Dihydrodiol dehydrogenases in guinea pig liver

Four major and four minor dihydrodiol dehydrogenases, with similar apparent molecular weights of 28,000 to 34,000 but with different charges, were purified from male guinea pig liver cytosol. One of the minor enzymes catalyzed only the oxidation of benzene dihydrodiol with a high Km value of 5.0 mM and was identified immunologically with aldehyde reductase. The other enzymes oxidized xenobiotic alicyclic alcohols and 17 beta-hydroxysteroids as well as benzene dihydrodiol. These enzymes exhibited higher affinity for 17 beta-hydroxysteroids than for alicyclic alcohols and benzene dihydrodiol, and immunologically cross-reacted with testosterone 17 beta-dehydrogenase purified from the same source. Four major enzymes and one minor with Km values for benzene dihydrodiol of about 0.2 mM, possessed specificity for 5 beta-androstane--17 beta-hydroxysteroids and dual cofactor requirement, whereas the other two minor enzymes with high Km values of over 5 mM showed apparent NADP and 5 alpha-androstane specificity. The dihydrodiol dehydrogenase activity was localized in the cytosol of liver. The results indicate that the hepatic oxidation of dihydrodiols in the guinea pig is mediated by cytosolic testosterone 17 beta-dehydrogenase isozymes and aldehyde reductase. Testosterone 17 beta-dehydrogenase immunologically identical to the liver enzymes was detected only in kidney, whereas aldehyde reductase was detected in all tissues of the guinea pig.     

Diminished alcohol preference in transgenic mice lacking aldehyde dehydrogenase activity

Aldehyde dehydrogenase (ALDH) 2 plays a major role in the detoxification of aldehyde and is known to be responsible for alcohol preference. A diminished enzyme activity due to mutation of the Aldh2 gene is associated with high alcohol sensitivity and a low alcohol tolerance in humans. The genomic background distinguishing an alcohol preference and avoidance in various inbred mouse strains is not clear. We created Aldh2-negative mice by transgenic knockout of the Aldh2 gene into the high alcohol preference C57BL/6 background. The Aldh2 gene targeting (Aldh-/-) mice exhibited an alcohol avoidance characteristic. After free-choice ethanol and water drinking, brain and liver acetaldehyde concentrations of Aldh2-/- mice were almost equal to those of wild-type (Aldh2+/+) mice although the Aldh2-/- mice drank less ethanol than the Aldh2+/+ mice. This result indicates that a direct effect of the Aldh2 genotype plays an important role on alcohol preference and acetaldehyde concentration in the brain is correlated with alcohol avoidance. This highlights the potential benefits of alcoholism and alcohol-related disease research in the animal model of ALDH2 alleles.     

Effect of gossypol on kinetics of mouse liver alcohol and aldehyde dehydrogenase

1. Gossypol, an antifertility ingredient of the cotton plant, altered specific activity of mouse liver alcohol dehydrogenase (L-ADH) and subcellular aldehyde dehydrogenase (L-ALDH) in mice of both sexes. 2. Intraperitoneal injection of a single gossypol dose, 50 mg/kg, inhibited both male and female L-ADH and cytoplasmic L-ALDH from saline controls 21 hr after drug treatment. 3. Gossypol inhibited female but not male mouse mitochondrial L-ALDH isoenzymes. 4. Gossypol-produced enzyme inhibition was determined as noncompetitive. 5. The results suggest gender-dependent sensitivity of mitochondrial L-ALDH to the gossypol inhibition. A toxic metabolic interaction between ethanol and gossypol has been indicated which suggests the contraindication of alcoholic beverages during gossypol use.     

Correlation between human erythrocyte aldehyde dehydrogenase activity and sensitivity to alcohol

Young healthy Japanese men were given 0.48 g ethanol/kg body weight orally. Those responding with a marked increase in heart rate after alcohol also exhibited facial flushing and had higher acetaldehyde levels than those not responding, in spite of similar blood alcohol levels. The activity of aldehyde dehydrogenase in erythrocytes was found tocorrelate significantly (r=?0.73, p<0.01) with the increase in heart rate after alcohol drinking. It is suggested that erythrocyte aldehyde dehydrogenase activity affects or reflects blood acetaldehyde levels and physiological response to alcohol, and may prove useful as a marker for alcohol sensitivity in Orientals.     

