Abstract
Free full text
Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model
Abstract
Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The Vmax of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.—Rivera-Meza, M., Quintanilla, M. E., Tampier, L., Mura, C. V., Sapag, A., Israel, Y. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.
Alcoholism is one of the most prevalent types of drug dependence in the world (1, 2). However, there is substantial evidence that individuals bearing certain polymorphisms in genes coding for enzymes involved in the metabolism of ethanol are greatly protected against alcoholism (3,4,5). In mammals, ethanol is metabolized mainly by hepatic alcohol dehydrogenase (ADH) to acetaldehyde, which is further oxidized to acetate by mitochondrial aldehyde dehydrogenase (ALDH2). Earlier studies have shown that individuals who carry an inactivating point mutation in the gene that codes for ALDH2 (ALDH2*2) are markedly protected against alcoholism (3, 4, 6,7,8). Upon ethanol ingestion, these individuals display high blood acetaldehyde levels, which are known to generate dysphoric effects (9) that lead to an aversion to ethanol.
Similarly, a number of studies (4, 10,11,12,13) have shown that in a gene that codes for human alcohol dehydrogenase (ADH1B*2), a certain point mutation that increases the activity of the enzyme also affords marked protection against alcoholism. Moreover, this mutation also protects against a number of adverse consequences of alcohol abuse, such as upper gastrointestinal tract and oropharyngolaryngeal cancers (14, 15). Although this point mutation (Arg47His) results in an alcohol dehydrogenase that is 100-fold more active than the wild type, subjects carrying this mutation show neither an accumulation of blood acetaldehyde (16,17,18) nor changes in the rate of ethanol elimination (19) on alcohol intake. In one study, a minor increase in ethanol elimination rate, of the order of only 10%, was reported (20). Therefore, the mechanism by which this polymorphism confers protection against alcoholism has remained elusive.
The aim of the present work was to investigate the mechanism by which the 47His ADH polymorphism protects against heavy alcohol intake. This mechanism was studied in rats bred for their high alcohol intake (21, 22), which underwent transduction with the rat analog of the human ADH1B*2 gene.
A characteristic of liver alcohol dehydrogenases is that their turnover is limited by the rate of release of bound NADH from the enzyme after NAD+ and ethanol have generated acetaldehyde and NADH (23, 24). Since ethanol metabolism per se increases the cytoplasmic NADH/NAD+ ratio, as seen by marked increases in the lactate/pyruvate ratio (25, 26), the initial rate of acetaldehyde generation, at the start of ethanol metabolism, is expected to be close to the Vmax of ADH. In contrast, a lower rate of acetaldehyde generation is expected at later times, when a high steady state NADH/NAD+ ratio has been established.
It was recently shown that an increased hepatic ADH activity in castrated male rats results in a marked surge of blood acetaldehyde after ethanol administration (27). This surge is supported by the initially high levels (in blood and liver) of pyruvate, which oxidizes NADH into NAD+ in a reaction catalyzed by lactate dehydrogenase. In the present work, we postulated that increasing the activity of rat liver ADH by in vivo transduction with the rat analog of human ADH1B*2 (rAdh-47His) would result in a short-lived “burst” in arterial acetaldehyde levels and in an aversion to ethanol in rats bred as heavy alcohol consumers. For this purpose, the cDNA of rat ADH that codes for Arg47 (rAdh-47Arg) was mutated to encode His47 (rAdh-47His). This cDNA variant was first incorporated into a suitable plasmid for transfection of ADH-null cells (embryonic human kidney cells), which were then used to characterize the kinetic properties of the resulting enzyme. Subsequently, the rAdh-47His gene was incorporated into an adenoviral vector and delivered to rat hepatoma cells to show its ability to be expressed in rat liver cells. Finally, the adenoviral vector was administered to rats selectively bred as high ethanol drinkers (21, 22) to determine liver ADH protein levels, ADH activity in liver and various tissues, blood acetaldehyde levels following ethanol administration, and voluntary ethanol consumption.
Acetaldehyde is not found in venous blood (i.e., blood that has perfused tissues that contain powerful aldehyde dehydrogenases) following alcohol intake by humans who carry an active aldehyde dehydrogenase (17, 28), and in the rat it does not exceed 2–3 μM (29). Therefore, in the present study acetaldehyde levels were determined in arterial blood rather than in venous blood. In addition, since the generation of acetaldehyde is most pronounced at early times during ethanol metabolism and later decreases (27), blood acetaldehyde levels were measured at different times starting shortly after ethanol administration.
