Liver and heart mitochondrial succinate dehydrogenase activity of newborn rats in anoxic hypoxia and starvation

Succinate dehydrogenase activity was determined in the liver and heart of newborn rats after 3 and 48 hours' exposure to anoxic hypoxia (10% O2) and after 48 hours' starvation. Control determinations were made on newborn animals of corresponding ages, full term foetuses (21 days), infantile (1 and 2 weeks) and full grown animals. Hypoxia for 3 h had no influence on succinate dehydrogenase activity at all in either the heart or liver mitochondria of the newborn animals. After 48 h no difference was observed in the liver between the hypoxic animals and the starved controls of the same age, though starvation itself had resulted in a significant increase in activity, as much as 42%. When liver mitochondrial succinate dehydrogenase in normal mitochondria was activated by preincubation mitochondria with the substrate, the activity increase obtained was greater than that resulting from starvation. The increase in activity in the heart of the hypoxic or starved animals was not significant (less than 10%).     

Orientation-dependent enhancement by H-NS of the activity of the type 1 fimbrial phase switch promoter in Escherichia coli

The role of 4-hydroxynonenal (HNE), a major lipid peroxidation product, in oxidative damage to mitochondrial cytochrome c oxidase (COX) was examined. Oxidative stress was induced in mitochondria isolated from livers of male Sprague-Dawley rats by tert-butylhydroperoxide (t-BHP). COX activity was inhibited, with a concomitant increase in endogenous HNE level in mitochondria. COX activity was also inhibited following incubation of mitochondria with 50-450 microM HNE. Blocking HNE degradation intensified COX inhibition by HNE and by t-BHP-induced oxidative stress, the latter accompanied by a simultaneous increase in endogenous HNE production. On the other hand, COX inhibition by HNE was markedly reduced by potentiating HNE degradation via enhancing conjugation of HNE with reduced glutathione (GSH). Incubation of purified COX with 10-400 microM HNE resulted in HNE adduct formation with specific subunits of COX, correlated with inhibition of the enzyme activity. These data suggest that HNE may inhibit mitochondrial COX by forming adducts with the enzyme, and that this could be one mechanism underlying mitochondrial damage caused by oxidative stress. The findings also illustrate a role for GSH in protecting mitochondria from the deleterious effects of HNE.     

Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.     

Fecal-oral transmission of viral hepatitis

The cumulative, subchronic and chronic toxicity of B-193 were studied on the rats and mice. It was found that this compound exerted weak tendency to cumulation in the body. Only the highest doses of B-193 (70, 40 mg/kg po for 12 weeks) caused the increase of animals mortality. Studies on subchronic and chronic toxicity have demonstrated, that B-193 administrated po or ip for 3 weeks, and po for 12 weeks, in general, neither affects the body weight gain nor the mass and morphology of heart, liver and kidneys, as well as spontaneous locomotor activity of animals. The weak depressant effect of B-193 on peripheral blood morphology was seen only after 3 weeks po or ip treatment with this compound. The moderate effect of B-193 on activity of alanine and aspartate transaminases (A1At and AspAt) and serum protein level found after 3 weeks of treatment, was no longer observed after 12 weeks of treatment. This could indicate that above effects of B-193 are reversible.     

Quantitation of mitochondrial DNA, RNA, and protein in starved and starved-refed rat liver

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved-5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of 3H-TTP into into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA sythesis.     

