Product Of Ethanol Metabolism Research Articles

ACETALDEHYDE INVOLVEMENT IN ETHANOL-INDUCED POTENTIATION OF RAT HEPATOCYTE DAMAGE DUE TO THE CARCINOGEN 1,2-DIBROMOETHANE

Previous experiments with hepatocytes isolated from ethanol-treated rats showed that alcohol potentiates the toxic action of 1,2-dibromoethane (DBE) by inhibiting its metabolism via glutathione-S-transferase. The aim of this study was to investigate whether acetaldehyde, the main product of ethanol metabolism, may be responsible for such inactivation. By pretreatment with 4-methylpyrazole, an inhibitor of acetaldehyde formation, the ethanol inactivation of glutathione transferase was actually prevented. As a consequence of this protective action, 4-methylpyrazole also prevented the high basal lipid peroxidation and the potentiated DBE toxicity observed in hepatocytes from ethanol-dosed animals. Finally, the inactivation of glutathione-S-transferase by concentrations of acetaldehyde likely to occur in the ethanol-intoxicated animal was confirmed in an in vitro model by direct aldehyde addition to hepatocyte suspensions.

Effects of acetaldehyde on glucose-6-phosphate dehydrogenase activity and mRNA levels in primary rat hepatocytes in culture

Ethanol has been shown to induce the activity of glucose-6-phosphate dehydrogenase (G6PDH). To clarify the mechanism behind this induction, we examined the role of acetaldehyde (AA), the first product of ethanol metabolism. In primary adult rat hepatocytes maintained in chemically defined medium, we examined the effect of AA on G6PDH activity, mRNA levels and lipid synthesis. We observe a 40% increase in G6PDH activity and a similar increase in mRNA levels, following exposure to 100 μM AA. The increase in activity was found to be maximal at 24 h while mRNA levels increased over controls as early as 3 h. The induction in G6PDH by AA was found to occur at lower concentrations and earlier time points than those reported using ethanol. The role of insulin, a known inducer of G6PDH activity was studied alone and in combination with AA on both G6PDH activity and mRNA levels as well as lipid biosynthesis. Insulin (300 ng/ml) was found to increase G6PDH activity, mRNA levels and [ 14C]-acetate incorporation into lipid. It was also shown to have an additive effect with AA on G6PDH activity, suggesting their actions are mediated via different mechanistic pathways. No change in [ 14C]-acetate incorporation into lipid, however, was observed with acetaldehyde alone.

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H(+) extrusion activity. This effect is strictly dependent on the presence of K(+) in the medium and is suppressed by the presence of the plasmalemma H(+)-ATPase inhibitor vanadate. Stimulation of H(+) extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K(+) influx. These results suggest that ethanol-induced H(+) extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H(+) extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H(+)-extruding mechanism.

Acetaldehyde decreases the antitryptic activity of α1-proteinase inhibitor

The trypsin-inhibiting activity of human serum is lowered upon addition of formaldehyde or acetaldehyde thereto. Acetaldehyde reacts with α −1-proteinase inhibitor ( α 1-PI) to decrease its trypsin-inhibiting ability. Acetaldehyde has only a slight effect on the tryptic hydrolysis of benzoyl- dl-arginine -p- nitroanilide . It did not decrease the inhibitory activity of the Kunitz inhibitor (Aprotinin) or soybean trypsin inhibitor. Since aldehydes form covalent products with primary amines, primary amides, arginine, tyrosine, and tryptophan in protein, as well as methylene bridges thereby crosslinking functional groups, it is proposed that one or more such interactions affect α 1-PI activity. It is further suggested that chronically high levels of acetaldehyde, as a metabolic product of ethanol, may be a contributory factor to the generation of pancreatitis in alcoholics by possibly lowering the effective α 1-PI level which is a natural protective element from proteolysis by trypsin.