The oxidation of acrolein by rat liver aldehyde dehydrogenases. Relation to allyl alcohol hepatotoxicity

The oxidation of acrolein by aldehyde dehydrogenase was studied in several subcellular fractions of rat liver by measuring acrolein-dependent production of NADH from NAD+. Mitochondrial and cytosolic fractions each contained two aldehyde dehydrogenase activities with Km values for acrolein of 0.4-0.7 mM and 0.015-0.025 mM. Microsomes demonstrated only a high Km (1.5 mM) activity. The low Km activities of mitochondria and cytosol differed in their sensitivity to inhibition by chloral hydrate and in their response to 1 mM MgCl2 (activation vs. inhibition). The metabolism of acrolein by low Km aldehyde dehydrogenase activities was markedly depressed in mitochondrial or cytosolic fractions from rats pretreated with cyanamide (2 mg/kg for 1 hr) or disulfiram (100 mg/kg for 24 hr). The effect of aldehyde dehydrogenase inhibition on allyl alcohol toxicity was determined by pretreating rats with cyanamide or disulfiram prior to treatment with allyl alcohol. Hepatotoxicity was assessed on the basis of elevated serum alanine aminotransferase and sorbitol dehydrogenase activities and the loss of microsomal cytochrome P-450. Pretreatment with the aldehyde dehydrogenase inhibitors enhanced the hepatotoxicity of allyl alcohol in both male and female rats. The results suggest that acrolein metabolism by rat liver aldehyde dehydrogenase isozymes is important for the inactivation of allyl alcohol-derived acrolein.     

Effect of almond and anis oils on mouse liver alcohol dehydrogenase, aldehyde dehydrogenase and heart lactate dehydrogenase isoenzymes

The effects of short-term intraperitoneal injection of diluted almond or anis oil on heart lactate dehydrogenase isoenzymes, liver alcohol dehydrogenase and subcellular aldehyde dehydrogenase were studied in the female mouse. Hepatic alcohol dehydrogenase was induced from control by administration of almond oil 3.2 g/kg/d for 7 days, or anis oil 1.6 g/kg/d for 7 days. Treatment with almond but not anis oil inhibited both cytoplasmic and mitochondrial liver aldehyde dehydrogenase. The mitochondrial isoenzyme with an apparently low Km was also inhibited by the almond oil trial. No significant changes occurred in heart lactate dehydrogenase isoenzymes by the treatments used. The enzymatic inhibition kinetics were found to be non-competitive. The apparent Km for almond-treated mouse aldehyde dehydrogenase was greater than the controls. This indicates lower substrate affinity for almond oil than for acetaldehyde. The results suggest adverse hepatic metabolic interaction between almond oil and alcohol.     

Inhibition of rat liver aldehyde dehydrogenases by acrolein

Acrolein, the lowest member of the ethylenic aldehyde series, has been widely studied as a result of the diverse toxicities associated with it. Previous investigations into the enzymatic process responsible for the detoxification of acrolein implicated rat liver aldehyde dehydrogenase (ALDH) in the oxidation of this aldehyde. Contrary to these reports, in our investigation we were unable to detect the oxidation of acrolein to acrylic acid by Sprague-Dawley rat liver mitochondrial or cytosolic ALDHs measured spectrophotometrically by the production of NADH, or by HPLC analysis for the production of acrylic acid. However, in the course of these experiments, it was demonstrated that acrolein is a potent inhibitor of rat liver ALDHs. Mitochondrial and cytosolic high affinity ALDHs are particularly sensitive to the inhibitory effects of acrolein. The type of inhibition exhibited by these high affinity ALDHs is primarily irreversible, with a slight degree of reversible noncompetitive inhibition. The inhibition is rapid with a 91 and 33% reduction in control mitochondrial and cytosolic ALDH activities, respectively, with a 5-sec preincubation of 30 microM acrolein prior to the addition of the aldehyde substrate. Significant inhibition of total (high plus low affinity isozymes) mitochondrial and cytosolic ALDHs occurs only at relatively high acrolein concentrations (greater than or equal to 50 microM). The inhibition displayed by the total mitochondrial and cytosolic ALDHs is mixed-type, with both reversible noncompetitive and irreversible inhibition demonstrated.     

