Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone

Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydro-testosterone (1 μM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 μM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.     

Interaction of ethanol oxidation with glucuronidation in isolated hepatocytes.

1. Glucuronidation of harmol, 2-naphthol, 4-methylumbelliferone and phenolphthalein in isolated hepatocytcs was inhibited up to 50 per cent in the presence of low concentrations of ethanol (10 mM). Sulphate conjugation was unaffected. The inhibitory effect of ethanol was reversed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase dependent ethanol oxidation. 2. The oxidation of harmine to harmol was not affected by 10 mM ethanol, but in hepatocytes isolated from phenobarbital-treated rats glucuronidation of the formed harmol was inhibited about 30 per cent in the presence of this amount of ethanol. 3. Ethanol increased the intracellular NADH/NAD⁺ ratio as did lactate and sorbitol. The latter two substances were also inhibitory to glucuronidation having no effect on the sulphate conjugation. 4. The synthesis of UDPglucuronic acid was inhibited by ethanol both in the presence and absence of a substrate undergoing glucuronidation. It is suggested that the inhibitory effect of ethanol on glucuronidation is due to a decreased UDPglucuronic acid synthesis caused by the increased NADH/NAD⁺ ratio resulting from the alcohol dehydrogenase dependent oxidation of ethanol.     

Effect of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism.

The hepatic activity of alcohol dehydrogenase was increased after 7, 14 and 42 days of stress induced by immobilization of rats for 2.5 $\text{hr}\text{day}$. A single immobilization had no effect on the activity of alcohol dehydrogenase. Immobilization for 14 days resulted in increases in the rates of ethanol metabolism. This was not associated with changes in the activity of the microsomal ethanol-oxidizing system, microsomal catalase, cytochrome P-450, or NADPH cytochrome c reductase. A decrease in the hepatic phosphorylation potential ([ATP]/[ADP][P_i]) was found to be due to a decrease in [ATP] and an increase in [P_i]; however, there were no changes in O₂ consumption by liver slices or in hepatic (Na⁺ + K⁺)-stimulated adenosine triphosphatase activity. The increased rate of ethanol metabolism after stress remains unexplained since alcohol dehydrogenase activity is not rate-limiting in ethanol oxidation and there were no increases in ethanol oxidation by microsomes or in mitochondrial oxidative rate.     

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 microM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the alpha 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD-/NADH and NADP++NADPH ratios and in the mitochondrial NAD+/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD+. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.     

ETHANOL METABOLISM IN THE LIVER

Summary: Ethanol elimination by the liver, a relatively constant process, may be increased at high concentrations of ethanol, when certain substances like fructose or pyruvate are metabolized together with ethanol or after prolonged exposure for ethanol. The metabolic effects on the liver are different at high and at low concentrations of ethanol. A number of enzymes or enzyme systems, – liver alcohol dehydrogenase, catalase and mixed-function oxidase – can in vitro catalyze the oxidation of ethanol, but little is known about the actual role of each enzyme at different metabolic conditions. The existence of more than one pathway for ethanol metabolism in the liver is now becomming increasingly evident, but whether the non-ADH mediated ethanol oxidation occurs via catalase, or mixed-function oxidase system, or both, cannot at present be decided. The activity of liver alcohol dehydrogenase cannot be induced by continuous use of ethanol. The ADH-mediated ethanol oxidation may be increased when the steady state concentration of free NADH in the cytoplasm is lowered. The rate of ethanol oxidation under in vivo conditions seems not to be determined by the rate of acetaldehyde elimination, although acetaldehyde is a product of ethanol metabolism. The reoxidation of NADH may proceed by NADH-linked substrate system, by transhydrogenation to NADP⁺ or by transporting reducing equivalents into mitochondria where they are oxidized. In this review an attempt is made to examine the effects of a number of substrates or metabolic conditions which may enhance ethanol oxidation (concentration of ethanol, fructose, pyruvate, D-glyceraldehyde, oxygen, CO₂ and 2,4-dinitrophenol) and to discuss the mechanisms involved in this action. It is concluded that the effect of pyruvate and fructose (maybe also CO₂ and D-glyceraldehyde) proceeds via ADH-mediated pathway (possibly involving the so-called “malic enzyme shuttle”) whereas the acceleration in ethanol metabolism, observed in rat liver preparations when high concentrations of ethanol are metabolized, is associated with the participation of non-ADH pathway (s) in ethanol metabolism.     

