Acetaldehyde levels during ethanol oxidation: a diet-induced change and its relation to liver aldehyde dehydrogenases and redox states.

3 different diets that had previously been observed to cause large differences in blood acetaldehyde levels of rats administered ethanol were compared with respect to their influence on liver enzymes metabolizing alcohol, on ethanol elimination and on the ethanol-induced changes in the hepatic content of metabolites that reflect the cytosolic or the mitochondrial redox state of the nicotine-amide dinucleotide couple. The results demonstrate that an unknown dietary factor affects the activity of liver aldehyde dehydrogenase, especially that of the low-K_m enzyme. It is suggested that these enzyme activity changes are reflected in the observed alterations in acetaldehyde levels, which in turn may be associated with the magnitude of the shift in the mitochondrial redox state during ethanol oxidation.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

The effect of methylene blue on the hepatocellular redox state and liver lipid content during chronic ethanol feeding in the rat.

Feeding of ethanol in a liquid diet to male Wistar rats caused decreases in the hepatic cytosolic and mitochondrial [NAD+]/[NADH] ratios. This redox-state change was attenuated after 16 days of feeding ethanol as 36% of the total energy intake. Supplementation of the ethanol-containing liquid diet with Methylene Blue largely prevented the ethanol-induced redox state changes, but did not significantly decrease the severity of the hepatic lipid accumulation that resulted from ethanol ingestion. Methylene Blue did not affect body-weight gain, ethanol intake or serum ethanol concentrations in ethanol-fed rats, nor did the compound influence the hepatic redox state or liver lipid content of appropriate pair-fed control animals. These findings suggest that the altered hepatic redox state that results from ethanol oxidation is not primarily responsible for the production of fatty liver after long-term ethanol feeding in the rat.     

Increased Levels of Monoamine-Derived Potential Neurotoxins in Fetal Rat Brain Exposed to Ethanol

Pregnant SD rats were exposed to ethanol (25 % (v/v) ethanol at 1.0, 2.0 or 4.0 g/kg body weight from GD8 to GD20) to assess whether ethanol-derived acetaldehyde could interact with endogenous monoamine to generate tetrahydroisoquinoline or tetrahydro-beta-carboline in the fetuses. The fetal brain concentration of acetaldehyde increased remarkably after ethanol administration (2.6 times, 5.3 times and 7.8 times as compared to saline control in 1.0, 2.0 and 4.0 g/kg ethanol-treated groups, respectively) detected by HPLC with 2,4-dinitrophenylhydrazine derivatization. Compared to control, ethanol exposure induced the formation of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol, Sal), N-methyl-salsolinol (NMSal) and 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline (6-OH-MTHBC) in fetal rat brains. Determined by HPLC with electrochemical detector, the levels of dopamine and 5-hydroxytryptamine in whole fetal brain were not remarkably altered by ethanol treatment, while the levels of homovanillic acid and 5-hydroxyindole acetic acid in high dose (4.0 g/kg) of ethanol-treated rats were significantly decreased compared to that in the control animals. 4.0 g/kg ethanol administration inhibited the activity of mitochondrial monoamine oxidase (51.3 % as compared to control) and reduced the activity of respiratory chain complex I (61.2 % as compared to control). These results suggested that ethanol-induced alteration of monoamine metabolism and the accumulation of dopamine-derived catechol isoquinolines and 5-hydroxytryptamine-derived tetrahydro-beta-carbolines may play roles in the developmental dysfuction of monoaminergic neuronal systems.     

Activities of hepatic enzymes related to ethanol oxidation and effects of ethanol on hepatic metabolites in rats with chronic liver injury.

