Effects of polymorphisms in untranslated regions of the class I alcohol dehydrogenase (ADH) genes on alcohol metabolism in Japanese subjects and transcriptional activity in HepG2 cells.

Human class I ADH is a dimmer formed by the random association of three types of subunits (alpha, beta and gamma) encoded by ADH1A, ADH1B, and ADH1C, respectively. Different kinetic properties were reported due to polymorphisms of ADH1B Arg47His and ADHIC Ile349Val. Besides these polymorphisms in the coding region, various mutations in the promoter region and 3' untranslated (UTR) region, which possibly affect expression and degradation rate, were recently reported. In this study, to asses the involvement of each genotype in alcohol metabolism in humans, our previously collected data set of blood EtOH and AcH changes were reanalyzed with regard to the ALDH2 Glu487Lys genotype. The effects of genotypes and haplo-types on transcriptional activity were also examined by a luciferase reporter assay by cloning the promoter region and 3' UTR corresponding to each polymorphism and transfecting into HepG2 cells. Among the nine polymorphisms, including ADH1B Arg47His and ADH1C Ile349Val, blood EtOH levels were significantly affected by polymorphisms ADH1B -451G>T, ADH1B +52A>G, ADH1B +531G>A, ADH1B +1176AG>del. and ADH1A -55C>T in ALDH2 Glu/Glu subjects. In the ALDH2 Glu/Lys genotype background, only ADH1C -254G>C and ADH1B His47Arg showed significant effects on blood EtOH. These five loci (and the two loci which had significant effect on blood EtOH in ALDH2 Glu/Glu and Glu/Lys subjects) also showed strong linkage disequilibrium. In comparison to the in vivo study on alcohol metabolism, significantly higher transcriptional activities in ADH1B -451T (rather than C) promoter and ADH1C-254 G (rather than C) promoter were observed in a luciferase assay in HepG2 cells. In conclusion, polymorphisms in the untranslated regions of ADH class I genes were demonstrated to clearly affect individual differences in alcohol metabolism. Especially, ADH1B -451G>T, ADH1C-254G>C polymorphisms were suggested to have functional significance with regard to transcriptional activity to the linkage equilibrium of polymorphisms ADH1B His47Arg and ADH1C Ile349Val.

Polymorphisms in the promoter region of the human class II alcohol dehydrogenase (ADH4) gene affect both transcriptional activity and ethanol metabolism in Japanese subjects.

Class II alcohol dehydrogenase (pi-ADH), encoded by alcohol dehydrogenase (ADH4), is considered to contribute to ethanol (EtOH) oxidation in the liver at high concentration. Four single nucleotide polymorphisms (SNPs) were found in the promoter region of this gene. Analysis of genotype distribution in 102 unrelated Japanese subjects revealed that four loci were in strong linkage disequilibrium and could be classified into three haplotypes. The effects of these polymorphisms on transcriptional activity were investigated in HepG2 cells. Transcriptional activity was significantly higher in cells with the -136A allele than in those with the -136C allele. To investigate whether this difference in transcriptional activity caused a difference in EtOH elimination, previous data on blood EtOH changes after 0.4 g/kg body weight alcohol ingestion were analyzed. When analyzed based on aldehyde dehydrogenase-2 gene (ALDH2) (487)Glu/Lys genotype, the significantly lower level of EtOH at peak in subjects with -136C/A and -136A/A genotype compared with subjects with -136C/C genotype indicated that -136 bp was a suggestive locus for differences in EtOH oxidation. This effect was observed only in subjects with ALDH2 (487)Glu/Glu. These results suggested that the SNP at -136bp in the ADH4 promoter had an effect on transcriptional regulation, and that the higher activity of the -136A allele compared with the -136C allele caused a lower level of blood EtOH after alcohol ingestion; that is, individuals with the -136A allele may consume more EtOH and might have a higher risk for development of alcohol dependence than those without the -136A allele.

A promoter polymorphism in the ALDH2 gene affects its basal and acetaldehyde/ethanol-induced gene expression in human peripheral blood leukocytes and HepG2 cells.

