High-dose vitamin B1 therapy prevents the development of experimental fatty liver driven by overnutrition.

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders.This article has an associated First Person interview with the joint first authors of the paper.

Design and synthesis of biotinylated Hexylselen as a probe to identify KGA allosteric inhibitors by a convenient biomolecular interaction assay.

Hexylselen is a novel submicromolar dual KGA/GDH inhibitor, which demonstrates potent inhibition of cancer cells with minimal toxicity. To further investigation its mechanism of action, we designed and synthesized its biotinylated derivative 2 as a novel probe. From commercially available starting material, 2 was obtained in 6 steps with 13.4% overall yield. It is notable that this practical synthetic route give a template for the preparation of unsymmetrical di-benzo[d][1,2]selenazol-3(2H)-ones. Based on probe 2, we developed a novel biomolecular interaction assay for convenient and reliable test of KGA allosteric inhibitors and confirmed that hexylselen as an allosteric inhibitor of KGA sharing the same binding pocket with BPTES but not with Ebselen via competitive experiments.

Parapyruvate, an Impurity in Pyruvate Supplements, Induces Senescence in Human Fibroblastic Hs68 Cells via Inhibition of the α-Ketoglutarate Dehydrogenase Complex.

Commercial dietary supplements of calcium pyruvate claim to be beneficial for losing weight, increasing muscle endurance, and regulating metabolism. Most industrial preparations have some impurities, including parapyruvate. Parapyruvate is an inhibitor of the α-ketoglutarate dehydrogenase complex (KGDHC). However, the effect and mechanism of parapyruvate on cell senescence and the content of parapyruvate in the dietary supplements of calcium pyruvate are unknown. In this study, we prepared pure parapyruvate with a purity of 99.8 ± 0.1% and investigated its ability to inhibit KGDHC activity and affect fibroblast senescence. Parapyruvate dose-dependently decreased KGDHC activity, with an IC50 of 4.13 mM and induced Hs68 cell senescence. Calcium ions, a KGDHC activator, antagonized the senescent effects of parapyruvate. The parapyruvate content was 1.4 ± 0.1% to 10.6 ± 0.2% in five brands of calcium pyruvate supplements. In this study, we showed that parapyruvate strongly induces Hs68 cell senescence by inhibiting KGDHC activity. Because of its KGDHC inhibition activity, the parapyruvate content should be an important issue for the food safety of calcium pyruvate supplements.

Role of thiamine in Huntington's disease pathogenesis: In vitro studies.

BACKGROUND: Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES: The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS: The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS: Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS: The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal Glycoalkaloid Biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16ß-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Mitochondrial Target of Nobiletin's Action.

Nobiletin is an 0-methylated flavonoid found in citrus peels that have anticancer, antiviral, neuroprotective; anti-inflammatory activities and depending on the cell types exhibits both pro- or anti-apoptotic properties We have found that nobiletin decreases oxygen consumption by bovine brain isolated mitochondria in the presence of glutamate and malate and increases in the presence of succinate. In paralleli nobiletin increases NADH: oxidation, a-ketoghitarate dehydrogenase activities and through matrix substrate-level phosphorylation elevates the a-ketoglutarate-dependent-production-of ATP. In addition, nobiletin reduces the production of peroxides in the presence of complex I substrates and slightly enhances succinate-driven H(2)0(2) formation. Besides, nobiletin induces transient elevation of membrane potential followed by mild depolarization. Affinity purified, nobiletin binding proteins revealed one major anti-NDUFVl positive protein with 52kD and NADH: ubiquinone oxidoreductase activity. This fraction can produce peroxide that is inhibited by nobiletin. We propose that nobiletin may act as a mild "uncoupler", which through activation of a-ketoglutarate dehydrogenase (a-KGDH)-complex and acceleration of matrix substrate-level phosphorylation maintains membrane potential at an abnormal level. This switch in mitochondrial metabolism could elevate succinate-driven oxygen consumption that may underlay in both pro- and anti-apoptotic effects of nobiletin.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

BACKGROUND: S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. METHODS: In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. RESULTS: We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. CONCLUSIONS: Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. GENERAL SIGNIFICANCE: Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Nobiletin restores impaired hippocampal mitochondrial bioenergetics in hypothyroidism through activation of matrix substrate-level phosphorylation.