Population heterogeneity of higher-plant mitochondria in structure and function

Mitochondria of rapidly developing mungbean seedlings were fractionated into four populations: two density classes, each from a 1500S and a 150S pellet. Each of the four populations exhibited cytochrome c oxidase (COX) activity and contained mitochondrial DNA and cardiolipin; plastid and glyoxysome content were found to be relatively low. Five mitochondrial membrane proteins, COXII/III, ATPase alpha/beta and porin, and a matrix enzyme, manganese superoxide dismutase (MnSOD), were detected by immunoblots in all four populations. Another matrix enzyme, pyruvate dehydrogenase was detected only in the two respiratory-competent 1500S populations. The two 150S populations contained a previously unidentified organelle that lacked demonstrable respiratory capability. This organelle, which we have tentatively referred to as "slow-sedimenting (ss-) mitochondrion", was small in size (below light-optics resolution, 70-300nm, majority < or =200nm) and possessed a peculiar looking boundary membrane, ribosomes, and an occasional prominent electron-dense spot. Characteristically, ss-mitochondria were almost always in contact with a filament-aligned membrane-like structure of varying length. Cristae structure, while undetected in small ss-mitochondria, appeared in larger individuals. Typical mitochondria were found in the denser 1500S population, while the lighter 1500S population consisted of 300-800 nm mitochondria exhibiting a varying degree of size-dependent inner membrane folding. Using electron microscopy (EM) immunolocalization and serial sectioning, we have identified in situ organelles resembling in size and in fine structure the ss-mitochondria, which also exhibit a size-dependent folding of the inner membrane. These results suggest that small ss-mitochondria may undergo a progressive development in situ. Taken together, our findings demonstrate the existence of a pattern of structure-function-coordinated gross heterogeneity among mitochondria. This pattern of mitochondrial heterogeneity, characterized both in isolated mitochondria and in situ, implies that small ss-mitochondria may represent a type of "nascent mitochondria" derived from a yet unidentified mitochondria-propagation mode operating during rapid seedling growth. Mitochondrial division by binary fission, characterized by the appearance of dumbbell-shaped intermediates, was also detected.     

Melatonin, a pineal hormone with antioxidant property, protects against adriamycin cardiomyopathy in rats

Adriamycin (ADR) is a potent, broad-spectrum chemotherapeutic agent whose clinical use is limited by its cardiotoxicity. Since the pathogenesis of ADR-induced cardiomyopathy may involve free radicals and lipid peroxidation, the antioxidant, melatonin (MEL) may protect against toxic effects of ADR. We therefore tested this hypothesis using a rat model of ADR-induced cardiomyopathy. Sprague-Dawley rats were given ADR (cumulative dose, 15 mg/kg), MEL (cumulative dose, 84 mg/kg), ADR+MEL, ADR plus probucol (PRB, cumulative dose, 90 mg/kg), or vehicle alone, according to known regimens. The rats were maintained for 3 weeks following treatment, after which their cardiac performance was measured. Following sacrifice, their myocardial ultrastructure was examined, and their myocardial lipid peroxidation was assessed. Mortality was observed only in rats treated with ADR alone. When compared to control rats, surviving rats in the ADR group showed significant decreases in ratio of heart to body weight, arterial pressure, and left ventricular fractional shortening as well as a significant accumulation of ascites. The amount of myocardial thiobarbituric acid reactive substances was significantly higher in ADR-treated than in control rats. Both antioxidants, MEL and PRB, significantly prevented these ADR-induced changes. Electron microscopic examination revealed myocardial lesions indicative of ADR-induced cardiomyopathy in the ADR-treated rats. In contrast, treatment of these rats with MEL or PRB preserved myocardial ultrastructure. By preventing lipid peroxidation, MEL may be highly effective in protecting against ADR-induced cardiomyopathy.     

Mitochondrial aldehyde reductase: identification and characterization in rat liver and kidney cortex