To study the effects of ethanol on liver chronically injured by CCl4, activities of hepatic enzymes related to ethanol oxidation, influences of ethanol on hepatic metabolites, and blood ethanol disappearance were observed. (1) Activities of alcohol dehydrogenase, low- and high-Km aldehyde dehydrogenase, microsomal ethanol-oxidizing system and drug-metabolizing enzyme were remarkably decreased in the injured liver. (2) Increases in lactate/pyruvate and beta-hydroxybutyrate/acetacetate ratios were shown in control liver 2 h after ethanol ingestion. Similar but less pronounced effects of ethanol on the 'redox state' were also seen in rats with chronic liver injury. (3) Delay in ethanol disappearance was not observed until 12 h after ethanol ingestion. The ethanol-induced changes in the redox state in the injured liver were similar to those in controls. Higher ethanol concentrations in blood from rats with chronic liver injury could be related to potentiate the injured liver.     

Ethane release during metabolism of aldehydes and monoamines in perfused rat liver

Infusion of aldehyde such as acetaldehyde, propionaldehyde or benzaldehyde to perfused rat liver leads to an increase in hepatic ethane production. Half-maximal effect was obtained with about 20 microM acetaldehyde, a concentration range found in plasma during ethanol metabolism. Compounds which metabolically generate aldehydes such as monoamines (benzylamine, phenylethylamine) as substrates for monoamine oxidase or ethanol as substrate for alcohol dehydrogenase [A. Müller and H. Sies (1982) Biochem. J. 206, 153-156] are also able to elicit ethane release. Results obtained with inhibitors of hepatic aldehyde metabolism (pargyline or cyanamide) or of monamine oxidase (pargyline or tranylcypromine) suggest that metabolism of the aldehydes is required for ethane production. Radical scavenging by the addition of the flavonoid, cyanidanol, or by pretreatment with vitamin E (alpha-tocopherol) abolished ethane release, in agreement with lipid peroxidation as a source of alkane production during aldehyde metabolism.     

Age-dependent changes in in vivo ethanol metabolism and in the activity of hepatic enzymes involved in ethanol oxidation and microsomal functions

The study of the influence of the age of the animals (13 to 53 weeks) on the rate of ethanol metabolism in vivo and the total activity of liver alcohol dehydrogenase and microsomal ethanol oxidizing system showed a progressive decline with age. These effects were observed concomitantly with a diminution in the content of cytochrome P-450 and microsomal functions related to oxidative and free-radical mediated reactions, namely, NADPH oxidase activity, NADPH-dependent oxygen uptake and NADPH-or t-butyl hydroperoxide-induced chemiluminescence. It is concluded that ageing is accompanied by a diminution in the total oxidative activity of the liver tissue, which would explain the depression in basal and ethanol-induced lipid peroxidation found in the oldest group of rats studied.     

Ethanol-induced redox imbalance in rat kidneys

This study reports the effects of long-term ethanol consumption on kidney redox status, in terms of enzymatic mechanisms involved in regulating the cytosolic [NADH]/[NAD(+) ] balance. Wistar rats were treated with ethanol (2 g/kg body weight/24 h) via intragastric intubation for 10 and 30 weeks, respectively. Ethanol administration induced an enhancement of alcohol dehydrogenase activities and affected the capacity of the kidney to prevent NADH accumulation in the cytosol. After 10 weeks, the excess of NADH was balanced by increased activities of malate dehydrogenase and aspartate transaminase. In the event of a longer period of ethanol intake, the kidney was not able to balance the NADH excess, even though an increase in malate dehydrogenase, lactate dehydrogenase, aspartate transaminase, and alanine transaminase activities was noted. The electrophoretic analysis of alcohol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase isoforms revealed differences between control and ethanol-treated animals. The results suggest that rat kidneys have a multicomponent metabolic response to the same daily dose of ethanol that functions to maintain the redox status and which varies with the length of the administration period.     

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.     