OBJECTIVE: Evaluation of the effect of citrus flavonoid - nobiletin on the bioenergetics of synaptic and non-synaptic mitochondria in the hippocampus of hypothyroid rats. METHODS: Male Wistar rats were divided into hypothyroid (methimazole-treated), nobiletin supplemented hypothyroid, thyroxine-treated hypothyroid, and euthyroid (control) groups. Synaptic and non-synaptic (cell) mitochondria were isolated from hippocampus. Oligomycin-sensitive, oligomycin-insensitive, α-ketoglutarate dehydrogenase-dependent synthesis of adenosine triphosphate (ATP), succinate dehydrogenase, and hexokinase activities were determined luminometrically and spectrophotometrically, respectively. RESULTS: Decreased synthesis of oligomycin-sensitive and oligomycin-insensitive ATP in hypothyroid rat hippocampus was observed in synaptic and non-synaptic mitochondria. Supplementation of hypothyroid rats with nobiletin increases oligomycin-insensitive and α-ketoglutarate-dependent production of ATP in both types of mitochondria. The activity of succinate dehydrogenase in non-synaptic mitochondria and the activities of hexokinase in both types of mitochondria were normalized in nobiletin-treated hypothyroid rats. DISCUSSION: Nobiletin restores reduced mitochondrial metabolism in hypothyroid rat hippocampus through acceleration of matrix substrate-level phosphorylation that may be important for the prevention of hypometabolic complications in neurological disorders.

Pharmacological effect of carvacrol on D: -galactosamine-induced mitochondrial enzymes and DNA damage by single-cell gel electrophoresis.

The present study aimed at investigating the effect of carvacrol on hepatic mitochondrial enzyme activities and DNA damage in D: -galactosamine (D: -GalN)-induced hepatotoxicity in male albino Wistar rats. The activities of hepatic mitochondrial enzymes such as isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADPH dehydrogenase and cytochrome c oxidase significantly decreased in D: -GalN-hepatotoxic rats, and administration of carvacrol brought these parameters towards normality. In D: -GalN-hepatotoxic rats, the hepatic mitochondrial concentration of thiobarbituric acid reactive substances significantly increased, and administration of carvacrol significantly reduced them towards normality. Furthermore, the activities of enzymatic antioxidants such as superoxide dismutase and glutathione peroxidase and the levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione decreased significantly in the liver mitochondria. Administration of carvacrol returned the enzymatic and non-enzymatic antioxidants towards normality. D: -GalN-hepatotoxic rats had increased DNA damage, which administration of carvacrol significantly decreased. These results suggest that carvacrol has liver mitochondrial antioxidant properties and possesses a defensive effect against mitochondrial enzymes and DNA damage in D: -GalN-induced rats.

Alpha-ketoisocaproic acid and leucine provoke mitochondrial bioenergetic dysfunction in rat brain.