Aldehyde reductase (EC 1.1.1.2) has been regarded so far as an exclusively cytosolic enzyme. The present investigation shows that mitochondria of rat liver, kidney cortex and, tentatively, heart also contain an enzyme catalyzing oxidation of NADPH by aldehydes, p-nitrobenzaldehyde, methylglyoxal and glyceraldehyde. Activity of the mitochondrial enzyme can only be measured after the organelles are disrupted by sonication or solubilized with nonionic detergents. Mitochondrial aldehyde reductase activity contributed to about 4.6% and 2.5% of the total cellular activity in liver and kidney cortex, respectively. However, the specific activity in liver mitochondria was about one third and in kidney cortex mitochondria one tenth of that in the cytosol of the corresponding organ. The mitochondrial enzyme resembled the cytosolic one by its absolute specificity towards NADPH as the electron donor, a similar profile of aldehydic electron acceptors and identical Km values. Mitochondrial aldehyde reductase differed from the cytosolic enzyme by low sensitivity to known inhibitors of cytosolic aldehyde reductase, AL-1576, AL-4114 and ONO-2235. In liver, about 60% of the mitochondrial activity was tightly bound to the membranes whereas about 40% was present in the mitochondrial matrix. The membrane-bound activity was inactivated by digestion of mitoplasts with trypsin, alpha-chymotrypsin or papain, thus pointing to exposition of the substrate-binding site at the external surface of the inner membrane. On the other hand, latency of the enzyme in intact mitochondria indicates that the NADPH-binding site is located at the inner surface. These data provide the first direct evidence for the existence of aldehyde reductase in mitochondria of some rat tissues.     

Early appearance of age-associated deterioration in mitochondrial function of diaphragm and heart in rats treated with doxorubicin

Age-associated deterioration of mitochondrial energy transduction seems to be a major contributory factor to age-related decline in organ function. Free radicals are likely to be involved in the age-related decline in mitochondrial function. This study was designed to elucidate whether or not doxorubicin, a radical generating drug that was administered to 7-week-old rats, affects age-associated mitochondrial functional changes in diaphragm, heart, and liver. Mitochondria from each tissue were prepared from rats aged 7, 13, 20, 28, 35, and 55 weeks, and the activities of four complexes in the mitochondrial energy transduction system were measured enzymatically. In diaphragm mitochondria of the control group, the complex I activity in 28-week-old rats declined to 82% of the activity in rats aged 7 weeks, and the complex IV activity in 55-week-old rats declined to 70% of the activity in rats aged 7 weeks. On the contrary, a significant decrease in the activity of complex I in rats aged 20 weeks (84%) and that of complex IV in rats aged 35 weeks (86%) were observed in the doxorubicin-treated group. In heart mitochondria, age-related changes in activities of complexes I and IV did not appear in rats aged up to 55 weeks, whereas significant decreases in the activities of complexes I (78%) and IV (90%) were observed in rats aged 35 weeks in the doxorubicin group. Age-related changes in liver mitochondria were not found in rats aged up to 55 weeks, and no deleterious effects of doxorubicin were observed in liver mitochondrial function. From these results, the early appearance of aging effects on mitochondrial function was observed in rats treated with doxorubicin particularly in postmitotic cells.     

HMG CoA reductase inhibitor accelerates aging effect on diaphragm mitochondrial respiratory function in rats

We examined effects of pravastatin on age-related changes in mitochondrial function in rats. Decline in the activity of complex I of the mitochondrial electron transport chain was observed in diaphragm and psoai major in rats aged 35 and 55 weeks, and that of complex IV in rats aged 55 weeks. Pravastatin accelerated significantly age-related decline in the activity of complex I of diaphragm mitochondria, though pravastatin did not show significant effect on normally observed age-associated decline in the activities of complex IV of psoai major and diaphragm mitochondria. Aging effect on mitochondrial respiratory function was not observed on heart muscle and liver in rats up to 55 weeks old, and pravastatin did not effect significantly heart and liver mitochondrial respiratory function. From these results, careful clinical examination on respiratory muscle function should be necessary in patients treated with pravastatin particularly in elderly patients.     