Subcellular distribution and properties of rabbit liver aldehyde dehydrogenases

In rabbit liver, both NAD⁺- and NADP⁺-dependent aldehyde dehydrogenases were identified. The activities were distributed among at least three major groups of isozymes identifiable by gel electrophoresis. These isozymes also differed in their substrate and coenzyme preferences, subcellular distributions, and/or responses to effectors. The NAD⁺-dependent aldehyde dehydrogenase activity was distributed among the mitochondrial, microsomal, and cytosolic fractions. The NADP⁺-dependent aldehyde dehydrogenase activity was largely microsomal, with little true cytosolic NADP⁺-dependent activity demonstrable. Aliphatic aldehydes were oxidized equally well by aldehyde dehydrogenases in all three fractions. Aromatic aldehydes, however, were preferentially oxidized by microsomal aldehyde dehydrogenases. Disulfiram significantly inhibited mitochondrial (45 per cent) and cytosolic (93 per cent) NAD⁺-dependent aldehyde dehydrogenase, but it did not cause significant inhibition of microsomal NAD⁺-dependent activity. Disulfiram inhibited the NADP⁺-dependent aldehyde dehydrogenase activity (>71 per cent) in all subcellular fractions. Diethylstilbestrol activated both NAD⁺- and NADP⁺-dependent aldehyde dehydrogenases in mitochondria and cytosol. Microsomal aldehyde dehydrogenases were not affected by diethylstilbestrol.     

Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

Effects of long-term ethanol treatment on aldehyde and alcohol dehydrogenase activities in rat liver.

Administration of intoxicating doses of ethanol by gavage for 3 weeks caused weight loss and reduced hepatic aldehyde dehydrogenase activity in the soluble, mitochondrial and microsomal fractions. Rats receiving equivalent amounts of ethanol as a constituent of a liquid diet for 5 weeks gained weight and showed no changes in aldehyde dehydrogenase activity. Alcohol dehydrogenase activity was decreased in the rats treated by gavage and unchanged in those given ethanol in the diet, but in spite of this the rate of ethanol elimination was accelerated in both groups. In the livers of two strains of rats genetically selected for their difference in voluntary alcohol consumption, the mitochondrial and microsomal aldehyde dehydrogenase activities had previously been shown to be significantly higher in the alcohol-consuming (AA) than in the alcohol-avoiding (ANA) rats. Similar differences were now found after long-term intragastric ethanol administration, although in both strains the absolute levels of aldehyde dehydrogenase were reduced. Profound reduction of mitochondrial low-K_m aldehyde dehydrogenase activity and high blood acetaldehyde were observed, especially in the ANA rats. This suggests a possible connection between the low activity of this enzyme and the increased acetaldehyde level.     

Species variations in cutaneous alcohol dehydrogenases and aldehyde dehydrogenases may impact on toxicological assessments of alcohols and aldehydes

Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1-3) and ALDH (1-3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V(max), nmoles/mg protein/min) was higher in liver (6.02-16.67) compared to skin (0.32-1.21) and lower in human skin (0.32-0.41) compared to mouse skin (1.07-1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to approximately 30-40% and approximately 2-10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.     

Effect of triiodothyronine on alcohol dehydrogenase and aldehyde dehydrogenase activities in rat liver. Implications for the control of ethanol metabolism

Treatment of rats with 20 micrograms of 3,3',5-triiodo-L-thyronine (T3) per 100 g body wt for a period of 6 days led to a 45% decrease in total liver alcohol dehydrogenase and a 36% decrease in total liver aldehyde dehydrogenase. Most of the latter decrease was directly attributable to a 57% fall in the level of the physiologically-important low Km mitochondrial isoenzyme. The high Km isoenzyme of the postmitochondrial and soluble fractions was much less affected by T3-treatment. T3, at concentrations up to 0.1 mM, did not inhibit the activity of aldehyde dehydrogenase in vitro. Despite these large losses of the two enzymes most intimately involved in ethanol metabolism, the rate of ethanol elimination in vivo was the same in T3-treated and control animals. Moreover, there was no difference between the two groups in the susceptibility of ethanol elimination to inhibition by 4-methylpyrazole, making it unlikely that an alternative route of ethanol metabolism had been significantly induced by treatment with T3. As it had been suggested that T3 might create a "hypermetabolic state" in which constraints normally imposed on alcohol dehydrogenase and aldehyde dehydrogenase are removed thereby compensating for any loss in total enzymic activity, 2,4-dinitrophenol (0.1 mmoles/kg body wt) was administered to rats in order to raise the general metabolic rate. However, the uncoupler proved to be lethal to T3-treated animals and did not stimulate ethanol elimination in controls. The results do not support the notion that ethanol elimination in vivo is normally governed either by the level of alcohol dehydrogenase or by that of hepatic aldehyde dehydrogenase. However, the mode of control remains unclear.     