Patients affected by maple syrup urine disease (MSUD) present severe neurological symptoms and brain abnormalities, whose pathophysiology is poorly known. In the present study we investigated the in vitro effects of leucine (Leu), alpha-ketoisocaproic acid (KIC) and alpha-hydroxyisovaleric acid (HIV), respectively, the branched-chain amino, keto and hydroxy acids that most accumulate in MSUD, on brain bioenergetic homeostasis, evaluating respiratory parameters obtained by oxygen consumption, membrane potential (Psim), NAD(P)H content, swelling and citric acid cycle enzyme activities in mitochondrial preparations from rat forebrain using glutamate plus malate, succinate or alpha-ketoglutarate as respiratory substrates. KIC increased state 4 and decreased the respiratory control ratio with all substrates, in contrast with Leu and HIV. Furthermore, KIC and Leu, but not HIV, decreased state 3 using alpha-ketoglutarate. A KIC-induced selective inhibition of alpha-ketoglutarate dehydrogenase activity was also verified, with no alteration of the other citric acid cycle activities. The ADP/O ratio and the mitochondrial NAD(P)H levels were also reduced by KIC using glutamate/malate and alpha-ketoglutarate. In addition, KIC caused a reduction in the Psim when alpha-ketoglutarate was the substrate. Finally, KIC was not able to induce mitochondrial swelling. The present data indicate that KIC acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor possibly through its inhibitory effect on alpha-ketoglutarate dehydrogenase activity, while Leu acts as a metabolic inhibitor. It is suggested that impairment of mitochondrial homeostasis caused by the major metabolites accumulating in MSUD may be involved in the neuropathology of this disease.

Oxidative stress and antioxidant defenses in asthmatic murine model exposed to printer emissions and environmental tobacco smoke.

Exposure to particulate emissions from printer and cigarette smoke affects the structure and function of mitochondria, which may account for the pathogenesis of respiratory diseases. The addition of charge for the pollutant aerosols may increase the toxicity by their deposition in the lower respiratory tract. The mitochondrial damage in the lung of asthmatic mice was assessed by examining the levels of reactive oxygen species (ROS), lipid peroxides, reduced glutathione, and the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, complexes I to IV, and cytochrome c. The oxidative phosphorylation (levels of adenosine triphosphatase) was evaluated for the assessment of mitochondrial functional capacity. We found highly significant elevated levels of ROS, lipid peroxides, and decreased levels of mitochondrial enzymes in the mice exposed to environmental tobacco smoke and printer emissions + environmental tobacco smoke (ETS). However, mice exposed to printer emissions alone exhibited slight significant variations in the parameters studied. From the results, we conclude that printer emissions exert a synergistic effect in the presence of ETS and induce intense damage to the lung mitochondria by disrupting the structural and functional integrity of the mitochondrial membrane.

Neuronal mitochondrial toxicity of malondialdehyde: inhibitory effects on respiratory function and enzyme activities in rat brain mitochondria.

Malondialdehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines so as to modify proteins and inactivate enzymes; it can also modify nucleosides so as to cause mutagenicity. Brain mitochondrial dysfunction is a major contributor to aging and neurodegenerative diseases. We hypothesize that MDA accumulated during aging targets mitochondrial enzymes so as to cause further mitochondrial dysfunction and additional contributions to aging and neurodegeneration. Herein, we investigated the neuronal mitochondrial toxic effects of MDA on mitochondrial respiration and activities of enzymes (mitochondrial complexes I-V, alpha-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH)), in isolated rat brain mitochondria. MDA depressed mitochondrial membrane potential, and also showed a dose-dependent inhibition of mitochondrial complex I- and complex II-linked respiration. Complex I and II, and PDH activities were depressed by MDA at >or=0.2 micromol/mg; KGDH and complex V were inhibited by >or=0.4 and >or=1.6 micromol MDA/mg, respectively. However, MDA did not have any toxic effects on complex III and IV activities over the range 0-2 micromol/mg. MDA significantly elevated mitochondrial reactive oxygen species (ROS) and protein carbonyls at 0.2 and 0.002 micromol/mg, respectively. As for the antioxidant defense system, a high dose of MDA slightly decreased mitochondrial GSH and superoxide dismutase. These results demonstrate that MDA causes neuronal mitochondrial dysfunction by directly promoting generation of ROS and modifying mitochondrial proteins. The results suggest that MDA-induced neuronal mitochondrial toxicity may be an important contributing factor to brain aging and neurodegenerative diseases.

