Alcohol Metabolizing Enzymes, Microsomal Ethanol Oxidizing System, Cytochrome P450 2E1, Catalase, and Aldehyde Dehydrogenase in Alcohol-Associated Liver Disease

Once ingested, most of the alcohol is metabolized in the liver by alcohol dehydrogenase to acetaldehyde. Two additional pathways of acetaldehyde generation are by microsomal ethanol oxidizing system (cytochrome P450 2E1) and catalase. Acetaldehyde can form adducts which can interfere with cellular function, leading to alcohol-induced liver injury. The variants of alcohol metabolizing genes encode enzymes with varied kinetic properties and result in the different rate of alcohol elimination and acetaldehyde generation. Allelic variants of these genes with higher enzymatic activity are believed to be able to modify susceptibility to alcohol-induced liver injury; however, the human studies on the association of these variants and alcohol-associated liver disease are inconclusive. In addition to acetaldehyde, the shift in the redox state during alcohol elimination may also link to other pathways resulting in activation of downstream signaling leading to liver injury

Protective Effects Of The Alcohol Dehydrogenase-Adh1b Allele

Alcohol dehydrogenase is a critical enzyme in the metabolism of alcohol. Expression of three alleles at the ADH1B locus results in enzymes that differ in turnover rate and affinity for alcohol. The ADH1B*3 allele, which appears to be unique to African Americans, is associated with more rapid alcohol metabolism than the more prevalent ADH1B*1 allele. It has been previously demonstrated that the presence of at least one maternal ADH1B*3 allele confers a protective effect against alcohol teratogenicity in African American infants and children. This study was conducted to determine whether the presence of the ADH1B*3 allele in the mother or fetus continues to be protective in alcohol-exposed individuals during adolescence. 186 adolescents and 167 mothers participating in the 14-year follow-up of the Detroit Longitudinal Cohort had been genotyped for ADH1B alleles. The frequencies of the ADH1B*3 allele were 17.6% in the mothers and 21.0% in the adolescents, which are consistent with the 22% expected for the African American population. Confirming previous studies, prenatal alcohol exposure was associated with increased attention problems and externalizing behaviors in adolescents born to mothers with two ADH1B*1 alleles but not in those whose mothers had at least one ADH1B*3 allele. The presence of an ADH1B*3 allele in the adolescent conveyed a less pronounced protective effect against fetal alcohol-related deficits at this age. This study is the first to demonstrate that the protective effects of the maternal ADH1B*3 allele continue to be evident during adolescence and suggests that differences in alcohol metabolism genes may help account for individual differences in the vulnerability of offspring to the effects of fetal alcohol exposure. The protective effect of the maternal ADH1B*3 allele may be due to the more rapid metabolism of alcohol that it confers on the mother, which presumably results in a reduction of the peak blood alcohol concentration to which the fetus is exposed during each drinking episode

Efecto ansiogénico del disulfiram en animales tratados con alcohol: Relevancia en la terapia farmacológica antialcohólica

Dotzenes Jornades de Foment de la Investigació de la FCHS (Any 2006-2007)El alcohol etílico, a dosis moderadas, produce efectos ansiolíticos, tanto en humanos como en roedores. Sin embargo, la acumulación de acetaldehído (primer metabolito del alcohol) en el organismo, está considerada aversiva y en humanos produce una respuesta vegetativa conocida como “sensibilidad al alcohol”. Este efecto aversivo, es la base de los tratamientos en alcohólicos mediante el disulfiram, un inhibidor de la Aldehído Deshidrogenada (ALDH), enzima que metaboliza el acetaldehído. La respuesta autonómica comparte muchos de los síntomas vegetativos que caracterizan a un episodio de ansiedad. En este trabajo estudiamos los posibles efectos ansiogénicos de la acumulación periférica de acetaldehído en ratones, utilizando el laberinto elevado en cruz, paradigma clásico para la evaluación de ansiedad en roedores. Como ha sido observado previamente, el alcohol aumentó el porcentaje de entradas en los brazos abiertos del laberinto elevado en cruz. Sin embargo, el pretratamiento con disulfiram produjo una reducción de estos efectos ansiolíticos del etanol a las dosis que fueron efectivas en inhibir la ALDH. Así mismo, el acetaldehído administrado periféricamente demostró reducir el porcentaje de exploración de los animales en los brazos abiertos. La locomoción no se vio modificada significativamente por estas dosis de acetaldehído, lo cual indica que los efectos ansiogénicos no son debidos a un cambio motor inespecífico