Rapid oxidation of NADH via the reconstituted malate-aspartate shuttle in systems containing mitochondrial and soluble fractions of rat liver: Implications for ethanol metabolism

In an attempt to assess whether hydrogen shuttle capacity might serve as the rate-limiting factor in the hepatic oxidation of ethanol, the malate-aspartate shuttle was reconstituted in systems containing mitochondrial and soluble fractions of rat liver. Oxidation of NADH was stimulated slightly by the addition of either glutamate or malate but when both substrates were added the stimulation was far stronger. This effect was greatly enhanced by aspartate indicating that. when not added to the system, extramitochondrial aspartate was limiting. It was found that the rate of oxidation of NADH was directly related to the amount of mitochondrial protein present but extramitochondrial reactions became restrictive when the ‘soluble protein/mitochondrial protein’ ratio fell below 0.8. When calculated on a whole tissue basis the maximum rate of oxidation of NADH by the reconstituted shuttle was substantially higher than reported rates of ethanol oxidation in vino. The results are discussed in relation to the normal control of ethanol metabolism.     

Role of the malate—aspartate shuttle in the metabolism of ethanol in vivo

In order to assess the role of the malate-aspartate shuttle during ethanol oxidation in vivo, the influence of aminooxyacetate administration was investigated on both the blood ethanol elimination rate and ethanol induced alterations of the liver redox state. Aminooxyacetate reduced by 50 per cent the blood ethanol elimination rate; it promoted at the same time the reduced state of the extramitochondrial space, whereas it induced a more oxidized state in the mitochondria. The comparison between the effect of either single or repeated administration of aminooxyacetate leads to the conclusion that the degree of inhibition of aspartate aminotransferase activity and the reduction of blood ethanol elimination rate are not directly connected. These findings suggest that an increased flux through other shuttle systems concerned with transport of reducing equivalents occurs when the malate-aspartate shuttle is inhibited.     

Effect of propranolol on ethanol metabolism-evidence for the role of mitochondrial NADH oxidation.

Ethanol metabolism in the rat as measured in vivo by ¹⁴CO₂ production or in vitro by the removal of ethanol by liver slices was inhibited approximately 30 per cent by propranolol. There was no inhibitory effect of propranolol on rat liver alcohol dehydrogenase, catalase. NADPH-dependent microsomal ethanol oxidation or formate oxidation to ¹⁴CO₂. Propranolol inhibited fatty acid oxidation to ¹⁴CO₂in vivo as well as by liver slices and isolated hepatic mitochondria. NADH oxidation by hepatic mitochondria was also reduced by propranolol. 2,4-Dinitrophenol treatment or chronic ethanol feeding of rats stimulated alcohol metabolism as well as hepatic mitochondrial NADH oxidation. These increases were abolished by propranolol. The effect of propranolol in blocking the increase in ethanol oxidation after chronic alcohol feeding appears to be related to its action on the mitochondrial re-oxidation of NADH to NAD. Propranolol inhibits mitochondrial NADH oxidation, while 2,4-dinitrophenol or chronic ethanol feeding stimulates this process. The present studies support the concept that the rate of hepatic ethanol metabolism is limited, at least in part, by the mitochondrial oxidation of NADH.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Effects of dichloroacetate on the lactate/pyruvate ratio and on aspartate and leucine metabolism in cultured rat skeletal muscle cells

