Takeshi Haseba

Alcohol metabolism in the body is a key theme in medical research on alcohol. It is primarily regulated by the alcohol dehydrogenase (ADH) and mitochondrial NADH reoxidation in the liver. Class I ADH1 is a well-known ADH isozyme and a key enzyme in alcohol metabolism, with the lowest Kms for ethanol and the highest sensitivity to pyrazole (Pz) among the ADH isozymes. However, a Pz-insensitive metabolic pathway also plays a role in systemic alcohol metabolism, with increasing metabolic contributions at higher blood alcohol concentrations (BACs) and under chronic alcohol consumption (CAC). The Pz-insensitive pathway is referred to as the non-ADH pathway—specifically, it is a non-ADH1 pathway—and is assumed to involve the microsomal ethanol oxidizing system (MEOS) or catalase, as both enzymes are insensitive to Pz and exhibit higher Kms than ADH1. The MEOS is a favored candidate for this pathway, as its activity markedly increases with the rate of alcohol metabolism under CAC. However, the role of the MEOS in alcohol metabolism has not been proven in vivo (even under CAC conditions), nor has that of catalase. Here, we report Class III ADH3 as a new candidate in the non-ADH1 pathway, as it also has a lower sensitivity to Pz and a higher Km. It is markedly activated by lowering Km following the addition of amphiphilic substances, which increases the solution’s hydrophobicity in the reaction medium; additionally, Nile red staining demonstrates a higher solution hydrophobicity in the cytoplasm of mouse liver cells. The rate of alcohol metabolism in ADH1 knockout (Adh1^−/−) mice—which depends solely on the non-ADH1 pathway—increased by more than twice under CAC and was significantly correlated with the amount of liver ADH3 protein, but not with CYP2E1 protein (a main component of the MEOS). The rate of alcohol metabolism in Adh3^−/− mice lacking ADH3 decreased in a dose-dependent manner compared with wild mice. The liver ADH3 protein in wild-type mice increased in line with the ADH1 protein under CAC. These data suggest that ADH3 contributes to alcohol metabolism in vivo as a non-ADH1 pathway and to the enhancement of alcohol metabolism under CAC through activation of the ADH1/ADH3/NADH reoxidation system. In alcoholic liver diseases, ADH1 activity decreased with the progression of liver disease, while ADH3 activity increased or was maintained even in alcoholic liver cirrhosis. Therefore, the role of ADH3 in alcohol metabolism may be increased in the context of alcoholic liver diseases, complementing the reduced role of ADH1. It has also been suggested that Class II ADH2, Class IV ADH4, and aldehyde dehydrogenase (ALDH) 2 play roles in alcohol metabolism in vivo under certain limited conditions. However, ADH2 and 4 may not contribute to the enhancement of alcohol metabolism through CAC.

Class III alcohol dehydrogenase (ADH3), primarily localized in the liver and kidney, contributes to alcohol metabolism during chronic alcohol consumption (CAC). However, its role in kidney function remains unclear. This study investigated renal morphological changes associated with ADH3-mediated alcohol metabolism. Nine-week-old male wild-type (WT) and ADH3-deficient (Adh3^-/-) mice were administered 10% ethanol for 1 month. Histological analyses were performed using periodic acid–Schiff (PAS) staining and electron microscopy. Serum biochemical parameters were also assessed. In WT mice, CAC induced an increase in cuboidal parietal epithelial cells (PECs) in Bowman’s capsule, along with elevated testosterone levels in both serum and urine. Adh3^-/- mice showed increased PECs even in the control group, with similarly elevated serum testosterone in both control and ethanol-treated groups. These findings suggest that ADH3 is involved in testosterone metabolism, and that that metabolism is suppressed by CAC because ADH3 shifts toward ethanol metabolism. The resulting testosterone elevation may contribute to PEC proliferation. An increase in PECs observed even in Adh3^-/- control mice may also be caused by the lack of testosterone metabolism via ADH3. Thus, renal ADH3 may protect kidney structure through testosterone metabolism, but its role may be disturbed by CAC. This study highlights the role of ADH3 in the relationship between physiological steroid metabolism and alcoholic pathological abnormality in the kidney.