Transcriptional regulation of the human alcohol dehydrogenases and alcoholism

Indiana University-Purdue University Indianapolis (IUPUI)Alcohol dehydrogenase (ADH) genes encode proteins that metabolize ethanol to acetaldehyde. Humans have seven ADH genes in a cluster. The hypothesis of this study was that by controlling the levels of ADH enzymes, cis-regulatory regions could affect the risk for alcoholism. The goal was thus to identify distal regulatory regions of ADHs. To achieve this, sequence conservation across 220 kb of the ADH cluster was examined. An enhancer (4E) was identified upstream of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell specific, as no enhancer activity was detected in a human lung cell line, H1299. The enhancer activity was located in a 565 bp region (4E3). Four FOXA and one HNF-1A protein binding sites were shown to be functional in the 4E3 region. To test if this region could affect the risk for alcoholism, the effect of variations in 4E3 on enhancer activity was tested. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold. A third variation had a small but significant effect. The effect of variations in the ADH1B proximal promoter was also tested. At SNP rs1229982, the C allele had 30% lower activity than the A allele. In addition to studying the regulatory regions of ADH genes, the effects of alcohol on liver-derived cells (HepG2) were also explored. Liver is the primary site of alcohol metabolism, and is highly vulnerable to injuries due to chronic alcohol abuse. To identify the effects of long term ethanol exposure on global gene expression and alternative splicing, HepG2 cells were cultured in 75 mM ethanol for nine days. Global gene expression changes and alternative splicing were measured using Affymetrix GeneChip® Human Exon 1.0 ST Arrays. At the level of gene expression, genes involved in stress response pathways, metabolic pathways (including carbohydrate and lipid metabolism) and chromatin regulation were affected. Alcohol effects were also observed on alternative transcript isoforms of some genes

Alcohol dehydrogenase and aldehyde dehydrogenase gene polymorphism in Turkish alcohololic people and control group

Thesis (Master)--İzmir Institute of Technology, Biotechnology and Bioengineering, İzmir, 2007Includes bibliographical references (leaves: 41-44)Text in English; Abstract: Turkish and Englishix, 44 leavesIn this study, alcohol dehydrogenase and aldehyde dehydrogenase gene, encoding alcohol metabolizing enzymes, polymorphisms were determined in alcoholic and nonalcoholic determination methods. The main objective in the study was to investigate the relationship of ADH2, ADH3 and ALDH2 gene polymorphism with the tendency of Turkish people to develop alcohol tolerance. The other significant objective was to compare polymorphism types of ADH2, ADH3 and ALDH2 seen in Turkish people and other ethnic groups or races in the world. In this present study, ADH3 genotypes of 141 alcoholic subjects, and also ADH2 and ALDH2 genotypes of 156 alcoholic subjects were assigned. The control group consisted of 80 healthy non-drinkers. Three different SNP genotyping methods were used in this study. ADH3 genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism method (RFLP-PCR). ADH2 genotyping was performed by allele specific primer extension method and ALDH2 genotyping was performed by multiplex PCR by using two allele specific primer pairs. The ADH2.1 genotype was the most common type of all ADH2 genotypes in both alcoholic and non alcoholic groups. However, there was no significant difference between alcoholic and non alcoholic groups for ADH2 genotyping.ADH3 genotyping of both groups suggested that the ADH3.2 genotype frequency was higher than ADH3.1. ADH3.2 was found to be more prevalent in alcoholics compared to control group, suggesting that alcoholics were more tolerant to alcohol. In all of the alcoholic and non-alcoholic subjects examined, the frequency of ALDH2.1 was found to be 100%. Finally,it can be inferred from that obtained results, ADH2 genetic variations seem not to be related to alcoholism. On the other hand, ADH3 and ALDH2 genetic variations can make Turkish people susceptible to alcohol dependency. If all the results are taken into consideration, it is inferred that Turkish people have the inherited variations of ADH and ALDH genes which do not protect them to have alcohol sensitivity and dependency. Obtained results in the study are consistent with the white race in the world including European people but not consistent with Oriental people as expected