AIMS: To assess the effect of the -360G/A polymorphism in the promoter region of the human aldehyde dehydrogenase-2 (ALDH2) gene on its transcription, basal and acetaldehyde/ethanol-induced gene expression was examined by in vivo and in vitro experiments. METHODS: Human peripheral blood leukocytes were collected before and after alcohol ingestion (0.4 g/kg body weight) in 21 healthy young Japanese volunteers with a deficient phenotype of ALDH2 ((487)Glu/Lys), and the levels of ALDH2 mRNA were quantified by real-time RT-PCR. The transcriptional activity of the ALDH2 promoter was investigated by a reporter assay using HepG2 cells in the presence or absence of acetaldehyde/ethanol. RESULTS: The basal level of ALDH2 mRNA was significantly higher in -360A heterozygous subjects than in -360G homozygous subjects. In all subjects, regardless of the genotype, ALDH2 mRNA increased following ethanol ingestion. The promoter activity of a reporter plasmid for -360G was significantly lower than that of a reporter plasmid for -360A. Exposure to acetaldehyde induced a significant increase in the transcriptional activity of the -360G reporter, but not the -360A reporter. CONCLUSIONS: In vivo and in vitro experiments showed that the -360G allele has lower basal transcriptional activity than the -360A allele, whereas acetaldehyde/ethanol-induced gene expression, in general, seems to be more enhanced in individuals homozygous for the -360G allele than in those with the -360A allele. Thus, the promoter polymorphism may be involved in individual differences in acetaldehyde elimination.

Effects of functional polymorphisms related to catecholaminergic systems on changes in blood catecholamine and cardiovascular measures after alcohol ingestion in the Japanese population.

BACKGROUND: The polymorphism of human aldehyde dehyrogenase-2 (ALDH2) Glu(487)Lys is well known to be a crucial factor underlying the genetic background for alcohol sensitivity in Asian populations. Subjects with the inactive Lys(487) allele show a marked increase in blood acetaldehyde level after alcohol intake, which results in facial flushing and various cardiovascular-related symptoms. However, other polymorphisms related to catecholaminergic systems that tightly regulate the activity of the sympathetic nervous system may also influence the physiological changes after acute alcohol intake. METHODS: We investigated whether, together with the ALDH2 Gly(487)Lys and ADH1B Arg(47)His genotype, putative functionally important polymorphisms, including 9 loci in 7 human genes, were associated with changes in blood catecholamine levels and cardiovascular measures after alcohol ingestion. Forty-nine young Japanese males were subjected to blood catecholamine analysis after alcohol ingestion. Among them, 28 were also subjected to heart rate variability and blood pressure analysis. The contribution of polymorphisms to the alcohol-induced response was analyzed by multiple regression analysis. RESULTS: Among the polymorphisms examined in this study, haplotypes of the phenylethanolamine N-methyltransferase (PNMT) promoter [(-182bpG/A)_(-387bpG/A)] and catechol-O-methyltransferase (COMT) exon 4 [(Ex4 + 119bpC/G)_(Ex4 + 138bpG/A), Leu(136)Leu_Val(158)Met] are suggested to have functionally important effects on alcohol-induced cardiovascular symptoms by affecting blood catecholamine levels. The neuropeptide Y (NPY) promoter C-1450T genotype is also suggested to be involved in the individual differences in regulation of catecholamine secretion. CONCLUSIONS: This study suggested that these common polymorphisms of genes related to catecholaminergic systems, as well as those of the alcohol metabolizing system, are significant for understanding the basis of individual differences in alcohol sensitivity.

Effect of -361 G/A polymorphism of aldehyde dehydrogenase-2 gene on alcohol metabolism and its expression in human peripheral blood leukocytes.

The deficiency in activity of aldehyde dehydrogenase-2 (ALDH2), commonly found in Asians, is due to a mutation at position 487 in exon 12, encoded by the ALDH2*2 allele, which is a crucial factor for deficient ability to acetaldehyde (AcH) oxidation. In addition to this locus, polymorphism in -361 G/A mutation of this gene at 5'flanking region, commonly found in multi-racial populations, is one of the suggestive polymorphisms which may affect on the enzyme activity because it has been reported to affect on the transcriptional activity in hepatoma cells. We aimed to examine the individual differences in alcohol metabolism in Japanese population based on the genotypes of both ALDH2 exon 12 and -361 G/A promoter region. Following genotyping of 2 loci, subject groups based on the promoter genotype was defined as variant A carrier (A+; A/A and G/A) or not (A-; G/G). Under the condition with 0.4 g/kg body weight of alcohol ingestion, significant differences in AcH peak levels, that reached at 30 or 60 minutes in most subjects, was not detected between promoter A+ and A- groups both in exon 12 ALDH2*1/*1 and ALDH2*1/*2 subjects. Furthermore, we developed a real-time RT-PCR method to detect and quantitate the ALDH2 mRNA levels in easily accessible peripheral blood leukocytes (PBLs) to examine whether this promoter mutation affects on the amount of ALDH2 mRNA in normal human tissue at pre- and post-alcohol ingestion phase in ALDH2*1/*1 subjects. Significant increase of mRNA was observed only in A- group at 2 hours after alcohol ingestion. Maximal changing rates of mRNA in PBLs within 3 hours after alcohol intake were +48 % and +17 % in A and A' groups, respectively. These results suggest that the individual differences in ALDH2 enzyme activity may be intricately regulated by the common polymorphisms in these two loci in Asian populations.