MATERIALS AND METHODS
Gene constructs encoding rat wild-type ADH (47Arg) and mutant ADH (47His)
Cloning of wild-type rat ADH (47Arg) cDNA
The wild-type rat ADH cDNA (30) was cloned and sequenced. Briefly, rat ADH cDNA was obtained by reverse transcription (RT) of total RNA from Lewis rat liver using a (dT)18 primer. The product was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Promega, Madison, WI, USA) and primers TG-267: 5′-AGCGAAGGACAGCATGAGCACAGC-3′ (forward; positions 66–89; GenBank NM_019286.3) and TG-268: 5′-TGTGATGTGGCTGGCGCTTGATTC-3′ (reverse; positions 1276–1253). The product (1211 bp) was cloned in inverted orientation downstream of the CMV promoter in the HincII site of the pAAV-MCS plasmid (Stratagene, Cedar Creek, TX, USA). The cDNA was released by digestion with XbaI and HindIII, and blunt ends were generated with Klenow (New England Biolabs, Ipswich, MA, USA). The resulting fragment (1233 bp), which contained nucleotides 66 to 1276 of the ADH cDNA, preceded by AGCTTCTGCAGGTC and followed by GACTCTAG, was cloned in the HincII site of pAAV-MCS; this plasmid was named pAAV-rADH-47Arg.
Site-directed mutagenesis of rat ADH-47Arg cDNA
To generate the mutant ADH cDNA encoding His at position 47, the G221A mutation, which changes the codon for aa 47 from CGC (Arg) to CAC (His), was incorporated into the cDNA of the wild-type rat ADH (rAdh-47Arg) by site-directed mutagenesis. An overlap extension method was used (31). For this aim, 2 mutagenic primers that carry the mutation (forward TG-370: 5′-AGTCTGCCACTCAGACGATC-3′ and reverse TG-371: 5′-GATCGTCTGAGTGGCAGACT-3′) and 2 primers anchored externally on the pAAV-rADH-47Arg plasmid (forward TG-241: 5′-CAACGTGCTGGTCTGTGTGC-3′ and reverse TG-242: 5′-CTGGAGTGGCAACTTCCAGG-3′) were synthesized. Employing pAAV-rADH-47Arg as template, 2 independent PCR reactions were performed to amplify the 5′ and 3′ regions of the mutant cDNA. Primers TG-371 and TG-241 were used to obtain the 5′ region (269 bp), and primers TG-370 and TG-242 were used to obtain the 3′ region (1177 bp). The mutant cDNA (rAdh-47His) was obtained by PCR amplification in a reaction in which both 5′ and 3′ regions were present, such that pairing and extension in the first cycle allowed amplification of the full-length cDNA in subsequent cycles with primers TG-241 and TG-242. The cDNA (rAdh-47His) was digested with EcoRI and HindIII and cloned downstream of the CMV promoter in pAAV-MCS/EcoRI, HindIII. This plasmid was named pAAV-rADH-47His, and the region obtained by PCR was sequenced to confirm the correct site-directed mutagenesis.
In vitro expression of rADH-47His cDNA and kinetic characterization of rADH-47His
Cell culture conditions
Human embryonic kidney (HEK-293) cells were used to express rADH-47His cDNA and to characterize rADH-47His. HEK-293 cells (ATCC CRL-1573) were obtained from the American Type Cell Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 mg/ml of glucose (Gibco BRL, Grand Island, NY, USA), 1.5 mg/ml of sodium bicarbonate, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.25 μg/ml of amphotericin-B. The culture medium was supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA).
Transfection of HEK-293 cells with plasmid constructs
To study the expression of the gene constructs, HEK-293 cells (which lack ADH activity) were plated on 6-well plates at 1 × 106 cells/well and transfected for 6 h with a mixture of 5 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 2 μg of either pAAV-rADH-47His, pAAV-rADH-47Arg, or the noncoding plasmid pAAV-MCS. Forty-eight hours after transfection, the cells were harvested, lysed in 1% Triton X-100 containing 0.33 mM dithiothreitol (DTT), and centrifuged at 20,800 g for 20 min at 4°C, and the supernatant was collected. Total protein concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA).