Saliva sampling of alcoholic participants using three saliva collection methods

The potential of using saliva as a diagnostic fluid is well documented. The aim of this study was to assess the quality and quantity of saliva DNA of alcoholic and non-alcoholic participants using three saliva collection methods; DNA-SalTM (Oasis Diagnostics, USA), Oragene-DNA (DNA Genotek Inc, Ontario, Canada) and whole saliva collection method. Saliva DNA of non-alcoholic (n=30) and alcoholic participants (n=10) age between 25 and 35 years was assessed qualitatively and quantitatively using spectrophotometry. Saliva DNA quantity was the highest for all participants when using the DNA-Sal TM saliva collection kit (p<0.05). The use of a mechanical scraper provided only in the DNA-Sal TM kit may have contributed to the highest DNA yield for all participants. The quantity of saliva DNA when assessed using spectrophotometer was found to be significantly lower (p<0.05) for the alcoholic (16±3.57 ng/μL) than non-alcoholic participants (19.92±6.18 ng/μL). To determine the integrity of the DNA samples, PCR amplification of the Alcohol Dehydrogenase gene, ADH1B was carried out and the PCR was found to be successful. For all participants, the DNA quality of the saliva collected using the three saliva collection methods was found to be in the acceptable range considered as pure DNA. The DNA quality and quantity of saliva collected from the three saliva collection methods were considered suitable for research purposes

A Study of Biochemical, Pharmacokinetic, Physiological and Psychomotor Variables and Ethanol Sensitivity after Low-dose Ethanol

The influence of ALDH2 and ADH2 genotype on the biochemical, physiological, psychomotor and subjective responses of Asian subjects to a challenge dose of ethanol were investigated. One hundred and ten healthy male and female subjects of fill] or partial North East Asian descent (with ancestral origins in China, Japan, Korea and Vietnam), who were living in Australia at the time of testing, were genotyped for alcohol dehydrogenase (ADHz) and aldehyde dehydrogenase (ALDHz) enzymes using a combination of the polymerase chain reaction (PCR) with restriction enzyme digestion, allele specific oligonucleotide probing, or constant denaturant gel electrophoresis methods. Volunteers were given a low oral dose (0.3 g kg^-1) of ethanol and were assessed subjectively for their degree of flush and for performance impairment using a battery of psychomotor tests (including divided attention, digit symbol coding, standing steadiness and critical flicker fusion frequency threshold). Self-report questionnaires were used to assess the subjects' perception of their intoxication and impairment. Blood ethanol concentrations (BECs) were monitored by breath analysis every fifteen minutes and blood samples were obtained from subjects before and at 15, 60 and 120 minutes afier ethanol administration. Measurements were made of the blood or plasma levels of acetaldehyde, acetate, pyruvate, lactate and ethanol. The blood pressure (systolic and diastolic), heart rate and facial temperature were also recorded at regular intervals. The effects of the ADHz genotype, ALDH; genotype, ALDHZ/ ADHz combination genotype, the degree of flush and gender on the psychomotor performance, physiological, biochemical, pharmacokinetic and subjective responses of the subjects were explored to determine their influence on the response to ethanol. When subjects were classified by ALDHz genotype, the BEC curve, acetaldehyde concentration, acetate concentration, facial temperature, heart rate, critical flicker fusion frequency threshold, digit symbol coding reaction time, standing steadiness and divided attention delay and excursion were all affected by whether the subject was ALDH; Homoll, Het or Hom022. The psychomotor performance of ALDHz Hom022 subjects was found to be more impaired in the divided attention delay, excursion and digit symbol coding reaction time tasks than in either Het or Homoll subjects. The standing steadiness epoch time and critical flicker fusion frequency threshold were also most affected by ethanol in the ALDHz Hom022 group. The effect of ethanol on the pyruvate concentration, heart rate, CFFF threshold and standing steadiness also differed significantly among subjects of different ADH; genotype. The ADHz Homozygote-22 (Hom022) subjects had a higher standing steadiness epoch time and critical flicker fusion frequency threshold than either Heterozygote (Het) or Homozygote—ll (Homol 1) subjects. The subjectively rated degree of flush was associated with differences in acetaldehyde concentration, acetate concentration, digit symbol coding reaction time, divided attention delay, facial temperature and heart rate measured. Psychomotor impairment in the divided attention delay task was greatest for the subjects who flushed with intermediate severity, although at two hours post-ethanol, the group which produced the most severe flushing was more severely affected. In the digit symbol coding reaction time, the subjects who produced the most severe flushing reactions were also most impaired