Thiamine deficiency-related brain dysfunction in chronic liver failure.

End-stage chronic liver failure results in thiamine deficiency caused principally by depletion of liver thiamine stores. Chronic liver failure also leads to increased brain ammonia concentrations. Both ammonia and thiamine deficiency result in decreased activity of alpha-ketoglutarate dehydrogenase, a rate-limiting tricarboxylic acid cycle enzyme. Loss of enzyme activity results in a mitochondrial oxidative deficit in brain and consequent increases in brain lactate, oxidative/nitrosative stress, cellular energy impairment and release of proinflammatory cytokines, all of which have been described in brain in end-stage chronic liver failure. Synergistic effects of ammonia exposure and thiamine deficiency could explain the diencephalic and cerebellar symptomatology described in patients with "hepatic encephalopathy". Unsuspected brain lesions due to thiamine deficiency could explain the incomplete resolution of neuropsychiatric symptoms following the use of ammonia-lowering agents or liver transplantation in patients with end-stage chronic liver failure. These findings underscore the need for prompt, effective thiamine supplementation in all patients with chronic liver failure.

High doses of nicotinamide prevent oxidative mitochondrial dysfunction in a cellular model and improve motor deficit in a Drosophila model of Parkinson's disease.

Nicotinamide, the principal form of niacin (vitamin B3), has been proposed to be neuroprotective in Parkinson's disease. However, the effects and mechanisms of nicotinamide on motor function in animals and on mitochondrial function in cellular systems have not been well studied. We hypothesized that niacin-derived NAD(P)H as antioxidants and enzyme cofactors could inhibit oxidative damage and improve mitochondrial function and thus protect neurodegeneration and improve motor function. In the present study, the effects of nicotinamide on mitochondrial function and oxidative stress were studied in a 1-methyl-4-phenylpyridinium (MPP(+))-induced cellular model of Parkinson's disease, and the effects of improving motor dysfunction were studied in an alpha-synuclein transgenic Drosophila Parkinson's model. Mitochondrial function was tested by measuring the activity of mitochondrial complex I and alpha-ketoglutarate dehydrogenase, and oxidative damage was tested by measuring reactive oxygen species, DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine and Comet assay), and protein oxidation (protein carbonyls) levels. Nicotinamide at a relatively higher concentration, that is, 100-fold of the level in the cell culture medium (101 mg/L), significantly protected SK-N-MC human neuroblastoma cells from an MPP(+)-induced decrease in cell viability, complex I and alpha-ketoglutarate dehydrogenase activity, and an increase in oxidant generation, DNA damage, and protein oxidation. In the Drosophila model, nicotinamide at 15 and 30 mg/100 g diet significantly improved climbing ability. These results suggest that nutritional supplementation of nicotinamide at high doses decreases oxidative stress and improves mitochondrial and motor function in cellular and/or Drosophila models and may be an effective strategy for preventing and ameliorating Parkinson's disease.

Identification of a novel PP2C-type mitochondrial phosphatase.

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.

Dactylorhin B reduces toxic effects of beta-amyloid fragment (25-35) on neuron cells and isolated rat brain mitochondria.

beta-amyloid is strongly implicated in Alzheimer's pathology, and mitochondria play an important role in neurodegenerative disorders. Dactylorhin B [short for bis(4-beta-D-glucopyranosyloxybenzyl)-2-beta-D-glucopyranosyl-2-isobutyltartrate (DHB)] is an active compound isolated from Coeloglossum viride. (L.) Hartm. var. bracteatum (Willd.) and may have neuroprotective effects. In the present study, we investigated the damage of rat brain mitochondrial function induced by beta-amyloid and the protective effect of DHB. Isolated rat brain mitochondria were incubated with amyloid-beta peptide (Abeta)(25-35) alone or together with DHB. reactive oxygen species production, pyruvate dehydrogenase complex activity, alpha-ketoglutarate dehydrogenase complex activity, cytochrome c oxidase activity, mitochondrial swelling, mitochondrial membrane potential and the release of cytochrome c from mitochondria were measured. Data showed that Abeta(25-35) directly disrupted mitochondrial function, inhibited the key enzymes and contributed to apoptosis and the deficiency of energy metabolism. Co-incubation of DHB attenuated Abeta(25-35)-induced pathological changes. The significance of DHB in the management of mitochondria-related disorders is discussed.