Involvement of Ad4BP/SF-1, DAX-1, and COUP-TFII transcription factor on steroid production and luteinization in ovarian theca cells

The link between chronic alcohol consumption and cardiovascular injury including hypertension is well known. However, molecular mediators implicated with alcohol-induced elevation in blood pressure (BP) remain elusive. The aim of this study was to investigate the relationship of chronic ethanol-induced endothelial injury and elevation in BP with angiotensin II levels in rats. Male Fisher rats were divided into two groups of seven animals each and treated as follows: (1) Control (5% sucrose, orally) daily for 12 weeks and (2) ethanol (4 g kg⁻¹, orally) daily for 12 weeks. The BP (systolic, diastolic, and mean) was recorded every week. The animals were anesthetized with pentobarbital after 12 weeks; blood and thoracic aorta were isolated and analyzed for aortic reactivity response, angiotensin II levels, and oxidative endothelial injury. The results show that the systolic, diastolic, and mean BP were significantly elevated 12 weeks after ethanol ingestion. The increased BP was related to elevated angiotensin II levels in the plasma and aorta of alcohol treated group compared to control. The aortic NADPH oxidase activity, ratio of oxidized to reduced glutathione (GSSG/GSH) and lipid peroxidation significantly increased, whereas nitric oxide (NO), endothelial NO synthase (eNOS), and vascular endothelial growth factor (VEGF) protein expressions were depressed in alcohol group compared to control. The phenylephrine-mediated vasoconstriction response was not altered, while acetylcholine-mediated vasorelaxation response was depressed in the aorta of ethanol treated rats compared to control. It is concluded that chronic ethanol ingestion induces hypertension which is correlated with elevated tissue angiotensin II levels, activation of NADPH oxidase activity causing endothelial injury, depletion of endothelial NO generating system, and impaired vascular relaxation in rats.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Isolation and characterization of phenylethylamine and phenylethanolamine from human brain

The Presence of endogenous 2-phenylethylamine in mammalian tissues has long been suspected, in view of the fact that L-phenylanine, a substrate for L-aromatic amino acid decarboxylase (Lovenberg , Weissbach and Udenfriend , 1962), is found in substantial amounts in many neural and non-neural tissues. It has been difficult to demonstrate the presence of phenylethylamine in tissues of untreated animals because this amine is an excellent substrate for monoamine oxidase (Mantegazza and Riva , 1963). Using paper chromatography and electrophoresis, Nakajima , Kakimoto and Sano (1964) tentatively identified phenylethylamine in many organs of animals pretreated with monoamine oxidase inhibitors. Phenylethylamine exerts, in animals pretreated with such inhibitors, behavioural stimulant effects similar to those induced by amphetamine (Mantegazza and Riva , 1963). These effects may in part be attributable to catecholamine release (Fuxe , Grobecker and Jonsson , 1967) and partly to a direct effect exerted by phenylethylamine itself (Fischer , Ludmer and Sabelli , 1967; Giardina , Pedemonte and Sabelli , 1972). The brain content of phenylethylamine in mice (Mosnaim and Sabelli , 1971), rabbits (Sabelli , Giardina , Mosnaim and Inwang , 1972) and rats (Fischer , Spatz , Heller and Reggiani , 1972) is increased by antidepressive treatments (imipramine, monoamine oxidase inhibitors, electroshock) and reduced by reserpine. The urinary excretion of phenylethylamine is decreased in depressed patients (Fischer , Heller and Miró , 1968; Boulton and Milward , 1971; Inwang , Sugerman , Mosnaim and Sabelli , 1972; Fischer et al., 1972). However, the presence of phenylethylamine in brain has not yet been conclusively demonstrated because the analytical procedures used in the above-mentioned investigations were not sufficiently specific. In the present study we isolated and identified, by a number of analytical procedures, phenylethylamine and its metabolite 2-hydroxy-2-phenylethylamine (phenylethanolamine) from human brain. Molinoff , Landsberg and Axelrod (1969) have shown by enzymatic methods the formation of phenylethanolamine following the administration of phenylethylamine.     

