Homozygous ALDH2 Research Articles

Abstract 491: E-cigarette Aerosol Elevates Cardiovascular Oxidative Stress in Mice With Aldehyde Dehydrogenase 2 Deficiency

Introduction: E-cigarette aerosol contains reactive aldehydes including acetaldehyde, formaldehyde, and acrolein when e-cigarette is heated. Approximately 560 million people worldwide cannot efficiently metabolize aldehydes present in e-cigarette aerosol, due to a genetic deficiency in aldehyde dehydrogenase 2 enzyme (ALDH2), known as ALDH2*2. Little is known how aldehyde exposure from e-cigarettes, coupled with genetic differences in aldehyde metabolism, affects cardiovascular oxidative stress both at a physiological and cellular level. Hypothesis: E-cigarette aerosol exposure will elevate heart rate and cellular oxidative stress more substantially in ALDH2*2 knock-in mice versus wild type ALDH2 mice. Methods: To measure aldehyde levels, e-cigarette Juul aerosols were collected and quantified by selective ion flow gas mass spectrometry. Further, age-matched male wild type and homozygous ALDH2*2 mice (8-10 weeks old, ~25g) were implanted with EKG telemeters. After surgical recovery, mice were paired by genotype (one wild type ALDH2 and one ALDH2*2 mice) and exposed to either Juul aerosol or room air 4 sessions per day for 10 days. For each session, 7 puffs/min were drawn for the first two minutes (a total of 14 puffs), and the whole body exposure to Juul aerosol lasted 7 minutes, continued with 23 minutes smoking-free intervals in each session. Mice EKG waveforms were recorded daily. After 10 days of exposure, heart homogenates were subjected to biochemical assays including lipid peroxidation, 4-HNE protein adduct formation, and protein carbonylation. Results: Quantification of reactive aldehyde levels in e-cigarettes revealed that Juul aerosol contained acetaldehyde (5.3±0.32 ppm), formaldehyde (0.20±0.02 ppm), and acrolein (0.09±0.01 ppm). When exposed to Juul aerosol, ALDH2*2 mice showed a maximal increase in heart rate unlike ALDH2 wild type mice (774.6±29.5 bpm versus 678.9±32.8 bpm respectively, * p <0.01, n=8) at day 6. Furthermore, heart homogenates from ALDH2*2 mice demonstrated exacerbated oxidative stress, including higher level of 4-HNE adducts (1.5-fold), protein carbonyls (1.5-fold) and lipid peroxides (2-fold) relative to hearts from wild type ALDH2 mice, when both genotype mice were exposed to Juul e-cigarette aerosol (n=4/group). Conclusions: These findings indicate e-cigarette aerosols contain reactive aldehydes, primarily acetaldehyde. A deficiency in reactive aldehyde metabolism by having an ALDH2*2 deficiency may contribute to increases in heart rate and oxidative stress within the cardiovascular system while smoking e-cigarettes.

Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population

Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2 * 2, CYP2E1 * 1 combined with genotype homozygous ALDH2 * 1 found in this study could be leading to the population to a potential risk to alcoholism.

OP228: Elucidating risk factors for oral leukoplakia affecting gingivae among a Japanese population