Dichloroacetate accelerates pyruvate and lactate metabolism in many tissues and has often been shown to increase the lactate/pyruvate (L/P) ratio, which is assumed to be in equilibrium with the cytosolic NADH/NAD⁺ ratio. The cause of the drug-induced increase in L/P ratio is not known, but abnormalities in the malate-aspartate hydrogen shuttle have been implicated in perfused skeletal muscle. In addition, indirect studies in perfused skeletal muscle suggest that dichloroacetate inhibits branched chain amino acid oxidation. In the present studies, cultured rat skeletal muscle cells were used to examine the effect of dichloroacetate on the L/P ratio and on the metabolism of aspartate and of leucine. Dichloroacetate increased the L/P ratio after only 60 min of incubation, and the process was saturable with an ED₅₀ of 1.0 mM. Drug treatment resulted in marked decreases in cell lactate, pyruvate, and alanine, but no significant differences were observed in cell ATP or in cell levels of compounds of the malate-aspartate shuttle (e.g. malate, aspartate, glutamate or α-ketoglutarate). In addition, dichloroacetate had no consistent effect on aspartate oxidation or on aspartate conversion into glutamate. However, action of the drug did result in an inhibition of leucine oxidation, an effect which contrasts with dichloroacetate action in heart or liver, wherein the drug stimulates leucine oxidation. These results confirm other studies showing that dichloroacetate raises the L/P ratio in skeletal muscle, but do not provide evidence for drug-induced alterations of the malate-aspartate cycle, the principal cytosolic hydrogen shuttle system.     

Inhibitory effect of propylthiouracil on the development of metabolic tolerance to ethanol

Chronic ethanol administration (4-5 weeks) to female spontaneously hypertensive (SH) rats led to a marked increase in the rate of ethanol metabolism. This was accompanied by an increase in hepatic alcohol dehydrogenase (ADH) and by an increase in the rate of oxygen consumption in perfused livers of these animals. Treatment with the antithyroid drug 6-n-propyl-2-thiouracil (PTU) during the last 9 days (40 mg/kg/day) of the chronic administration of ethanol reduced hepatic oxygen consumption, resulting in a net diminution of the metabolic tolerance to ethanol, despite a further elevation in ADH activity. In these animals, microsomal ethanol-oxidizing system (MEOS) activity was not affected by chronic ethanol administration or by treatment with PTU. Data strongly suggest that in the female SH rat all the metabolic tolerance to ethanol proceeds via the ADH pathway, and that the increase in hepatic oxygen consumption is more important in the development of metabolic tolerance to ethanol than the increased ADH levels.     

Effects of clofibrate on mitochondrial function

The effects of ethyl and sodium clofibrate on mitochondrial function were studied. Both compounds exerted similar effects, the ethyl derivative being more potent. State 3 respiration was inhibited; the order of inhibitory effectiveness was NAD⁺-dependent substrates > succinate > ascorbate. State 4 NAD⁺-linked oxidation was not significantly affected, but state 4 oxidations of succinate and ascorbate were stimulated by ethyl clofibrate. Energy production was inhibited, as evidenced by the decrease in the respiratory control ratio, the P/O ratio and the ATP-³²P exchange reaction. Energy utilization, assessed by substrate or ATP-supported energy-linked Ca²⁺ uptake, was also inhibited. By contrast, energy-independent Ca²⁺ uptake was not affected. Clofibrate interfered with the integrity of the mitochondrial membranes, since it stimulated ATPase activity and increased the normally low permeability of intact mitochondria toward NADH. The transfer of reducing equivalents into the mitochondria, catalyzed by the α-glycerophosphate, fatty acid or malate-aspartate shuttles, was inhibited by sodium clofibrate. These results may explain our previous finding that the reconstituted a-glycerophosphate shuttle was not stimulated in rats fed clofibrate, despite an increase in the activity of mitochondrial α-glycerophosphate dehydrogenase.     

Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.     