Effect of alcohol intoxication and aldehyde dehydrogenase inhibitors on lipid peroxidation in rat liver

A single intraperitoneal administration of ethanol (3.5 g/kg) to rats induced a marked increase in lipid peroxidation and a decrease of antioxidative activity in the liver after 1 h when assessed by chemi-luminescence in liver homogenates. The pretreatment with aldehyde dehydrogenase inhibitor, disulfiram (200 mg/kg 24 hr before ethanol), caused a 10-fold elevation of the blood acetaldehyde levels, with no effect on the hepatic lipid peroxidation compared to control. Cyanamide (50 mg/kg, 2 h before the ethanol) increased approximately 100-fold the acetaldehyde levels, however, the changes in lipid peroxidation were not significantly different from that produced by ethanol alone. The present results suggest, that the metabolism of acetaldehyde and not acetaldehyde itself is responsible for the in vivo activation of lipid peroxidation during acute alcohol intoxication. Disulfiram prevents the ethanol-induced lipid peroxidation in the rat liver.     

Aldehyde dehydrogenase activity and level of dopamine in certain sections of the brain of rats preferring and refusing ethanol

Aldehyde dehydrogenase activity (KF 1.2.1.3) of cytosol fractions of brain structures (hypothalamus, midbrain and new cortex) as well as dophamine content in these structures were studied in comparative aspect in rats preferring and rejection ethanol. It has been shown that there were two isoforms of aldehyde dehydrogenases (aldehyde dehydrogenase 1 and aldehyde dehydrogenase 2) in cytosol fractions of all investigated brain structures of animals preferring ethanol while only aldehyde dehydrogenase 2 has been found in the new cotex of rats rejecting ethanol. Thus, aldehyde-dehydrogenase activity is higher in the animals preferring ethanol than in those ones rejecting ethanol. Content of dophamine in the rats preferring ethanol is higher than in those ones rejecting ethanol both in the hypothalamus and new cortex. Differences between the studied groups of animals can underlie the pathologic attraction to alcohol.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

Ethanol potentiates in vivo hepatotoxicity of endosulfan in adult male rats.

In an attempt to evaluate the effect and interaction of ethanol on endosulfan-induced hepatotoxicity in vivo to adult male rats, both, endosulfan (7.5 mg/kg body wt) and ethanol (1.5 g/kg body wt) were studied separately as well as in combination after a chronic oral exposure of 30 days. When fed separately, both the agents were found to induce microsomal mixed function oxidase (MFO) system in treated animals. A simultaneous induction in the activity of cytosolic GSH-s-transferase was found to be associated with significantly induced ascorbate-induced microsomal lipid peroxidation. Both endosulfan and ethanol showed increasing trends in the activities of reducing equivalent (NADPH)-generating enzymes in liver. The activity of hepatic alcohol dehydrogenase was, however, found to be relatively unaffected. When ethanol was administered in combination with endosulfan, the observed effects on the activities of major drug metabolizing enzymes, microsomal lipid peroxidation and NADPH generation were further pronounced. Findings demonstrated the MFO inducing capability of both endosulfan and ethanol, and showed further that chronic ethanol ingestion might potentiate the in vivo hepatotoxicity of endosulfan if administered in combination.     

Hepatic redox state: attenuation of the acute effects of ethanol induced by chronic ethanol consumption

The effects of an acute dose of a diet containing ethanol (3g/kg) on hepatic redox state was compared in rats fed ethanol for 25 days and in their littermates given isocaloric carbohydrate. In both groups, cytoplasmic and mitochondrial redox states of pyridine nucleotides shifted to a more reduced level, but the changes were much less extensive in rats chronically fed ethanol. This metabolic adaption may reflect the oxidation of ethanol by a pathway not involving alcohol dehydrogenase, such as the microsomal ethanol osidizing system, which increases in activity after chronic ethanol ingestion.     