Purpose Population screening studies in Japanese have confirmed that oral leukoplakia (OL) affecting the gingivae (gingival leukoplakia: GL) is the most frequent location of OL. We investigated a cohort of patients with GL and their clinic-pathological features were compared with patients with OL affecting other sites (n-GL). Methods Among 204 subjects diagnosed with OL (GL:114, n-GL:90) during 09-11 in 4 city hospitals in Japan, 151(74%) subjects (GL:88, n-GL:63) (age: 64.4 ± 12.7) were recruited. OL was diagnosed by WHO criteria (Warnakulasuriya et al., J Oral Pathol Med, 2007). They were interviewed for risk factors and investigated by patch testing for dental materials, biopsy for dysplasia recording and detection of aldehyde dehydrogenase (ALDH2) polymorphism in saliva by Real-Time PCR for SNP genotyping (rs671) and screened for HPV-DNA by GP5+/6+PCR from paraffin-embedded tissues among selected subjects ( n = 31). Results There were no significant differences by age (mean ages: GL, 65.9; n-GL, 62.3) or in tobacco and alcohol use in the two groups. Eighty-one percent of GL were seen in association with prosthesis and 27% close to amalgam fillings. Metal allergy test was positive in 58% of the GL but was not significantly higher compared to the n-GL (41%). Higher dysplasia grades were found in n-GL ( P = 0.018). Alcohol intolerance (flush reaction) was higher in n-GL (73%) ( P = 0.035). The homozygous ALDH2∗2/2 and 2∗1/2, which work less efficiently in alcohol metabolism was higher in n-GL (55%) than in GL (41%). HPV-DNA was not detected in any of the samples. Conclusions GL had lower dysplasia scores compared to n-GL, but the risk factors appear similar. Oral leukoplakia in n-GL may be associated with aberrant oxidative pathway of alcohol metabolism in Japanese. There was no evidence to support a causal relationship between HPV-DNA detection and GL nor n-GL (Funded by Sasakawa GB Foundation).

Increased frequencies of micronucleated reticulocytes and T-cell receptor mutation in Aldh2 knockout mice exposed to acetaldehyde

Aldehyde dehydrogenase-2 (ALDH2) metabolizes acetaldehyde produced from ethanol into acetate and plays a major role in the oxidation of acetaldehyde in vivo. About half of all Japanese people have inactive ALDH2. We generated homozygous Aldh2 null (Aldh2-/-) mice by gene targeting knockout as a model of ALDH2-deficient humans. To investigate the mutagenicity of acetaldehyde, a micronucleus assay and a T-cell receptor (TCR) gene mutation assay were performed in Aldh2-/- mice and wild-type (Aldh2 +/+) mice exposed to acetaldehyde. The mice were continuously exposed to 125 and 500 ppm of acetaldehyde vapor for 2 weeks. Another group was orally administered 100 mg/kg once a day for 2 weeks continuously. The mice were killed after 2 weeks of exposure to acetaldehyde, and the frequency of micronucleated reticulocytes was measured by flow cytometry. We also observed the incidence of TCR gene mutations in T-lymphocytes by measuring the variant CD3(-CD4+) expression by flow cytometry. The frequency of micronucleated reticulocytes induced by acetaldehyde was significantly increased in Aldh2 -/- mice, but not in Aldh2 +/+ mice. TCR mutant frequency was also associated with acetaldehyde exposure in Aldh2-/ - mice, especially after oral administration; however, it was not associated with acetaldehyde exposure in Aldh2 +/+ mice. In conclusion, Aldh2 -/- mice showed high sensitivity in the micronuclei and TCR mutation assays compared with Aldh2 +/+ mice after exposure to acetaldehyde.

Alcohol dehydrogenase and aldehyde dehydrogenase polymorphisms and colorectal cancer: The Fukuoka Colorectal Cancer Study

Alcohol dehydrogenase and aldehyde dehydrogenase are key enzymes in alcohol metabolism and therefore may be of importance to colorectal cancer development. The present case-control study was conducted to determine the influence of ADH2, ADH3 and ALDH2 polymorphisms in Fukuoka, Japan, with 685 incident cases of histologically confirmed colorectal adenocarcinomas and 778 community controls selected randomly from the study area. Alcohol use was ascertained by in-person interview. Statistical adjustment was made for sex, age class, area, and alcohol use. Individuals with the allele 47Arg of the ADH2 polymorphism (slow metabolizers) had a statistically significant increase in risk, with an adjusted OR of 1.32 (95% CI = 1.07-1.63), compared with those having the ADH2*47His/His genotype. This association was not affected by the level of alcohol consumption. The ADH3 polymorphism showed no measurable association with the risk of colorectal cancer on either overall analysis or stratified analysis with alcohol use. The heterozygous ALDH2*487Glu/Lys genotype was not associated with an increase in the risk of colorectal cancer (adjusted OR 0.89, 95% CI = 0.71-1.13) compared with the ALDH2*487Glu/Glu genotype. Rather unexpectedly, the homozygous ALDH2*487Lys/Lys genotype was related to a statistically significantly decreased risk of colorectal cancer (adjusted OR 0.55, 95% CI = 0.33-0.93). It is unlikely that acetaldehyde metabolism determined by ALDH2 polymorphism contributes to the risk of colorectal cancer, whereas the role of ADH2 polymorphism deserves further investigation.