ГЕНЕТИКА И ГЕНОМИКА ЧЕЛОВЕКА. ПОПУЛЯЦИИ И ЭТНОСЫ В ПРОСТРАНСТВЕ И ВРЕМЕНИ: ЭВОЛЮЦИОННЫЕ И МЕДИЦИНСКИЕ АСПЕКТЫ

Генетическая адаптация популяций человека к локальным условиям среды может быть представлена как возникновение новых аллелей в результате мутаций и последующее изменение частот аллелей в поколениях вследствие естественного отбора признаков, ассоциированных с этими аллелями и важных для выживания и успешной репродукции человека. Помимо процессов адаптации, на изменение частот аллелей влияют генетический дрейф и миграции. Исследование генетической адаптации человека занимает одно из центральных мест в биологической антропологии, генетике человека и эволюционной биологии и уже дало значительный вклад в понимание взаимодействия средовых и генетических факторов, влияющих на здоровье человека.Ниже будут описаны подходы к поиску аллелей, определяющих приспособленность человека к действию факторов окружающей среды. Эти подходы потенциально приложимы не только к человеку, но и к другим животным и растениям. Исходной посылкой для применения этих подходов является перекрывание ареала действия конкретного фактора среды и ареала повышенной популяционной частоты аллеля, что позволяет предполагать их связь,хотя и не доказывает ее. Такое совпадение ареалов, после вычитания вклада случайных причин (генетический дрейф, генетическая подразделенность популяций), является основой для выдвижения и дальнейшей проверки гипотезы о роли данного фактора отбора в повышении частоты данного аллеля в популяциях человека. Прямое доказательство таких гипотез может быть получено, если удастся показать связь генотипа и фенотипа с приспособленностью к данному фактору отбора (выживаемость или репродуктивный успех) (Hancock et al., 2008, 2010а).Мы привыкли сравнивать генетические особенности индивидов друг с другом. Индивид как единица учета был и остается необходимым, но позволяет установить лишь наличие аллеля, но не его частоту. Для придания «смысла» гену и конкретному аллелю нужно применить эволюционный подход. Для этого единицей учета должна стать популяция, а учитываемым признаком станет частота аллеля. При этом сравнивать друг с другом нужно признаки популяций (условия среды и частоты аллелей), а не признаки индивидов

Functional Analysis of Corazonin and Its Receptor in Drosophila melanogaster

Corazonin (Crz) is an amidated undecapeptide originally isolated from the American cockroach. It has been shown to affect diverse physiological functions in a species-specific manner. However, the functionality of Crz in Drosophila melanogaster has not yet been determined. To gain insight into the role of Crz signaling in vivo, Crz and CrzR null alleles were obtained by transposable element mobilization. Flies carrying a deficiency uncovering Crz and pr-set7 loci were generated via P-element excision, and the latter was rescued by wild-type pr-set7 transgene. A mutation of Crz receptor (CrzR) was generated by Minos-element mobilization from GRHRIIMB00583 [GRHRIIMB00583] allele. The mutant flies showed normal circadian rhythm and fecundity as compared with the wild-types. Using these mutants, the functions of the Crz signaling system in alcohol metabolism were investigated. Two major enzymes involved in alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In this study, Crz expressing neurons and CrzR were identified as important regulators of these enzymes. Flies lacking Crz neurons or CrzR display significantly delayed recovery from ethanol-induced sedation, which is causally associated with fast accumulation of acetaldehyde in the CrzR01 [CrzR01] mutant following ethanol exposure. Consistent with this, Crz and CrzR were found to be required for normal ALDH activity. In addition, CrzR shows a down-regulatory effect on ADH activity, which is transcriptionally initiated through the protein kinase A (PKA)-dependent pathway. Such transcriptional regulation was found to be exclusive to ADH, but not ALDH mRNA. To gain the evolutionary aspect of CrzR, CrzR from the Housefly, Musca domestica (MdCrzR) was cloned and characterized. MdCrzR deduced from the full-length cDNA sequence is a 655-amino acid polypeptide that contains seven trans-membrane (TM) domains and other motifs that are characteristics of Class-A G-protein coupled receptors. Although the TMs and loops between the TMs are conserved in other CrzRs, N-terminal extracellular domain is quite dissimilar. Tissue-specific RT-PCR revealed a high level of MdCrzR expression in the larval salivary glands and a moderate level in the CNS. In adults, the expression was broadly observed without significant gender difference, suggesting multifunctionality of the Crz signaling system