Efficacy of levo carnitine and alpha lipoic acid in ameliorating the decline in mitochondrial enzymes during aging.

BACKGROUND: Mitochondria are central to energy production and are therefore fully integrated into the rest of the cell's physiological responses to stress. The age-related decline of capacity of each cell to manufacture energy (as ATP) is due to the progressive loss of structural integrity of mitochondria. It is apparent that as the body ages, the cells become less and less able to maintain threshold levels of cellular energy production. METHODS: In the present study we have evaluated the efficacy of carnitine, a mitochondrial metabolite and lipoic acid, a potent antioxidant on the activities of the tri carboxylic acid (TCA) cycle enzymes like succinate dehydrogenase, malate dehydrogenase, alpha-ketoglutarate dehydrogenase, Isocitrate dehydrogenase and electron transport complex I-IV in young and aged heart mitochondria. RESULT: We observed that there was an age-dependent decrement in the levels of the TCA cycle enzymes and electron transport chain complexes. Supplementation of carnitine (300 mg/kg bw/day) and lipoic acid (100 mg/kg bw/day) for 30 days brought the activities of these enzymes to almost near normal levels. CONCLUSION: These findings suggest that the combination of these drugs raises the mitochondrial energy producing capabilities by reversing the age-associated decline in mitochondrial enzyme activities and thereby protecting mitochondria from aging.

Cu2+ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo.

Wilson's disease results from mutations in the P-type Cu(2+)-ATPase causing Cu(2+) toxicity. We previously demonstrated that exposure of mixed neuronal/glial cultures to 20 microM Cu(2+) induced ATP loss and death that were attenuated by mitochondrial substrates, activators, and cofactors. Here, we show differential cellular sensitivity to Cu(2+) that was equalized to 5 microM in the presence of the copper exchanger/ionophore, disulfiram. Because Cu(2+) facilitates formation of oxygen radicals (ROS) which inhibit pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), we hypothesized that their inhibition contributed to Cu(2+)-induced death. Toxic CU(2+) exposure was accompanied by early inhibition of neuronal and hepatocellular PDH and KGDH activities, followed by reduced mitochondrial transmembrane potential, DeltaPsi(M). Thiamine (1-6 mM), and dihydrolipoic acid (LA, 50 microM), required cofactors for PDH and KGDH, attenuated this enzymatic inhibition and subsequent death in all cell types. Furthermore, liver PDH and KGDH activities were reduced in the Atp7b mouse model of Wilson's disease prior to liver damage, and were partially restored by oral thiamine supplementation. These data support our hypothesis that Cu(2+)-induced ROS may inhibit PDH and KGDH resulting in neuronal and hepatocellular death. Therefore, thiamine or lipoic acid may constitute potential therapeutic agents for Wilson's disease.

Chemopreventive efficacy of selenium against N-nitrosodiethylamine-induced hepatoma in albino rats.

The chemopreventive/chemotherapeutic effect of sodium selenite on tricarboxylic acid cycle key enzymes was investigated against hepatoma induced by environmental carcinogen N-nitrosodiethylamine. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in hepatoma and surrounding tissues of hepatoma-bearing rats were observed. Upon selenium supplementation the above biochemical changes were reverted in a dose- and duration-dependent manner. This study further confirms the chemopreventive/chemotherapeutic effect of sodium selenite which is found to be more effective in the initiation phase of carcinogenesis.

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria.

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.