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria

Administration of dehydroepiandrosterone (DHEA), a steroid hormone of the adrenal cortex which acts as a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from the brain. The effect of DHEA on rat liver mitochondrial lipid peroxidation became discernible after feeding steroid-containing diet (0.6% w/w) for 3 days, and reached maximal levels between 1 and 2 weeks. DHEA in the concentration range 0.001-0.02% did not significantly increase lipid peroxidation compared to the control. Lipid peroxidation was significantly enhanced in animals given a diet containing > or = 0.05% DHEA. The addition of DHEA in the concentration range 0.1-100 microM to mitochondria isolated from control rats had no effect on lipid peroxidation. It seems, therefore, that the steroid effect is mediated by an intracellular process. Our data indicate that induction of mitochondrial membrane lipid peroxidation is an early effect of DHEA administration at pharmacological doses.     

Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.     

Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging

Oxygen free radicals have been hypothesized to play an important role in the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of oxidative protein damage and lipid peroxidation in the brain and liver, and activities of antioxidant enzymes in the brain, liver, heart, kidney, and serum from the Fisher 344 rats at ages of 1, 6, 12, 18, and 24 months. The results showed that the level of oxidative protein damage (measured as carbonyl content) in the brain and liver was significantly higher in older animals than in young animals. No statistical difference was observed in the lipid peroxidation of the liver and brain between young and old animals. The activities of antioxidant enzymes in most tissues displayed an age-dependent decline. Superoxide dismutases in the heart, kidney, and serum, glutathione peroxidase activities in the serum and kidney, and catalase activities in the brain, liver, and kidney, significantly decreased during aging. Cytochrome c oxidase, an enzyme involved in electron transport in mitochondria, initially increased, but subsequently decreased in the aged brain, whereas no significant alteration was observed in the liver mitochondrial antioxidant enzymes. The present studies suggest that the accumulation of oxidized proteins during aging is most likely to be linked with an age-related decline of antioxidant enzyme activities, whereas lipid peroxidation is less sensitive to predict the aging process.     

Enzymic analysis of the structure of oxidized potato starches

The binding of TNP-ATP (2' or 3'-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 microM) and one with lower affinity (Kd = 0.9 microM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 microM). The binding of [35S]ATP alpha S was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 microM) and six at the liver enzyme (Kd = 12 microM). The Paracoccus enzyme had only one binding site (Kd = 16 microM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e-stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature.     

Reversibility of hepatic mitochondrial damage in rats with long-term cholestasis

BACKGROUND/AIMS: Long-term bile duct ligation in rats is associated with secondary biliary cirrhosis and metabolic alterations, e.g. mitochondrial dysfunction. We performed the current studies to characterize the reversibility of hepatic mitochondrial dysfunction after reversing biliary obstruction by Roux-en-Y anastomosis. METHODS: Rats were studied after 4 weeks of bile duct ligation, and after 5 or 14 days of reanastomosis. Control rats were pair-fed to treated rats and all rats were studied after starvation for 24 h. Mitochondria were isolated by differential centrifugation and enzyme activities determined by spectrophotometric methods. RESULTS: In comparison to controls, plasma beta-hydroxybutyrate concentrations were decreased in bile duct ligated rats (200+/-70 vs. 790+/-200 micromol/l) and remained decreased after relief of biliary obstruction. In contrast, plasma free fatty acids were not different between controls and treated rats. Oxidative metabolism of L-glutamate, succinate and duroquinol was decreased in liver mitochondria from bile duct ligated rats. After relief of biliary obstruction, the metabolism of L-glutamate and duroquinol normalized quickly, whereas succinate metabolism remained impaired. Similar results were obtained for the mitochondrial oxidases in disrupted mitochondria. The activities of complex I, II, III and V of the respiratory chain were reduced in bile duct ligated rats. After relief of biliary obstruction, complex I and III normalized quickly, whereas complex II and V remained impaired. Oxidative metabolism of long-chain fatty acids by isolated liver mitochondria was decreased in bile duct ligated rats and did not recover after relief of biliary obstruction. CONCLUSIONS: Long-term cholestasis in the rat is associated with a decrease in specific functions of liver mitochondria which recover only partially after Roux-en-Y anastomosis. The persistence of decreased mitochondrial fatty acid metabolism cannot be explained by impaired activity of the respiratory chain, but is more likely due to alterations in mitochondrial beta-oxidation.     