Effects of castration and testosterone administration on rat liver alcohol dehydrogenase activity

The effects of castration and testosterone administration on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in male Sprague-Dawley rats. Castration increased liver alcohol dehydrogenase activity. The total liver activity in castrated animals was 2.37 ± 0.229 (S.E.) $\text{mmoles}\text{hr}$ as compared with a value of 1.39 ± 0.125 $\text{mmoles}\text{hr}$ in sham-operated controls (P < 0.01). Testosterone administration partially suppressed the enhanced activity of liver alcohol dehydrogenase produced by castration. By contrast, in control animals testosterone administration resulted in a small paradoxical increase in liver alcohol dehydrogenase. The increase in the enzyme activity in castrated animals was associated with a parallel increase in the rate of ethanol elimination. Castrated and control animals showed decreases in free cytosolic and mitochondrial NAD⁺/NADH ratios after ethanol administration. These observations suggest that testosterone (and probably other as yet unknown factors modified by castration) affects liver alcohol dehydrogenase activity, and that the total enzyme activity can be a principal limiting factor in ethanol elimination.     

Effect of chronic clofibrate administration on mitochondrial fatty acid oxidation.

The effect of chronic clofibrate administration on fatty acid oxidation by isolated liver and skeletal muscle mitochondria was studied to determine if the hypolipidemic action of clofibrate may be mediated by reducing levels of fatty acyl substrates via enhanced fatty acid oxidation. Oxygen consumption and CO₂ production associated with the oxidation of fatty acids were decreased 30 per cent in liver mitochondria from clofibrate-treated rats. By contrast, CO₂ production from acetate and citric acid cycle intermediates was not significantly affected. This indicates impairment of β-oxidation of fatty acids to the level of acetyl CoA, an interpretation supported by the findings of a decrease in ketone body production. In liver mitochondria, oxygen consumption associated with the oxidation of glutamate, succinate and ascorbate was depressed. The per cent decrease was comparable in the absence or presence of ADP or dinitrophenol, suggesting impairment of the respiratory chain. There was no effect on energy production or utilization, as evidence by unchanged respiratory control, ADP/O ratio, ATP?³²P exchange reaction, and substrate- or ATP-supported Ca²⁺ uptake. Unlike isolated liver mitochondria, there were no effects on oxygen uptake or fatty acid oxidation by muscle mitochondria. It is unlikely that the hypolipidemic effects of clofibrate are mediated by reducing fatty acyl substrate levels via enhanced fatty acid oxidation.     

Effect of carbidine on parameters of ethanol oxidation in experimental alcoholic intoxication

The effect of carbidine on enzymes of ethanol and acetaldehyde oxidation, the rate of ethanol elimination and the parameters of ethanol consumption were studied during long-term alcoholic intoxication. Carbidine administration was shown to increase the activity of alcohol dehydrogenase of the liver tissue, to decrease the activity of aldehyde dehydrogenase with a low Km to acetaldehyde. Also, the rate of ethanol elimination and a relative amount of consumed ethanol increase at the expense of an increase of the volume of consumed liquid.     

Contribution of non-ADH pathways to ethanol oxidation in hepatocytes from fed and hyperthyroid rats. Effect of fructose and xylitol

The metabolism of (1R)[1-3H]ethanol, [2-3H]lactate or [2-3H]xylitol was studied in hepatocytes from fed or T3-treated rats in the presence or absence of fructose or xylitol. The yields of tritium in ethanol, lactate, water, glycerol and glucose were determined. A simple model, describing the metabolic fate of tritium from these substrates is presented. The model allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-proR-hydrogen of ethanol and the hydrogen bound to carbon 2 of lactate (or xylitol) under identical condition. In our calculations we have taken into account that the reactions catalyzed by lactate dehydrogenase and alcohol dehydrogenase are reversible and that lactate or ethanol labelled during the metabolism of the other tritiated substrates will contribute to the tritium found in water. The contribution of non-ADH pathways to ethanol oxidation varied from 10 to 50% and was correlated to changes in the lactate/pyruvate ratio from 80 to 500. In T3-treated rats the activity of non-ADH pathways were greater than in fed rats for the same lactate/pyruvate ratio.     