Aldehyde dehydrogenase 2 genotypes, alcohol flushing symptoms and drinking patterns in Thai men

The drinking behavior, alcohol-induced facial flushing and ALDH2 genotypes were determined in 283 Thai men comprising 85 who were alcohol-dependent, 62 hazardous/harmful drinkers and 136 non-drinkers or infrequent drinkers. A structured interview questionnaire, containing the ‘tri-level’ method and the Alcohol-Use Disorders and Associated Disabilities Schedule, was used to determine the quantity of drinking and the number of alcohol-related adverse experiences. The study revealed the mutant ALDH2*2 allele in 44 (15.5%) subjects. The risks of being alcohol-dependent and of having hazardous/harmful drinking were lower in individuals with heterozygous ALDH2*1/*2, compared with homozygous ALDH2*1/*1 [relative probability ratios (95% CI) 0.14 (0.05–0.41) and 0.23 (0.08–0.61), respectively]. Eighty percent of those who were heterozygous and 28% of those who were homozygous ALDH2*1 reported flush symptoms after drinking alcohol. Twenty-nine percent of homozygous ALDH2*1 individuals, but only 9% of heterozygous subjects, drank almost everyday (24–30 days/month). Similarly, higher percentages of people drinking beyond the safety limit (>60 g/day) and having alcohol-related problems were observed in homozygous ALDH2*1 compared with heterozygous individuals: 32% vs. 5% and 27% vs. 12%, respectively. Overall, the study supports the role of the mutant ALDH2*2 allele in preventing high alcohol consumption and the development of alcohol dependence in a Thai population.

Association between polymorphisms of ethanol-metabolizing enzymes and susceptibility to alcoholic cirrhosis in a Korean male population.

Alcohol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH) and cytochrome P-4502E1 (CYP2E1), and then to acetate by aldehyde dehydrogenase (ALDH). Polymorphisms of these ethanol-metabolizing enzymes may be associated with inter-individual difference in alcohol metabolism and susceptibility to alcoholic liver disease. We determined genotype and allele frequencies of ALDH2, CYP2E1, ADH2, and ADH3 in male Korean patients with alcoholic cirrhosis (n=56), alcoholics without evidence of liver disease (n=52), and nondrinkers (n=64) by using PCR or PCR-directed mutagenesis followed by restriction enzyme digestion. The prevalences of heterozygous ALDH2*1/*2 plus homozygous ALDH2*2/*2 in patients with alcoholic cirrhosis (7.1%) and alcoholics without evidence of liver disease (3.8%) were significantly lower than that in nondrinkers (45.3%). The c2 allele frequencies of the CYP2E1 in alcoholic cirrhosis, alcoholics without evidence of liver disease, and nondrinkers were 0.21, 0.20, and 0.20, respectively. Allele frequencies of ADH2*2 in the three groups were 0.78, 0.74, and 0.77 and those of ADH3*1 were 0.94, 0.98, and 0.95. Therefore, we confirmed the observation that the ALDH2*2 gene protects against the development of alcoholism. However, the development of cirrhosis in Korean alcoholic patients was not associated with polymorphisms of ethanol-metabolizing enzymes.

A Novel Polymorphism (‐357 G/A) of the ALDH2 Gene: Linkage Disequilibrium and an Association With Alcoholism