The Role of Acetaldehyde in the Pathogenesis of Acute Alcoholic Pancreatitis

Acetaldehyde (AA), the first product of ethanol metabolism, has been suggested as an important mediator in alcoholic pancreatitis, but experimental evidence has not been convincing. Prior work using the isolated perfused canine pancreas preparation has suggested that toxic oxygen metabolites generated by xanthine oxidase (XO) may mediate the early injury in pancreatitis. Xanthine oxidase is capable of oxidizing AA, and during this oxidation free radicals are released. The hypothesis that acute alcoholic pancreatitis may be initiated by AA in the presence of active XO (converted from xanthine dehydrogenase [XD]) was tested in the authors' experimental preparation by converting XD to XO by a period of ischemia, and infusing AA. Control preparations remained normal throughout the 4-hour perfusion (weight gain, 7 +/- 4 g; amylase activity, 1162 +/- 202 U/dL). One hour of ischemia or infusion of AA at 25 mg/hr or at 50 mg/hr without ischemia did not induce changes in the preparation. Acetaldehyde at 250 mg/hr induced minimal edema and weight gain (16 +/- 4 g; p less than 0.05), but not significant hyperamylasemia. Changes also were not observed when 1-hour ischemia was followed by a bolus of ethanol (1.5 g) or sodium acetate (3.0 g), or by infusion of 25 mg/hr of AA. One hour of ischemia followed by infusion of AA at 50 mg/hr or at 250 mg/hr induced edema, hemorrhage, weight gain (22 +/- 7 g [p less than 0.05] and 26 +/- 17 g [p less than 0.05]) and hyperamylasemia (2249 +/- 1034 U/dL [p less than 0.05] and 2602 +/- 1412 U/dL [p less than 0.05]). Moreover infusion of AA at 250 mg/hr after 2 hours of ischemia potentiated the weight gain (62 +/- 20 g versus 30 +/- 14 g [p less than 0.05]), but not the hyperamylasemia (3404 +/- 589 U/dL versus 2862 +/- 1525 U/dL) as compared with 2 hours of ischemia alone. Pancreatitis induced by 1 hour of ischemia followed by AA at 50 mg/hr could be inhibited by pretreatment with the free radical scavengers superoxide dismutase and catalase and ameliorated with the XO inhibitor allopurinol. The authors conclude that AA, in the presence of active XO, can initiate acute pancreatitis in the isolated canine pancreas preparation and may be important in the initiation of acute alcoholic pancreatitis in man. Toxic oxygen metabolites appear to play an important intermediary role.

THE INFLUENCE OF ETHANOL ON HEPATIC TRANSMETHYLATION

One of the most important biochemical pathways in the organism is the biosynthesis of methionine from the methylation of homocysteine. Two different reactions are responsible for this methylation, one utilizing N5-methyltetra-hydrofolate as a methylating agent and the other using betaine as the methyl donor. This paper reviews some recent findings in this laboratory, which demonstrate that ethanol-feeding to rats impairs the folate-induced reaction. Our findings also show that this impairment is compensated for through the adaptive increase in the enzyme using betaine in the biosynthesis of methionine. Further studies indicate that the mechanism of action in the impairment may occur through the formation of individual adducts between the folate-induced enzyme (methionine synthetase), its essential cofactors and acetaldehyde, a metabolic product of ethanol. These findings suggest a basis for why rats are more resistant to alcoholic liver injury than humans and may offer a means of protecting against alcoholic liver injury in man.

Depression of iron uptake from transferrin by isolated hepatocytes in the presence of ethanol is a pH-dependent consequence of ethanol metabolism.

Incubation of freshly isolated rat hepatocytes with highly purified radiolabeled rat transferrin in weakly buffered medium in the presence of 10 mM ethanol resulted in a marked diminution of iron uptake by these cells, associated with a greater pH depression than in ethanol-free control studies. This effect on iron uptake persisted, even when the cells were preincubated for 90 min with ethanol before the addition of transferrin. Increasing the buffering capacity of the system or the addition of a metabolic inhibitor of alcohol dehydrogenase (4-methylpyrazole) returned iron uptake to control values. Acetaldehyde, acetate, lactate (products of ethanol metabolism), and 3-butanol (an alcohol not metabolized by alcohol dehydrogenase) had no influence on iron uptake. Further investigation of iron uptake over the pH range 6-8.5 revealed a marked dependency of iron uptake on the extracellular pH. Leucine incorporation into cell protein was also found to be pH dependent. It is suggested that, in the light of current understanding of transferrin recycling by other cell types, the disturbances of iron homeostasis observed in alcoholics can be partially accounted for by alterations in their acid-base metabolism.