Protective effect of cysteine on the inhibition of mitochondrial functions by acetaldehyde.

Acetaldehyde, the primary metabolite of ethanol oxidation, inhibited a number of mitochondrial functions in vitro. Cysteine, in vitro, afforded protection against the depression of CO₂ production from palmitate, octanoate and α-ketoglutarate by acetaldehyde. Relief occurred when the concentrations of cysteine and acetaldehyde were equimolar; greater relief was produced in the presence of excess cysteine. Acetaldehyde had no effect on glutamate-linked state 4 oxygen consumption, whereas the state 3 rate was inhibited. Cysteine almost completely relieved the inhibition of state 3 oxygen uptake, while the state 4 rate was slightly increased. Similar results were obtained with several other NAD⁺-dependent substrates. The oxidation of succinate was inhibited only by much higher concentrations of acetaldehyde than those which inhibited the oxidation of NAD-dependent substrates. This inhibition was not affected by cysteine. Thiols containing free amino and free sulfhydryl groups in close proximity were the most effective in relieving the inhibition by acetaldehyde. Although a small protective effect was observed, cysteine did not significantly prevent the inhibition of oxidative phosphorylation by acetaldehyde. However, the mitochondria remained coupled in the presence of acetaldehyde plus cysteine. The ability of cysteine and acetaldehyde to interact was demonstrated by several criteria. Cysteine may exert its protective effect by forming a complex with acetaldehyde, thereby preventing acetaldehyde from interacting with the mitochondria.     

Ethanol oxidation by isolated hepatocytes from fed and starved rats and from rats exposed to ethanol,phenobarbitone or 3-amino-triazole: No evidence for a physiological role of a microsomal ethanol oxidation system

In isolated hepatocytes from fed and starved rats, basal rates of ethanol oxidation were 1.15 and 0.71 μmoles/g wet wt, respectively, and were unchanged over the ethanol concentration range 8–96 mM. The addition of 4-methyl pyrazole (4 mM), a competitive inhibitor of alcohol dehydrogenase, largely abolished ethanol oxidation from 8mM ethanol, while at an ethanol concentration of 96 mM, the oxidation rate was inhibited by 87 per cent. Pyrazole was a less effective inhibitor of alcohol oxidation than 4-methyl pyrazole. In hepatocytes isolated from rats treated with ethanol, phenoharbitone or 3-amino-triazole, basal rates of ethanol oxidation were the same at ethanol concentrations of 8–96 mM and the rates were similar to, and never exceeded, the rate found in hepatocytes from normal fed rats. 4-Methyl pyrazole inhibited ethanol oxidation to the same extent in all liver cell preparations. regardless of the treatment the donor animal had received. Pyruvate stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation, together with the ethanol-induced accumulation of lactate, was abolished by 4-methyl pyrazole. This suggests that the capacity for alcohol oxidation in isolated liver cells is generally limited by the lack of suitable acceptors for the hydrogen generated in the cytoplasm by the alcohol dehydrogenase-catalysed oxidation of ethanol to acetaldehyde. Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration, irrespective of the prior treatment of the donor animal. This enhancement of ethanol oxidation and respiration was prevented by 4-methyl pyrazole. These artificial electron acceptors appear to act by circumventing normal pathways for the oxidation of cytoplasmic NADH generated in the conversion of ethanol to acetaldehyde. In cells from each treatment group, antimycin was more effective than rotenone as an inhibitor of ethanol oxidation; inhibition of ATP formation by oligomycin had least effect on alcohol oxidation. Ethanol oxidation by cells from alcohol-treated rats was most affected by these inhibitors of mitochondrial respiration. These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidizing system to ethanol oxidation in isolated, intact liver cells appears minimal. Thus they cast doubt on a physiological role for this system in vivo.     

Silencing of cytosolic NADP<Superscript>+</Superscript>-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells

It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells’ vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.