Metabolic pharmacokinetics of early chronic alcohol consumption mediated by liver alcohol dehydrogenases 1 and 3 in mice.

BACKGROUND AND AIM: Alcohol dehydrogenases (ADHs) 1 and 3 are responsible for systemic alcohol metabolism. The current study investigated the contribution of liver ADH1 and ADH3 to the metabolic pharmacokinetics of chronic alcohol consumption (CAC). METHODS: The 9-week-old male mice of different ADH genotypes (wild-type [WT], Adh1-/- , and Adh3-/- ) were administered with 10% ethanol solution for 1 month, followed by acute ethanol administration (4.0 g/kg). The alcohol elimination rate (AER), area under the blood alcohol concentration curve (AUC), and the maximum blood alcohol concentration (Cmax ) were calculated. The liver content, activity, and mRNA levels of ADH were evaluated. RESULTS: Chronic alcohol consumption increased the AER and reduced the AUC in all ADH genotypes. The increased ADH1 content was correlated with AER in WT mice but not in the Adh3-/- mice. Similarly, the increased ADH3 content was also correlated with AER in both WT and Adh1-/- mice. The Cmax was significantly higher in Adh3-/- control mice than in WT control mice. It decreased in the Adh1-/- mice by CAC along with an increase in the ADH3 content. CONCLUSIONS: Alcohol dehydrogenases 1 and 3 would accomplish the pharmacokinetic adaptation to CAC in the early period. ADH1 contributes to the metabolic pharmacokinetics of CAC with a decrease in AUC in conjunction with an increase of AER by increasing the enzyme content in the presence of ADH3. ADH3 also contributes to a decrease in AUC in conjunction with not only an increase in AER but also a decrease in Cmax by increasing the enzyme content.

Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris.

Recombinant protein production under the control of the PADH3 was compared with Pichia pastoris PAOX1 and PGAP. The single-copy-clones expressing Aspergillus niger xylanase (XylB) gene with the three different promoters were tested in shake flask and 5 L fed-batch fermentation processes. Recombinant protein production with PADH3, PAOX1 and PGAP were initiated by addition of ethanol, methanol and glucose, respectively in the culture medium. The fermentation process was carried out for 72 h at 30 °C, pH 5 and 30% dissolved oxygen. Extracellular protein production yield for PADH3 (3725 U/mL) was higher than for PAOX1 (2095 U/mL) and PGAP (580 U/mL) at fermentor scale under the conditions tested. These results show that the PADH3 promoter is a promising tool for large scale production of recombinant proteins and can be an alternative to the PAOX1 and PGAP.

Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

Genetic polymorphisms in alcohol dehydrogenase, aldehyde dehydrogenase and alcoholic chronic pancreatitis susceptibility: a meta-analysis.

PURPOSE: This study was aimed to determine the relationship of alcohol-metabolizing enzymes ADH2, ADH3, and ALDH2 polymorphisms with the susceptibility to alcoholic chronic pancreatitis (ACP). METHODS: Meta-analyses that evaluated the association of ADH2, ADH3, and ALDH2 variations with ACP were performed. RESULTS: Eight case-control studies were selected for analysis. The overall data revealed a significant association of ADH2 polymorphism (OR=1.56, 95% CI=1.42-1.72, P=0.000 for dominant model; OR=1.63, 95% CI=1.55-1.71, P=0.000 for homozygote comparison model; OR=1.11, 95% CI=1.01-1.22, P=0.030 for allelic contrast model), ADH3 polymorphism (OR=0.95, 95% CI=0.86-1.06, P=0.389 for dominant; OR=0.64, 95% CI=0.44-0.93, P=0.020 for homozygote comparison; and OR=0.87, 95% CI=0.77-0.99, P=0.039 for allelic contrast model) and ALDH2 polymorphism (OR=0.57, 95% CI=0.40-0.81, P=0.002 for dominant; OR=0.50, 95% CI=0.23-1.08, P=0.079 for homozygote comparison; and OR=0.58, 95% CI=0.41-0.84, P=0.003 for allelic contrast model) with ACP risk. The subgroup analyses suggested that the variant ADH2*2/*2+*1/*2, ADH2*2/*2 genotype and ADH2*2 allele significantly increased ACP risk among Asian individuals; the variant ADH3*2/*2 genotype and ADH3*2 allele significantly decreased ACP risk among non-Asian individuals; and the variant ALDH2*2/*2+*1/*2 genotype and ALDH2*2 allele significantly decreased ACP risk among Asians. CONCLUSIONS: ADH2, ADH3 and ALDH2 polymorphisms may be susceptibility facts of ACP, and it may be ethnic and race-dependent.

A role for glutathione, independent of oxidative stress, in the developmental toxicity of methanol.

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6h later in embryonic ROS formation, measured by 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2'-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.

Pichia pastoris Promoters.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.

The Contribution of Alcohol Dehydrogenase 3 to the Development of Alcoholic Osteoporosis in Mice.

