120 +/- 9.0 relative fluorescence per g dry wt). Parallel with these changes was a decrease in the level of vitamin E in the serum and an increase in the pancreas. The concentration of vitamin E in the pancreas after 6 h was 162 +/- 8.5 ng/mg dry mass vs 128.1 +/- 6.1 ng/mg dry mass in control animals. The effect of heparin on vitamin E redistribution induced by acute pancreatitis was also investigated. It was found that heparin at a dose of 100 U/kg body mass prevents the drop of the vitamin E level in the serum as well as the increases in the concentration in the pancreas tissue. It was concluded that acute pancreatitis induced redistribution of vitamin E in the rat body. Moreover, we studied the effects of heparin treatment on oxidative stress in the pancreas tissue. Acute pancreatitis caused an increase in lipofuscin accumulation, and a decrease in protein sulfhydryl groups in citrate synthetase (CS) and in malate dehydrogenase (MDH) activity. Heparin treatment that protected vitamin E accumulation in the pancreas tissue did not influence the changes in the level of lipofuscin and proteins sulfhydryl.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of indolinic and quinolinic aminoxyls on protein and lipid peroxidation of rat liver microsomes.

A study on peroxyl radical induced oxidation of rat liver microsomal membranes in the presence of different indolinic and quinolinic aminoxyls (Scheme 1) was carried out in order to test their efficiency as antioxidants in lipid and protein peroxidation. The extent of lipid peroxidation was quantified by the amount of malondialdehyde (MDA) produced, and the measurement of carbonyl residues was used as an index of microsomal protein oxidation. The results obtained suggest that lipid soluble indolinic and quinolinic aminoxyls are efficient in protecting lipids and proteins of biological membranes against oxidation. The efficacy of these aminoxyls as protectors of lipids and proteins was much higher than the water soluble TEMPOL. Moreover, the hydrophobic aminoxyls were more effective in preventing protein than lipid oxidation at low concentrations (1-20 microM). However, at high concentration (100 microM), lipid as opposed to protein oxidation was almost completely inhibited. The data supports the hypothesis that proteins probably have a different oxidation pattern from lipids.

Further studies on physicochemical properties of mitochondrial membranes during the formation process of megamitochondria in the rat liver by hydrazine.

Physicochemical properties of lipids extracted from hepatic mitochondria of rats fed with 1% hydrazine diet for 3 days (3H-mitochondria) and 7 days (7H-mitochondria) were studied using differential scanning calorimetry and the fluorescence polarization measurements. Results obtained from lipids extracted from 3H-mitochondria were as follows: (1) Thermotropic phase transition temperatures (Tm) were elevated with a distinct increase in transition enthalpy (delta H) compared to those of the control due to increases in relative amounts of phosphatidylethanolamine (PE) and phosphatidylserine (PS) in phospholipid species. (2) Tm in lipids and phospholipids extracted from 3H-mitochondria was elevated with decreases in delta H after the addition of Ca2+, whereas delta H of the control was increased after the addition of Ca2+. (3) Phosphatidylcholine (PC) liposomes containing PE or PS at concentrations corresponding to PE/PC or PS/PC ratio in 3H-mitochondria showed elevations of transition temperatures with decreases in delta H values after the addition of Ca2+, whereas those containing PE or PS ratio at concentrations corresponding to PE/PC or PS/PC in the control mitochondria showed increases in delta H values after the addition of Ca2+. (4) There were essentially no changes in membrane fluidity between lipids extracted from 3H-mitochondria and those extracted from the control mitochondria. Addition of Ca2+ to lipids extracted from 3H-mitochondria, however, caused elevation of membrane fluidity while those extracted from the control mitochondria showed no changes in membrane fluidity after the addition of Ca2+. (5) Increases in the ratio of unsaturated fatty acids to saturated fatty acids (U/S ratio) in phospholipids extracted from 3H-mitochondria were found to be mainly due to those in PS. These data may suggest that hydrazine modifies the membrane of mitochondria favorable for the membrane fusion via enhanced acidic phospholipid-Ca2+ interactions.

Indolinonic and quinolinic aminoxyls as protectants against oxidative stress.

A study on thermally and peroxyl radical induced oxidation of linolenic acid micelles in the presence of different concentrations of aminoxyls was carried out in order to test their efficiency as antioxidants in lipid peroxidation. The extent of peroxidation was measured by the malondialdehyde (MDA) produced and by oxygen consumption evaluated using an oxygraph. The results obtained indicate that indolinonic and quinolinic aminoxyls synthesized by us could be used as effective antioxidants in biological systems.

Effects of alkyl alcohols and related chemicals on rat liver structure and function. III. Physiochemical 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 calorimetry 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 unsaturated/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 (delta H) was revealed by differential scanning calorimetry. 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.

Effects of alkyl alcohols and related chemicals on rat liver structure and function. II. Some biochemical properties of ethanol-, propanol- and butanol-treated rat liver mitochondria.

Functional changes of mitochondria in the liver obtained from rats given 32% ethanol, 32% propanol and 6.9% butanol in drinking water for up to 3 months were investigated. Animals were also fed a liquid diet containing ethanol for comparison. Results obtained were as follows: 1) Animals given ethanol in drinking water consumed twice as much ethanol daily as those fed a liquid diet containing ethanol, while ultrastructural changes of hepatic mitochondria were essentially the same between the former and the latter animals: the co-existence of megamitochondria and small mitochondria with poorly developed cristae. 2) Effects of alkyl alcohols tested on the respiratory rates and coupling efficiency of mitochondria were variable, depending on the kind of alkyl alcohols, the duration of experiments and oxidizable substrates used. 3) There was essentially no difference between the heavy and the light mitochondrial fractions obtained from alkyl alcohol-treated rat livers in terms of respiratory rates and coupling efficiencies. 4) Decreases in the content of cytochrome aa3 and the activity of activity of cytochrome oxidase, and increases in MEOS activity were most distinct in ethanol-treated rat livers. A possible role of chronic relative oxygen deficiency inside the hepatocyte caused by the metabolization of alkyl alcohols is discussed in order to interpret such peculiar ultrastructural changes of mitochondria.