Age-dependent changes in electroencephalographic responses to alcohol consumption in subjects with aldehyde dehydrogenase-2 genetic variations.

BACKGROUND: We have recently reported that alcohol consumption resulted in a significant increase in alpha power of the EEGs in aldehyde dehydrogenase-2 (ALDH2)-normal (NN) subjects but not in ALDH2-deficient heterozygote (ND) subjects. The purpose of the present study was to investigate interactive effects of individual factors such as age and ALDH2 genotype on alcohol-induced EEG changes. METHODS: We examined EEG power spectral changes induced by 0.4 ml/kg of alcohol ingestion in 53 NN and 21 ND subjects of two different age groups: younger and older groups. Blood ethanol and acetaldehyde levels were also determined in 17 NN and 13 ND subjects in separate studies. RESULTS: Alcohol consumption markedly increased EEG power in the NN subjects of the older group, especially in theta and slow alpha power, whereas only slight increases were noted in fast alpha and beta power in the NN subjects of the younger group. However, no such differences between the two age groups were observed in the ND subjects. It should be noted that there were no differences in blood ethanol and acetaldehyde level at 30 min after alcohol ingestion between the different age groups in both genotypes. However, there was a significant increase in frequency of alcohol intake in the older group of both genotype groups. The multiple regression analysis indicated that both alcohol use habits and genotype, as well as aging, significantly modulated EEG changes after alcohol ingestion. CONCLUSIONS: The results suggest that both ALDH2 genotype and age as well as alcohol use habits modify alcohol sensitivity in the central nervous system, resulting in greater increases in EEG energy in response to alcohol intake in the older group of the NN subjects.

Effects of aldehyde dehydrogenase-2 genotype on cardiovascular and endocrine responses to alcohol in young Japanese subjects.

The present study was designed to investigate the effects of aldehyde dehydrogenase-2 (ALDH2) genotype on cardiovascular and endocrine responses to alcohol ingestion in young, healthy Japanese subjects. For this purpose, we monitored changes in the electrocardiogram (ECG), blood pressure (BP), finger blood flow (BF) and facial skin temperature (FST) during and after alcohol ingestion (0.4 ml/kg body weight). Spectral analyses of beat-to-beat variations of heart rate (HR), BP and BF were applied. Two major spectral components were examined at low frequency (LF: 0.04-0.15 Hz) and high frequency (HF: 0.15-10.4 Hz) bands for HR and BP variability (BPV). Significant effects for ALDH2 genotype were observed in HR variability (HRV) analysis; HF power of HRV was markedly depressed and the LF/HF ratio was significantly higher with alcohol in ALDH2-deficient (ND) subjects, while ALDH2-normal (NN) subjects did not display such changes. Analysis of BP variability showed increased LF and HF power after alcohol ingestion in the NN subjects, but there were no significant differences between genotypic groups. We also examined BF variability (BFV) in six major spectral components; power of the 0.8-2.2 Hz frequency band was significantly affected by genotype and higher power was observed in the ND subjects. Plasma concentrations of both epinephrine and norepinephrine increased after alcohol ingestion only in the ND subjects. Furthermore, plasma concentrations and urinary excretion of epinephrine, but not norepinephrine, were higher after alcohol ingestion in the ND than in the NN subjects. Blood acetaldehyde levels were about 10-fold higher in the ND than in the NN subjects although blood alcohol levels similarly increased in the ND and NN subjects. Our results also indicated that alcohol ingestion increased secretion of pituitary-adrenal hormones including ACTH, beta-endorphin and cortisol in the ND subjects. The present results along with previous studies suggest that alcohol-induced tachycardia in the ND subjects was probably mediated by acetaldehyde-induced rise in epinephrine secretion from the adrenal medulla and/or changes in the autonomic nervous system. Alcohol-induced relative predominance of cardiac sympathetic activity in the ND subjects might be ascribed partly to increased norepinephrine secretion from sympathetic nerve terminals. Effects of acetaldehyde on these cardiovascular and endocrine systems were discussed in terms of their effects on the central nervous system.