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Alcohol metabolism in vivo cannot be explained solely by the action of the classical alcohol dehydrogenase, Class I ADH (ADH1). Over the past three decades, attempts to identify the metabolizing enzymes responsible for the ADH1-independent pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. In this study, we used Adh3-null mutant mice to demonstrate that Class III ADH (ADH3), a ubiquitous enzyme of ancient origin, contributes to alcohol metabolism in vivo dose-dependently resulting in a diminution of acute alcohol intoxication. Although the ethanol oxidation activity of ADH3 in vitro is low due to its very high Km, it was found to exhibit a markedly enhanced catalytic efficiency (kcat/Km) toward ethanol when the solution hydrophobicity of the reaction medium was increased with a hydrophobic substance. Confocal laser scanning microscopy with Nile red as a hydrophobic probe revealed a cytoplasmic solution of mouse liver cells to be much more hydrophobic than the buffer solution used for in vitro experiments. So, the in vivo contribution of high-Km ADH3 to alcohol metabolism is likely to involve activation in a hydrophobic solution. Thus, the present study demonstrated that ADH3 plays an important role in systemic ethanol metabolism at higher levels of blood ethanol through activation by cytoplasmic solution hydrophobicity.

The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol intake increases. Furthermore, ADH3 is induced in damaged cells that have greater hydrophobicity, whereas ADH1 activity is lower when there is severe liver disease. These data suggest that chronic binge drinking and the resulting liver disease shifts the key enzyme in alcohol metabolism from low-Km ADH1 to high-Km ADH3, thereby reducing the rate of alcohol metabolism. The interdependent increase in the ADH3/ADH1 activity ratio and AUC may be a factor in the development of alcoholic liver disease. However, the adaptive increase in ADH3 sustains alcohol metabolism, even in patients with alcoholic liver cirrhosis, which makes it possible for them to drink themselves to death. Thus, the regulation of ADH3 activity may be important in preventing alcoholism development.

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To study the cytophysiological effects of ethanol systematically, L929 cells, a fibroblastic cell line derived from mouse connective tissue, were exposed to various concentrations of ethanol (12.5, 50, 100 and 200 mM) for short (3 and 6 h) and longer (24 or 26 h) durations. Ethanol-induced cellular responses were analysed by a combination of the following assays: number of cells, amounts of DNA and protein, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cell cycle. Ethanol dose-dependently suppressed these cellular functions, except that 12.5 mM exposures for both 6 and 26 h increased the amount of protein in spite of almost no change in other cellular functions, compared to the control. The most marked dose-dependency was observed in a reduction of formazan product in an MTT assay after both 6 and 26 h exposures to ethanol, being independent of the number of cells and probably reflecting dose-dependent depression of mitochondrial respiration. A G2 + M block in the cell cycle, an inhibition of cell division, was induced after short-term exposures (3 and 6 h) to 100 and 200 mM ethanol, but the block was released before 24 h had passed. Alternatively, prolonged exposures (24 h) to 50-200 mM ethanol induced a G0/G1 block, resulting in a decrease in the amount of DNA below the control value. Moreover, the percentage of the S phase was decreased gradually and dose-dependently throughout the 24 h exposure. Thus, high concentrations of ethanol (50, 100 and 200 mM) perturbed the cell cycle progression by causing both a transient G2 + M block (an inhibition of mitosis) and a continuous G0/G1 block, though the latter was masked by the G2 + M block during short-term exposure. The cells seem finally to acquire some tolerance to ethanol so as to pass through mitosis, but much less tolerance to pass through the checkpoint from the G1 to the S phase, which results in a decline in proliferation.

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