Human mitochondrial aldehyde dehydrogenase (ALDH2) is a major enzyme responsible for the oxidation of acetaldehyde derived from ethanol metabolism. The human ALDH2 gene shows genetic polymorphism at position 1510 with a G to A transition in exon 12. This mutation leads to ALDH2 enzyme deficiency and protection against alcoholism. As yet, no polymorphism for the promoter region of the ALDH2 gene has been reported. We analyzed 600 nucleotides of the promoter region in addition to exon 12 from 571 Japanese, 68 Chinese, 80 Myanmar, 60 Mongolians, and 82 North-American Caucasians using single-strand conformational change polymorphism (SSCP) analysis and the polymerase chain reaction (PCR). PCR products that showed an aberrant banding pattern detected by the SSCP analysis were subjected to PCR direct sequencing. A novel polymorphism at -357 with a G to A substitution was found in all the population groups, including North-American Caucasians. In addition, the polymorphic status in the promoter and exon 12 suggested linkage disequilibrium between the two loci, which indicated that among Japanese, the ALDH2*2 allele is linked to the G promoter allele, and theALDH2*1 allele is linked to the A allele. A total of 206 healthy male controls and 185 alcoholic male patients with the homozygous ALDH2*1 genotype were analyzed for the polymorphism in the promoter. Genotypic frequencies of GG, GA, and AA for alcoholics were 54.1%, 44.3%, and 1.6%, and those for controls were 52.9%, 40.3%, and 6.8%, respectively. The A allele frequencies for alcoholics and controls were 0.24 and 0.27, respectively. A chi2 test for the entire 3 x 2 table indicated significant variations in the three genotypes (chi2 = 6.40, p < 0.05). However, no significant difference in allelic frequencies between the two groups was observed. This new polymorphism in the ALDH2 promoter is present in all populations studied. Further analysis in other ethnic groups is necessary to establish this as an additional risk factor for alcoholism.

Characteristics of Japanese alcoholics with the atypical aldehyde dehydrogenase 2*2. I. A comparison of the genotypes of ALDH2, ADH2, ADH3, and cytochrome P-4502E1 between alcoholics and nonalcoholics.

We examined the genotypes of the aldehyde dehydrogenase (ALDH)-2, alcohol dehydrogenase (ADH)-2, ADH3, and P-4502E1 loci of 53 alcoholics and 97 nonalcoholics. All of the subjects fulfilled the DSM-III-R criteria for alcohol dependence. The control group consisted of 97 subjects who were either hospital staff or students. We also compared the frequencies of homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes in alcoholics. Our study revealed differences in the allelic frequencies of the ALDH2, ADH2, and ADH3 loci between alcoholics and nonalcoholics. For alcoholics with both homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes, it was found that ADH2 and ADH3 played important rates. Alcoholics with the heterozygous ALDH2*1/2 genotype showed a significantly higher frequency of ADH2*1/1 than ones with the homozygous ALDH2*1/1 genotype. We assume ADH2*1 plays an important role in the development of alcoholism in alcoholics with the heterozygous ALDH2*1/2 genotype.

Investigation of the role of polymorphisms at the alcohol and aldehyde dehydrogenase loci in genetic predisposition to alcohol-related end-organ damage

Little is known about factors determining individual susceptibility to the physical complications of alcohol abuse but genetically determined differences in ethanol metabolism may be important. The oxidative metabolism of alcohol is catalyzed by alcohol and aldehyde dehydrogenase. Polymorphisms have been observed at two of the five loci encoding alcohol dehydrogenase subunits: ADH2 (producing three beta subunits) and ADH3 (producing two tau subunits) and also at the locus encoding the metabolically important form of aldehyde dehydrogenase, ALDH2. We have compared ADH2, ADH3 and ALDH2 allele frequencies in patients with alcohol-related cirrhosis (n = 59) and chronic pancreatitis (n = 13) with 79 local healthy control subjects. The different alleles were detected with allele-specific oligonucleotide probes after amplification of leukocyte DNA by the polymerase chain reaction. All patients and all but one control subject were homozygous ADH2*1, encoding the beta 1 subunit. No ADH2*3 alleles were detected. All 34 patients and 39 control subjects tested were homozygous ALDH2*1 encoding the active enzyme. ADH3 allele frequencies were different in patients and control subjects. ADH3*1 frequency: control subjects, 55.1%; cirrhotic patients, 62.7%; chronic pancreatitis patients, 65.4%. The difference between the patient groups combined and the control subjects was significant (p less than 0.05; G-test of Sokal and Rohlf) if it was assumed that the allele frequency in our control population was a reasonable estimate of our local population allele frequency. These results suggest that genetically determined differences in alcohol metabolism may, in part, explain predisposition to alcohol-related end-organ damage.