Genetic polymorphism and activities of human lung alcohol and aldehyde dehydrogenases: Implications for ethanol metabolism and cytotoxicity

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) exhibit genetic polymorphism and tissue specificity. ADH and ALDH isozyme phenotypes from 39 surgical Chinese lung specimens were identified by agarose isoelectric focusing. The identity of the lung β-ADHs was further demonstrated by their characteristic pH-activity profiles for ethanol oxidation,K _m values for NAD and ethanol, and inhibition by 4-methylpyrazole or 1,10-phenanthroline. The β₂ allele, coding for β₂ polypeptide, was found to be predominant in the lung specimens studied. The ADH activities in the lungs with the homozygous phenotype ADH₂ 2-2 (exhibiting β₂β₂) and ADH₂ 1-1 (exhibiting β₁β₁) and the heterozygous phenotype ADH₂ 2-1 (exhibiting β₂β₂, β₂β₁, and β₁β₁) were determined to be 999±77, 48±17, and 494±61 nmol/min/g tissue, respectively. Fifty-one percent of the specimens studied lacked the ALDH2 activity band on the isoelectric focusing gels. The activities in the lung tissues with the ALDH2-active phenotype and the inactive phenotype were determined to be 30±3 and 17±1 nmol/min/g tissue, respectively. These findings indicate that human pulmonary ethanol-metabolizing activities differ significantly with respect to genetic polymorphism at both theADH ₂ and theALDH ₂ loci. The results suggest that individuals with highV _max β₂-ADH and deficient in low-K _m mitochondrial ALDH2, accounting for approximately 45% of the Chinese population, may end up with acetaldehyde accumulation during alcohol consumption, rendering them vulnerable to tissue injury caused by this highly reactive and toxic metabolite. This work was supported by Grants NSC 77-0412-B016-58 and NSC 80-0412-B016-21 from the National Science Council, Republic of China.     

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.     

菌群、乙醇代谢与急性酒精中毒的关系研究

酒是生活中常见的饮品,过度饮酒会对机体产生毒害作用。要防治急性酒精中毒首要的就是了解乙醇的代谢途径以及致病机制,从而找到加速乙醇代谢,减轻危害的方法。因为菌群与乙醇代谢相关,并可以通过菌群修复乙醇带来的损伤。本研究以乙醇代谢和损伤机制为基础,对菌群调节乙醇代谢及对酒精中毒的缓解作用进行综述。     

In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

The role of acute ethanol (2.5 g/kg i.p.) and phenylethylamine (100 mg/kg i.p.) on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behaviour were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver whilst at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effects described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioural indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals. The results suggest that some of the toxic actions of ethanol in rats may be mediated through the activation of monoamine oxidase type B, with the consequent depletion of the endogenous levels of phenylethylamine. The data appear to support the concept of phenylethylamine involvement in affective disorders.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

In vivo metabolism of 4'-deoxypyridoxine in rat and man.

In rats 80 to 95% of 4'-deoxypyridoxine administered intraperitoneally, intravenously, intramuscularly, or subcutaneously was excreted in the urine within 7.5 hours. Orally administered deoxypyridoxine was also rapidly eliminated. Over one-half of the excreted material appeared as deoxypyridoxine-3-(hydrogen sulfate) and the remainder as unchanged deoxypyridoxine. Tissue concentrations of deoxypyridoxine 5'phosphate were comparable to those of pyridoxal 5'phosphate. In normal men about 50% of a single oral dose (3 to 7.5 mg/kg of body weight) appeared in the urine within 6 hours. 4'Deoxy-5-pyridoxic acid accounted for 50 to 100% of the excreted material. The remainder was unchanged deoxypyridoxine. No deoxypyridoxine-3-(hydrogen sulfate) was detected in human urine and no 4'-deoxy-5-pyridoxic acid was found in rat urine. Deoxypyridoxine 5'-phosphate was not detected in the urine of either species. The complexity of deoxypyridoxine metabolism indicated by these data suggests the use of caution in extrapolating data obtained with deoxypyridoxine to B6 metabolism in the absence of deoxypyridoxine, and particularly in extrapolating results from the rat to man. Synthesis for 4'-deoxypyridoxine-3-(ethyl carbonate), 4'-deoxypyridoxine 5'-acetate, 4'-deoxy-3-0-(2-sulfoethyl)-pyridoxine, and the metabolites are presented. These synthesis were facilitated by using ethylchloroformate conjugates and N-methylpiperazine hydrolysis to block and unblock the phenol group.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver.

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

Role of cytosolic rat liver aldehyde dehydrogenase in the oxidation of acetaldehyde during ethanol metabolism in vivo.

The activity of a high-Km aldehyde dehydrogenase in the liver cytosol was increased by phenobarbital induction. No corresponding increase in the oxidation rate of acetaldehyde in vivo was found, and it is concluded that cytosolic aldehyde dehydrogenase plays only a minor role in the oxidation of acetaldehyde during ethanol metabolism.     

Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism

Aldehydes are highly reactive molecules that are intermediates or products involved in a broad spectrum of physiologic, biologic and pharmacologic processes. Aldehydes are generated from chemically diverse endogenous and exogenous precursors and aldehyde-mediated effects vary from homeostatic and therapeutic to cytotoxic, and genotoxic. One of the most important pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs). Oxidation of the carbonyl functional group is considered a general detoxification process in that polymorphisms of several human ALDHs are associated a disease phenotypes or pathophysiologies. However, a number of ALDH-mediated oxidation form products that are known to possess significant biologic, therapeutic and/or toxic activities. These include the retinoic acid, an important element for vertebrate development, gamma-aminobutyric acid (GABA), an important neurotransmitter, and trichloroacetic acid, a potential toxicant. This review summarizes the ALDHs with an emphasis on catalytic properties and xenobiotic substrates of these enzymes.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.     