BACKGROUND: Alcohol dehydrogenase 3 (ADH3) plays major roles not only in alcohol metabolism but also in nitric oxide metabolism as S-nitrosoglutathione reductase (GSNOR). ADH3/GSNOR regulates both adipogenesis and osteogenesis through the denitrosylation of peroxisome proliferator-activated receptor γ. The current study investigated the contribution of ADH3 to the development of alcoholic osteoporosis in chronic alcohol consumption (CAC). METHODS: Nine-week-old male mice of different ADH genotypes [wild-type (WT) and Adh3-/-] were administered a 10% ethanol solution for 12 months. The femurs were evaluated by histochemical staining and computed tomography-based bone densitometry. The mRNA levels of ADH3 were evaluated in the WT mice by reverse transcription-quantitative polymerase chain reaction. RESULTS: The Adh3-/- control mice exhibited increased activities of both osteoblasts and osteoclasts and lower bone masses than the WT control mice. CAC exhibited no remarkable change in osteoblastic and osteoclastic activities, but decreased bone masses were observed in WT mice despite an increase in the mRNA levels of ADH3. Conversely, bone masses in the Adh3-/- control mice were not reduced after CAC. CONCLUSIONS: The Adh3-/- control mice exhibited a high turnover of osteoporosis since osteoclastogenesis dominated osteoblastogenesis; however, bone resorption was not enhanced after CAC. In comparison, CAC lead to alcoholic osteoporosis in WT mice, accompanied by increased mRNA levels of ADH3. Hence, ADH3 can prevent osteoporosis development in normal ADH genotypes with no alcohol ingestion. However, ADH3 contributes to the development of alcoholic osteoporosis under CAC by participating in alcohol metabolism, increasing metabolic toxicity, and lowering GSNO reducing activity.

The Analysis of Polymorphism of Alcohol Dehydrogenase 3 (ADH3) Gene and Influence of Liver Function Status in Indonesia.

Indonesian culture actually has no historical record of behaviors in consuming alcohol, but there are many recent reports of alcohol abuse among Asian people involving their traditional drink. In genotype studies, the damage of the liver caused by consuming alcohol is influenced by the presence of the polymorphism enzyme gene. The lack of study regarding such topic is a signal to further investigate ADH3 gene distribution and its effect on liver function status. The total of 197 research subjects of Javanese descent received alcohol dehydrogenase 3 (ADH3) genetic polymorphism and liver status tests in the city of Yogyakarta, Indonesian. An analytical study with a cross-sectional design was then conducted on the subjects, with the resulting isolated DNAs amplified through polymerase chain reaction (PCR). The genotype of ADH3 was determined by means of restriction fragment length polymorphism (RFLP) using Ssp1 restricting enzyme. Liver function status was assessed by measuring serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT) and gamma glutamyl transferase (GGT) using a photometric system. Gene types of ADH3*1 (2.1%), ADH3*2 (82.7%) and ADH3*1/3*2 (15.2%) on the subjects were concluded, finding that there is no difference between the gender. In conclusion most of the ADH3 gene polymorphism of the subjects were ADH3*2 (82.7%). The influence of genetic polymorphisms on the status of liver function in the subjects showed significant difference according to GGT measurement, but the same cannot be said on the other two values measuring SGOT and SGPT.

A novel mechanism for endogenous formaldehyde elevation in SAMP8 mouse.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people worldwide. Increasing evidence suggests that formaldehyde might be one of the various pathological mechanisms involved in the process of AD onset. Here, we use an AD mouse model, senescence accelerated mouse-prone 8 strain (SAMP8), to study the relationship between endogenous formaldehyde and impairment of cognition. The Morris water maze test was used to evaluate the spatial learning and memory ability of 3-month-old SAMP8 mice, and we correlated the results with endogenous formaldehyde concentrations in the brain. To investigate the underlying reasons for formaldehyde elevation in neurodegenerative diseases, the expression levels of enzymes involved in formaldehyde metabolism were analyzed, including (anabolic) semicarbazide sensitive amine oxidase (SSAO) and (catabolic) alcohol dehydrogenase III (ADH3). When compared with age-matched SAMR1 mice, we found that in 3-month-old SAMP8 mice the capacity for spatial learning and memory was lower, while brain formaldehyde levels were higher. By using real-time PCR, western blotting, enzyme assay, and immunohistochemistry techniques, we discovered that SSAO expression levels were increased, whereas ADH3 exhibited reduced expression levels of mRNA, protein, and enzyme activity. The imbalance of these metabolic enzymes may represent a causal explanation for the observed formaldehyde elevation in the SAMP8 brain. Such increase could be responsible for the observed tau hyperphosphorylation assumed to result in protein aggregation, ultimately leading to cognitive impairment. Taken together, our study gives new insights into the role of metabolic enzymes in age-related accumulation of formaldehyde, and thus the establishment of neurodegenerative diseases.

A new view of alcohol metabolism and alcoholism--role of the high-Km Class III alcohol dehydrogenase (ADH3).

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 (beta, 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.