Incubation of rat hepatic tumor cells with ethanol and acetaldehyde in vitro: effects on growth rate, albumin secretion and cellular protein content.

The in vitro response (defined as changes in growth rate, cellular protein content, and albumin secretion) of liver epithelial cells to the putative hepatotoxins ethanol and acetaldehyde were evaluated using the well-characterized 32IIIA 6/2d rat liver tumor clonal cell line. Exposure of hepatic tumor cells to ethanol (50-100 mM) for a period of 3 days reduced final population density (apparently due to a drug-induced increase in mean cell cycle transit time), reduced secretion of albumin, and increased the mean cellular protein content. Since these ethanol-associated effects were also observed in cells cultured in growth medium containing acetaldehyde (0.1 mM), and were inhibited by simultaneous addition of pyrazole, the changes in the parameters measured in this study appear to be induced by products of ethanol metabolism. These data complement recent in vivo studies implicating acetaldehyde as an inhibitor of hepatocyte secretory function. The 32IIIA 6/2d liver cell system, thus, responds to certain hepatotoxic compounds in a manner analogous to the in vivo organ and may facilitate future analysis of molecular mechanisms underlying alcohol-induced liver disease under defined culture conditions.

Effect of ethanol on choline transport in rat jejunum.

The effect of ethanol on choline transport across the rat jejunum was studied by intraluminal perfusion in vivo and by influx measurement across the brush-border membrane in vitro. Acute ethanol administration (4 g/kg) through a gastric tube caused an increase in net choline absorption within 1 h. The increase was prevented by pretreatment with pyrazole, an inhibitor of ethanol metabolism. Chronic ethanol administration also caused an increase in choline absorption, the effect being unrelated to the nutritional changes that occur with ethanol ingestion. In contrast, direct instillation of 0.65 M ethanol through the perfusate caused no changes in choline absorption, and the perfusion of a 1.14 M solution even decreased absorption. The in vitro influx of choline across the mucosal membrane of the isolated rat jejunum was also enhanced by pretreatment with ethanol given by gavage 1 h prior to experimentation. Similarly, the ethanol-related increase in the influx rate was inhibited by pyrazole but was unaffected by acetaldehyde or acetate. Like ethanol, pretreatment of rats with methanol stimulated the choline influx rate. The results suggest that ethanol metabolism, rather than the direct effect of ethanol by itself, stimulates the absorption and influx of choline into the rat jejunum. The effect is not produced by the primary metabolites of ethanol, acetaldehyde, or acetate but is very likely related to stimulation by other products of ethanol metabolism.

Inhibition of testosterone production by rat Leydig cells with ethanol and acetaldehyde: prevention of ethanol toxicity with 4-methylpyrazole.

The toxic effects of ethanol and acetaldehyde on testosterone biosynthesis were examined in vitro using isolated Leydig cells prepared from adult rat testes. The ability of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, to prevent the toxic effects of ethanol on testosterone production was investigated. Ethanol was found to inhibit gonadotropin-stimulated testosterone production in a dose dependent fashion. Concentrations of ethanol (25 mg/100 reduce testosterone levels by 44% as compared to the controls. Acetaldehyde at micromolar concentrations also inhibited testosterone biosynthesis. The addition of 4-methylpyrazole to the culture medium prevented the toxic effects of ethanol as determined by testosterone production. These studies suggest that ethanol per se may not directly inhibit testosterone biosynthesis. Rather, it would appear that acetaldehyde, the first product of ethanol metabolism, may be responsible for the toxic effects of ethanol upon Leydig cells at least in vitro.

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.