Lipid accumulation in the liver due to chronic alcohol consumption (CAC) is crucial in the development of alcohol liver disease (ALD). It is promoted by the NADH/NAD ratio increase via alcohol dehydrogenase (ADH)-dependent alcohol metabolism and lipogenesis increase via peroxisome proliferator-activated receptor γ (PPARγ) in the liver. The transcriptional activity of PPARγ on lipogenic genes is inhibited by S-nitrosylation but activated by denitrosylation via S-nitrosoglutathione reductase (GSNOR), an enzyme identical to ADH3. Besides ADH1, ADH3 also participates in alcohol metabolism. Therefore, we investigated the specific contribution of ADH3 to ALD onset. ADH3-knockout (Adh3-/-) and wild-type (WT) mice were administered a 10% ethanol solution for 12 months. Adh3-/- exhibited no significant pathological changes in the liver, whereas WT exhibited marked hepatic lipid accumulation (p < 0.005) with increased serum transaminase levels. Adh3-/- exhibited no death during CAC, whereas WT exhibited a 40% death. Liver ADH3 mRNA levels were elevated by CAC in WT (p < 0.01). The alcohol elimination rate measured after injecting 4 g/kg ethanol was not significantly different between two strains, although the rate was increased in both strains by CAC. Thus, ADH3 plays a key role in the ALD onset, likely by acting as GSNOR.

The important roles of retinols and their metabolites have recently been emphasized in the interactions between hepatic stellate cells (HSCs) and natural killer (NK) cells. Nevertheless, the expression and role of retinol metabolizing enzyme in both cell types have yet to be clarified. Thus, we investigated the expression of retinol metabolizing enzyme and its role in liver fibrosis. Among several retinol metabolizing enzymes, only alcohol dehydrogenase (ADH) 3 expression was detected in isolated HSCs and NK cells, whereas hepatocytes express all of them. In vitro treatment with 4-methylpyrazole (4-MP), a broad ADH inhibitor, or depletion of the ADH3 gene down-regulated collagen and transforming growth factor-β1 (TGF-β1) gene expression, but did not affect α-smooth muscle actin gene expression in cultured HSCs. Additionally, in vitro, treatments with retinol suppressed NK cell activities, whereas inhibition of ADH3 enhanced interferon-γ (IFN-γ) production and cytotoxicity of NK cells against HSCs. In vivo, genetic depletion of the ADH3 gene ameliorated bile duct ligation- and carbon tetrachloride-induced liver fibrosis, in which a higher number of apoptotic HSCs and an enhanced activation of NK cells were detected. Freshly isolated HSCs from ADH3-deficient mice showed reduced expression of collagen and TGF-β1, but enhanced expression of IFN-γ was detected in NK cells from these mice compared with those of control mice. Using reciprocal bone marrow transplantation of wild-type and ADH3-deficient mice, we demonstrated that ADH3 deficiency in both HSCs and NK cells contributed to the suppressed liver fibrosis. Conclusion: ADH3 plays important roles in promoting liver fibrosis by enhancing HSC activation and inhibiting NK cell activity, and could be used as a potential therapeutic target for the treatment of liver fibrosis. (Hepatology 2014;60:1044–1053)

It is said that blood alcohol concentrations (BAG) are higher in female than in male due to the smaller distribution volume of alcohol in female, whereas the rate of alcohol metabolism is faster in female than in males due to a higher activity of liver alcohol dehydrogenase (ADH) in female. However, it is also known that alcohol metabolism varies depending on drinking conditions. In this study, we evaluated the dose effect of alcohol on sex differences in alcohol metabolism in daily drinking conditions, where young adults (16 males, 15 females) with ALDH2*1/1 genotype drunk beer at a dose of 0.32g or 1.0g ethanol/kg body weight with a test meal (460kcal). This study was conducted using a randomized cross-over design. In the considerable drinking condition (1.0g/kg), BAG was significantly higher in females than in males, whereas the rate of alcohol metabolism (beta) was higher in female than in male. In the moderate drinking condition (0.32g/kg), however, no sex differences in alcohol metabolism including BAG were seen. These results suggest that an increased first pass metabolism through liver ADH in female, which may be caused by the reduction of gastric emptying rate due to the meal intake, contribute to the vanishing of sex difference in BAC in the moderate drinking condition.