Effects of methylxanthine derivatives on antitumor activity and toxic side effect of adriamycin induced by inhibition of DNA biosynthesis

The antitumor activity of adriamycin (ADR) was enhanced by combination with theobromine or pentoxifylline. Theobromine increased the concentration of ADR in the tumor without any effects on that in the heart and the liver. The influence of the combination of theobromine or pentoxifylline with ADR on the lipid peroxide level (indicating the ADR-induced side effect) and on DNA biosynthesis (indicating the side effect and antitumor activity) were examined. When ADR was administered into mice, the lipid peroxide level in the liver and the heart increased. However, the combination of theobromine or pentoxifylline did not enhance the ADR-induced increment of the lipid peroxide level in the liver, and moreover, it inhibited that in the heart. The decrease of DNA biosynthesis in the liver and the heart, induced by ADR, were not enhanced by combination with theobromine or pentoxifylline. On the other hand, the combination of theobromine with ADR significantly increased the inhibition of DNA biosynthesis in the tumor. These findings indicate that the combination of theobromine or pentoxifylline with ADR have no effect on the side effects of ADR in the liver and the heart, with the increase of antitumor activity of ADR in the tumor, and it is suggested that these drugs will be of value as a biochemical modulator of ADR.     

A study of hepatic mitochondrial respiration and microsomal cytochrome P450 content in mice infected with the liver fluke Fasciola hepatica

Previous studies of the effects of infection of Wistar rats with the common liver fluke, Fasciola hepatica, on liver bioenergetic and drug metabolism have demonstrated a loss of respiratory control in isolated mitochondria and reduced microsomal cytochrome P450 content, respectively, from 2 weeks post-infection throughout the acute phase of the infection. In the present study male Balb/c mice infected with F. hepatica showed a loss of respiratory control in isolated liver mitochondria only at 4 weeks post-infection. A similar time course was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. Preparations from infected CBA mice showed similar changes to Balb/c mice but mitochondrial respiration in preparations from infected Swiss outbred mice was normal. A host difference between strains of mice and between mice and rats is therefore evident in the timing and extent of liver mitochondrial dysfunction and in the timing of the decrease in the cytochrome P450 content of hepatic microsomes. This difference between hosts may be related to the reported differences in cellular inflammatory responses to the migrating juvenile flukes in the livers of rats and mice.     

Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Forward (-->ATP) and reverse (-->CrP) fluxes through the creatine kinase reaction were determined in isolated rat and bovine heart mitochondria and with soluble MM-CK from rabbit skeletal muscle, using 31P-saturation transfer NMR. With soluble MM-CK forward and reverse fluxes were identical in the absence and presence of BSA or rat liver mitochondria. Addition of liver mitochondria decreased fluxes with increasing mitochondria concentration. The fluxf/Vmax(f) ratio was 0.006 with 10 mg BSA and 0.04 with 10 mg rat liver mitochondria, respectively. With heart mitochondria, fluxr was considerably higher than fluxf and the fluxf/Vmax(f) ratio was 1.7 for rat heart and 0.22 for bovine heart. It is concluded that in the presence of isolated mitochondria, the flux through the creatine kinase is driven by the mitochondrial ATP-ADP turnover. Therefore the fluxf/Vmax(f) ratio is highest for rat heart mitochondria with a high ATP-ADP turnover, intermediate for bovine heart mitochondria and low for MM-CK in the presence of liver mitochondria. It is lowest with MM-CK alone, where the creatine kinase reaction is at equilibrium and external ATP-ADP turnover is absent. The higher reverse than forward fluxes of mitochondrial creatine kinase determined at steady state by saturation transfer NMR, are caused mainly by a high ATP<-->Pi exchange in heart mitochondria preparations, having a high ATPase activity, compared to liver mitochondria.