Effects of alkyl alcohols and related chemicals on rat liver structure and function. I. Induction of two distinct types of megamitochondria.

The effects of alkyl alcohols and related chemicals on the ultrastructure of mitochondria in the rat hepatocyte were studied. The following three different groups of chemicals were tested: Group 1: alkyl alcohols with straight carbon chains (ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-octanol, 1-dodecanol and 1-octadecanol); Group 2: tert- and cyclo-compounds (tert-butanol, cyclo-pentanol, and cyclo-hexanol); and Group 3: polyhydroxy alcohols (ethylene glycol, propylene glycol, 1, 3-propanediol, glycerol and pentaerythritol). Results obtained were summarized as follows: 1) Ethanol, 1-propanol, 1-butanol, 1-pentanol and 1-octanol had essentially the same effects on the mitochondrial ultrastructure: a mixed population of small and enlarged mitochondria with poorly developed cristae; 1-dodecanol induced ultrastructural changes of mitochondria of two distinct types: a mixed population of small and enlarged mitochondria with poorly developed cristae in some hepatocytes and remarkably enlarged mitochondria with well-developed cristate in others; and 1-octadecanol induced remarkably enlarged mitochondria in all hepatocytes. 2) Chemicals belonging to group 2 and group 3 induced essentially the same changes as those induced by 1-octadecanol. More than one month was required to induce those changes. The mechanism by which those ultrastructural mitochondrial changes were induced is not clear, but the present results may suggest that the hydroxy group (-OH) common to all these chemicals in some way accounts for the phenomenon.

Tightly coupled respiration in rat brain homogenates.

"Respiratory control", a typical feature of well coupled mitochondria, was found to be higher in rat brain homogenate than in isolated mitochondria. This observation points to the possibility of studying the coupling between respiration and ADP phosphorylation, as well as mitochondrial metabolism, directly in homogenates and not in isolated mitochondria, using very small samples of brain tissue.

Human skeletal muscle: participation of different metabolic activities in oxidation of L-lactate.

The pure mitochondrial fraction obtained from human skeletal muscle did not show coupled L-lactate (+ NAD) oxidation, but this function could be restored by addition of LDH. Thus the "direct", coupled oxidation of L-lactate described earlier (Popinigis et al., 1990. International Perspectives in Exercise Physiology, Human Kinetics Books, pp. 132-133) should be attributed to contaminations.

Direct oxidation of glutamate by mitochondria from porcine adrenal cortex.

1. Mitochondria isolated from porcine adrenal cortex under State 3 conditions oxidized succinate with a rate of 47 +/- 4.48 na oxygen/min/mg/protein and with ADP:O ratio 0.98 +/- 0.09. In the presence of 15 microM deoxycorticosterone the rate of succinate oxidation was 36.8 +/- 3.08 na oxygen/min/mg/protein. 2. Under the same conditions the rate of glutamate oxidation was 22.8 +/- 2.21 and 16.8 +/- 0.65 na oxygen/min/mg/protein, respectively. ADP:O ratio was 1.45 +/- 0.14. 3. Introduction of trace amounts of malate into the mitochondria oxidizing glutamate only slightly increased the rate of O2 uptake. 4. The glutamate dehydrogenase activity in these mitochondria was 12.5 +/- 0.69 nmol/min/mg.

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.

Oxidation of NADH via an "external" pathway in skeletal-muscle mitochondria and its possible role in the repayment of lactacid oxygen debt.

Mitochondria isolated from skeletal muscle of rat catalyse oxidation of the external NADH (in the presence of rotenone, antimycin A and cytochrome c) at a rate of 15 natoms O2/min/mg protein by a pathway sensitive to mersalyl. In a medium supplemented with commercial lactate dehydrogenase, or when mitochondria were incubated in the presence of a cytoplasm, the NADH oxidation could be arrested by pyruvate. The inhibitory effect of pyruvate could be released by lactate. In the presence of NAD and cytochrome c, the reconstructed system containing skeletal muscle mitochondria plus cytoplasmic fraction was active in oxidation of L-lactate despite of the presence of rotenone and antimycin A. The lactate oxidation was sensitive to mersalyl and cyanide.

Transaminative pathway of glutamate oxidation in adrenal-cortex mitochondria.

Mitochondria isolated from adrenal cortex of beef do oxidize glutamate if the amino group acceptor-oxaloacetate (or its precursor-malate) is present in the incubation medium. The glutamate (plus oxaloacetate) oxidation was enhanced by ADP or deoxycorticosterone, indicating that this respiration can support both oxidative phosphorylation and 11 beta-hydroxylation of deoxycorticosterone to corticosterone. Avenaciolide (inhibitor of glutamate entry into the mitochondria), aminooxyacetate (inhibitor of aspartate aminotransferase activity) and arsenite (inhibitor of 2-oxoglutarate dehydrogenase) when introduced into the incubation media before respirating substrates, inhibited the ability of ADP or deoxycorticosterone to stimulate the rate of glutamate (plus oxaloacetate) oxidation.