Mouse liver dihydrodiol dehydrogenases. Identity of the predominant and a minor form with 17 beta-hydroxysteroid dehydrogenase and aldehyde reductase

A major and a minor form of dihydrodiol dehydrogenase were co-purified with 17 beta-hydroxysteroid dehydrogenase and aldehyde reductase, respectively, to apparent homogeneity from liver cytosol of male ddY mice. The activities of dihydrodiol dehydrogenase and testosterone dehydrogenase or aldehyde reductase of the two enzyme forms comigrated electrophoretically. The major form of the enzyme oxidized 17 beta-hydroxysteroids and nonsteroidal alicyclic alcohols and reduced 17-ketosteroids and various synthetic carbonyl compounds, showing higher affinity for steroids than for xenobiotics. The activity of this enzyme form toward benzene dihydrodiol and testosterone exhibited identical thermostability and susceptibility to inhibition by quercitrin, SH-reagents, nonsteroidal estrogens and anti-inflammatory agents. On the other hand, the minor form of the enzyme, which oxidized benzene dihydrodiol but not 17 beta-hydroxysteroids, also reduced various aldehydes well and was specifically inhibited by barbiturates and sorbinil. These results indicate that the major form of dihydrodiol dehydrogenase is identical to 17 beta-hydroxysteroid dehydrogenase and the minor enzyme form to aldehyde reductase.     

Phenylacetaldehyde oxidation by freshly prepared and cryopreserved guinea pig liver slices: the role of aldehyde oxidase

Phenylacetaldehyde is formed when the xenobiotic and biogenic amine 2-phenylethylamine is inactivated by a monoamine oxidase-catalyzed oxidative deamination. Exogenous phenylacetaldehyde is found in certain foodstuffs such as honey, cheese, tomatoes, and wines. 2-Phenylethylamine can trigger migraine attacks in susceptible individuals and can become fairly toxic at high intakes from foods. It may also function as a potentiator that enhances the toxicity of histamine and tyramine. The present investigation examines the metabolism of phenylacetaldehyde to phenylacetic acid in freshly prepared and in cryopreserved guinea pig liver slices. In addition, it compares the relative contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase in the oxidation of phenylacetaldehyde using specific inhibitors for each oxidizing enzyme. The inhibitors used were isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase. In freshly prepared liver slices, phenylacetaldehyde was converted mainly to phenylacetic acid, with traces of 2-phenylethanol being present. Disulfiram inhibited phenylacetic acid formation by 80% to 85%, whereas isovanillin inhibited acid formation to a lesser extent (50% to 55%) and allopurinol had little or no effect. In cryopreserved liver slices, phenylacetic acid was also the main metabolite, whereas the 2-phenylethanol production was more pronounced than that in freshly prepared liver slices. Isovanillin inhibited phenylacetic acid formation by 85%, whereas disulfiram inhibited acid formation to a lesser extent (55% to 60%) and allopurinol had no effect. The results in this study have shown that, in freshly prepared and cryopreserved liver slices, phenylacetaldehyde is converted to phenylacetic acid by both aldehyde dehydrogenase and aldehyde oxidase, with no contribution from xanthine oxidase. Therefore, aldehyde dehydrogenase is not the only enzyme responsible in the metabolism of phenylacetaldehyde, but aldehyde oxidase may also be important and thus its role should not be ignored.     

Effect of Ukrain on human liver alcohol dehydrogenase activity in vitro

In the present study it was found that Ukrain inhibits the ethanol oxidation process catalyzed by human alcohol dehydrogenase (ADH). Ukrain at a concentration of 10(-6), 2.5 x 10(-6), and 5.0 x 10(-6) M decreased liver ADH activity in the presence ethanol (approximately 8.41%, 13.28%, 16.69%, respectively) as well as in the presence of methanol (approximately 13.29%, 19.07% and 42.20%, respectively).     

N-Hydroxylation of 2-aminofluorene in the guinea pig and by guinea pig liver microsomes in vitro

Summary N-hydroxy-2-aminofluorene was found in the urine of guinea pigs intraperitoneally injected with 2-aminofluorene. The hydroxylamine was oxidized to the nitroso analogue and this was identified and determined in the carbon tetrachloride extract by its characteristic UV absorption, by thin-layer chromatography, and by the formation of a diazo compound in the reaction with nitrous acid. Only a small fraction of the 2-aminofluorene injected appeared in the urine as N-hydroxy derivative.Guinea pig liver microsomes were observed to N-hydroxylate 2-aminofluorene rather rapidly, the reaction proceeding at least as rapidly as the N-hydroxylation of aniline.The results of this paper were presented at meetings of the Deutsche Pharmakologische Gesellschaft in Mainz, April 26 to 28, 1965 (Kampffmeyer and Kiese) and Göttingen, September 27 to 30, 1965 (von Jagow, Kiese, Renner, and Wiedemann).