METABOLIC CONSEQUENCES OF ETHANOL OXIDATION IN BRAINS FROM MICE CHRONICALLY FED ETHANOL

Abstract— Effects of the acute and chronic administration of ethanol have been investigated in mouse brain on the redox-state, citric acid cycle function, levels of adenine nucleotides and other metabolites. Cerebral oxidation of ethanol, activity of alcohol dehydrogenase and the permeability of brain and liver mitochondrial preparations after chronic ethanol administration have been also investigated. Acute or chronic administration of ethanol resulted in a small but significant increase in the reduced components of certain dehydrogenase-linked substrate pairs in brain. Pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced changes in brain. ¹⁴CO₂ production from several substrates was inhibited in brains from chronically ethanol-fed animals. Addition of pyrazole, however, prevented the ethanol-mediated inhibition of ¹⁴CO₂ production. Chronic administration of ethanol resulted in decreased levels of ATP and creatine phosphate in the brain, and increased contents of ADP and AMP. The cerebral activities of alcohol dehydrogenase and succinic dehydrogenase, oxidation of ethanol, mitochondrial oxidation of a-glycerophosphate, and levels of NADH remained unaffected by the chronic administration of ethanol. In contrast to liver, where chronic administration of ethanol increased the contribution of 'substrate shuttles'resulting in increased oxidation of ethanol; in brain, the contribution of these 'shuttles'remained unaffected.     

In vivo inhibition of liver alcohol dehydrogenase by ethanol administration

The activity of liver alcohol dehydrogenase (LADH) from rats sacrificed two hours after the administration of ethanol 3, 4 or 5 g/kg intraperitoneally was significantly inhibited compared to the activity of LADH from control rats. LADH activity was inversely related to the dose of ethanol administered, to the concentration of ethanol in the liver, and to the concentration of ethanol in the blood. The clearance of blood ethanol in rats was dose-dependent and was inversely related to the dose administered. The half-life of ethanol elimination increased as the dose of ethanol increased. These results suggest that ethanol-induced inhibition of LADH can occur in vivo and that the level of activity of this enzyme determines the rate of oxidation of ethanol.     

Involvement of iron and iron-catalyzed free radical production in ethanol metabolism and toxicity

Lipoperoxidation, a degradative process of membranous polyunsaturated fatty acids, has been suggested to represent an important mechanism in the pathogenesis of ethanol toxicity on the liver and possibly also on the brain. Catalysis by transition metals, especially iron, is involved in the biosynthesis of free radicals contributing to lipid peroxidation. Although the exact nature of the redox-active iron implicated in this catalysis is still unknown, it has been well established that lipid peroxidation can be prevented in vitro by iron chelators such as desferrioxamine. Deprivation of redox-active iron through desferrioxamine inhibits by about 50% the microsomal oxidation of ethanol in vitro and reduces very significantly in vivo the overall ethanol elimination rate in rats. Administration of desferrioxamine together with ethanol also reduces the ethanol-induced disturbances in the antioxidant defense mechanisms of the hepatocyte. It also reduces in mice both the severity of physical dependence on ethanol and lethality following the acute administration of a narcotic dose of ethanol. Chronic overloading of rats with iron results, on the opposite, in an increased rate of ethanol elimination, although alcohol dehydrogenase and catalase activities are reduced and cytochrome P-450 depleted in the liver of such iron-overloaded animals. The magnitude of the ethanol-induced increase in lipid peroxidation and decrease in the major membranous antioxidant, alpha-tocopherol, is exacerbated in iron-overloaded rats. Several disturbances of iron metabolism have been reported in human alcoholics. Their contribution to ethanol toxicity appears very likely in the case of hepatic siderosis associated with alcohol abuse. Ethanol could however disturb iron metabolism even in the absence of gross abnormalities of the total iron stores. It is suggested that ethanol intoxication could increase cellular redox-active iron, thus contributing to an enhanced steady-state concentration of reactive-free radicals. This oxidative stress would lead to lipoperoxidative damage and cellular injury.