A comparative study of the inhibition of hepatic aldehyde dehydrogenases in the rat by methyltetrazolethiol, calcium carbimide, and disulfiram

Methyltetrazolethiol (1-methyl-5-mercapto-1,2,3,4-tetrazole, MTT) is a heterocyclic substituent of the cephalosporin antibiotics, cefamandole, cefoperazone, and moxalactam. Pretreatment of rats with MTT has been reported to increase blood acetaldehyde concentration after ethanol administration. The time course of MTT-induced inhibition of hepatic aldehyde dehydrogenases (ALDH) was determined in adult, male Sprague-Dawley rats in comparison with the hepatic ALDH inhibition induced by calcium carbimide (calcium cyanamide, CC) and disulfiram (D). The apparent onset of maximal inhibition of hepatic low Km ALDH occurred at 2 h for 50 mg/kg MTT (subcutaneous, s.c.) and 7 mg/kg CC (oral) and at 24 h for 300 mg/kg D (oral). The relative magnitude of maximal inhibition of low Km ALDH was CC greater than D greater than MTT. The relative duration of enzyme inhibition was D greater than MTT greater than CC. High Km ALDH was only inhibited by CC. Hepatic low Km ALDH was selectively inhibited by s.c. and oral administration of 125 mg/kg MTT. For s.c. administration of 125 mg/kg MTT, the magnitude of maximal enzyme inhibition and the duration of inhibition were greater than for the 50 mg/kg dose. Oral administration of 125 mg/kg MTT produced similar inhibition of hepatic low Km ALDH compared with s.c. administration of the same dose. The time course of blood ethanol and acetaldehyde concentrations was determined for the intravenous infusion of two 0.3-g/kg doses of ethanol to rats that were pretreated orally with saline (1 h), MTT (125 mg/kg, 2 h), or CC (7 mg/kg, 1 h). The relative increase in blood acetaldehyde concentration compared with saline pretreatment was CC greater than MTT. The elimination of ethanol from blood was slower in the MTT- and CC-pretreated animals, and this effect was more pronounced for CC pretreatment. Overall, the data demonstrate that the characteristics of hepatic ALDH inhibition for MTT are different from those of the known ALDH inhibitors, CC and D.     

Comparison of phenobarbital-and carcinogen-induced aldehyde dehydrogenases in the rat

There is a genetically determined variation in the inducibility of a high-K_m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo-pdioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.     

No effect of chronic ethanol administration on the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the near-term pregnant guinea pig

The objective of this study was to determine the effect of chronic maternal administration of moderate-dose ethanol on alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the guinea pig at near-term pregnancy. The activity of each enzyme in the maternal liver, fetal liver, and placenta of the guinea pig at 59 days of gestation (term, 66 days) was determined spectrophotometrically following chronic daily oral administration of two doses of 1 g ethanol/kg maternal body weight or isocaloric sucrose solution. There was no experimental evidence of ethanol-induced malnutrition in the mother or growth retardation in the fetus. There was a statistically significant increase (65%) in the microsomal cytochrome P-450 content of the maternal liver for the ethanol treatment compared with the sucrose treatment. The alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the maternal liver, fetal liver, and placenta were not statistically different for the ethanol-treated compared with the sucrose-treated animals. This also was the case for the maternal blood and fetal blood ethanol and acetaldehyde concentrations, determined at 2h after maternal administration of 1 g ethanol/kg maternal body weight. These data demonstrate that the ethanol- and acetaldehyde-oxidizing enzyme activities in the maternal-placental-fetal unit of the guinea pig at near-term pregnancy were not changed by chronic administration of moderate-dose ethanol.     

Redox interactions between catalase and alcohol dehydrogenase pathways of ethanol metabolism in the perfused rat liver

Alcohol metabolism via alcohol dehydrogenase (ADH) and catalase was studied in perfused rat livers by measuring the oxidation of methanol and butanol, selective substrates for catalase and ADH, respectively. In livers from fasted rats, basal rates of methanol uptake of 15 +/- 1 mumol/g/h were decreased significantly to 8 +/- 2 mumol/g/h by addition of butanol. Concomitantly, pyridine nucleotide fluorescence detected from the liver surface was increased by butanol but not methanol. Both effects of butanol were blocked by an inhibitor of ADH, 4-methylpyrazole, consistent with the hypothesis that elevation of the NADH redox state by butanol inhibited H2O2 production via NAD+-requiring peroxisomal beta-oxidation, leading indirectly to diminished rates of catalase-dependent methanol uptake. In support of this idea, both butanol and butyraldehyde inhibited H2O2 generation. The NADH redox state was also elevated by xylitol, causing a 75% decrease in rates of methanol uptake by livers from fasted rats. This effect was not observed in livers from fed rats unless malate-aspartate shuttle activity was reduced by infusion of the transaminase inhibitor aminooxyacetate. Taken together, these data indicate that generation of reducing equivalents from ADH in the cytosol inhibits H2O2 generation leading to significantly diminished rates of peroxidation of alcohols via catalase. This phenomenon may represent an important physiological mechanism of regulation of ethanol oxidation in intact cells.