Studies on metabolisms of alcohol and the metabolites (e.g.:acetaldehyde) after drinking give basic and important information to recognize the physiological influence of drinking to human bodies. The aims of these studies were to clarify the influences of ALDH2 genotype difference, kinds of alcohol beverages, and fasted or prandial state to alcohol metabolisms at moderate drinking. The studies were conducted by a randomized cross-over design. After overnight fast, fifteen of ALDH2*1/*1 (Experiment 1) and twenty of ALDH21/*2 (Experiment 2) in Japanese healthy men aged 40 to 59 years old drank beer or shochu at a dose of 0.32g ethanol / kg body weight with or without test meal (460 kcal). The peak of blood ethanol (C(max)) was higher with shochu than with beer in the fasted condition in both ALDH2 genotypes, however, the difference between two types of alcohol beverages went out in the prandial condition. Simultaneous ingestion of test meal with alcohol beverage significantly decreased blood ethanol concentrations and increased ethanol disappearance rate (EDR) in the both genotypes. EDR values were significantly higher in ALDH2*1/*1 type than in ALDH2*1/*2 type in the both beverages with and without meal, whereas beta values showed no significant difference between two genotypes. The concentrations of blood acetaldehyde in ALDH2*1/*2 type were higher in prandial condition than in fasted condition with shochu. These results indicate that meal modified the differences of alcohol metabolism between beer and shochu and also between ALDH2 genotypes. Thus, alcohol metabolism in daily drinking is shown to be regulated by various combinatorial drinking conditions.

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 K_m 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 K_m, the catalytic efficiency (k_cat/K_m) 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 (b, CL_T, 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-K_m ADH1 to high-K_m 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.

The maturation of distilled spirits is known to change constituent congeners to improve the qualities of smell and taste. However, it has been largely unknown how maturation modifies the pharmacokinetics or neuropharmacological effects of ethanol. We used single malt whiskies to investigate the effects of spirit maturation on ethanol metabolism and drunkenness. Mice were injected with 5-year (5-y) or 20-year (20-y) aged single malt whisky with a concentration of 20% (w/v) ethanol at a dose of 3 g/kg. The concentrations of ethanol and its metabolites in the blood and the duration of loss of righting reflex (LORR) were compared between the 2 whisky groups. In addition, the effects of nonvolatile congeners in whisky on the biomedical reactivities of ethanol were investigated by administering a nonvolatile fraction added to a 20% ethanol solution, whose fraction was prepared by evaporating 16-y whisky. Liver alcohol dehydrogenase (ADH) activity was measured with whisky as the substrate or in the presence of nonvolatile congeners with ethanol as the substrate. The rate of ethanol elimination (mmol/kg/h) was smaller in the 20-y whisky group than in the 5-y group (p<0.01 by Fisher's protected least significant difference), which resulted in lower concentrations of blood acetaldehyde and acetate in the former group than in the latter group (p<0.01 by ANOVA). Nonvolatile congeners added to the ethanol solution also depressed the rate of ethanol elimination in mice. In vitro studies demonstrated that liver ADH activity measured with whisky as the substrate was decreased as a function of the age of the whisky, and that the activity measured with ethanol as the substrate was strongly inhibited by nonvolatile congeners. The duration of LORR was longer in the 20-y group than in the 5-y group (p<0.01). Nonvolatile congeners also prolonged the duration of ethanol-induced LORR, when administered together with ethanol. Maturation of whisky delayed ethanol metabolism to lower the level of blood acetaldehyde and acetate with increasing inhibition of liver ADH activity by nonvolatile congeners. It also prolonged drunkenness by enhancing the neurodepressive effects of ethanol, due to increases in the amount of nonvolatile congeners. These biomedical effects of whisky maturation may reduce aversive reactions and cytotoxicity due to acetaldehyde, and may also limit overdrinking with the larger neurodepression.