Western blot analysis of ADH
Samples (3–15 μg of total protein) were subjected to electrophoresis in denaturing 10% polyacrylamide gels. Proteins were transferred to a Trans-Blot nitrocellulose membrane (Bio-Rad) and subjected to immunoblot analysis with rabbit polyclonal antibodies against ADH (sc-22750) or α-tubulin (sc-5546) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bands were detected by chemoluminescence generated by horseradish peroxidase coupled to a goat anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology) and the ECL Western blotting substrate from Pierce (Rockford, IL, USA).
Assay of alcohol dehydrogenase activity
The activity of ADH was determined spectrophotometrically by the measurement of absorbance (340 nm) of NADH generated from NAD+. The assays were performed in duplicate in a final volume of 0.8 ml at 37°C in 0.5 M Tris-HCl buffer (pH 10.0) containing 0.33 mM DTT, 24 mM semicarbazide, and 5 mM NAD+. After 5 min of stabilization, the reaction was initiated by the addition of 10 mM ethanol. A blank cuvette containing 10 mM pyrazole (ADH inhibitor) instead of ethanol was run, and the results were corrected for the blank reaction. The ADH activity was expressed as nanomoles of NADH per minute per milligram of protein.
Determination of kinetic constants of ADH in cell lysates
To determine the Km of ADH for NAD+ in HEK-293 cells transfected with the plasmids, the ADH activity (initial velocity) was measured in cell lysate supernatants at pH 7.0 using 0.1, 0.5, 1, 2, and 3 mM NAD+. To obtain the Km (NAD+), the reciprocals of the initial activities were plotted against the reciprocals of the substrate concentrations (NAD+) by the method of Lineweaver and Burk (32). To determine the Ki for NADH, the ADH activity (initial velocity in the forward direction) was measured at a fixed NAD+ concentration (0.3 mM) and 0, 25, 50, and 100 μM NADH. To obtain the Ki (NADH), the reciprocals of the initial velocities were plotted against the inhibitor concentrations (NADH) according to Dixon (33).
Production of adenoviral vectors coding for rADH-47His and rADH-47Arg
The adenoviral vectors AdV-rADH-47His, AdV-rADH-47Arg, and the control vector AdV-noncoding were generated according to He et al. (34) with the AdEasy System (American Type Culture Collection). To obtain AdV-rADH-47His and AdV-rADH-47Arg, NotI fragments containing the rADH-47His and the rADH-47Arg expression cassettes were excised from pAAV-rADH-47His and pAAV-rADH-47Arg and cloned in the NotI site of AdEasy pShuttle. To obtain the AdV-noncoding virus, a NotI fragment containing the CMV promoter and the human β-globin intron was excised from pAAV-MCS and cloned in the NotI site of pShuttle. All the pShuttle expression plasmids were linearized with PmeI and used for recombination with pAdEasy-1 in the Escherichia coli BJ5183 rec+ strain to generate the plasmids containing the recombinant adenovirus genomes. The viruses were propagated in HEK-293 cells, purified in 2 consecutive CsCl gradients, and dialyzed for 24 h against 10 mM Tris-HCl, 2 mM MgCl2, and 5% sucrose (storage buffer). Total viral particles (vp) were estimated by absorbance at 260 nm (35). Adenoviral vectors were kept at −80°C in storage buffer.
Expression of rAdh-47His and rAdh-47Arg cDNAs in rat hepatoma cells
Cell culture conditions
Rat hepatoma cells (H4-II-E-C3) were used to study the in vitro expression of the rAdh-47His and rAdh-47Arg cDNAs in hepatic cells. H4-II-E-C3 cells (ATCC CRL-1600) were obtained from the American Type Culture Collection and grown as indicated for HEK-293 cells. The culture medium was supplemented with 10% equine serum and 5% fetal bovine serum (HyClone).
Transduction of H4-II-E-C3 cells with adenoviral vectors
The hepatoma cells were plated on 6-well plates at 2 × 106 cells/well and transduced with each of the adenoviral vectors AdV-rADH-47His, AdV-rADH-47Arg, or AdV-noncoding at a viral dose of 1 × 103 vp/cell. Forty-eight hours after the transduction, the cells were harvested, lysed in 1% Triton X-100 with 0.33 mM DTT, and centrifuged at 20,800 g for 20 min at 4°C, and samples of the supernatant were collected. Total protein concentrations in the samples were determined using the Bio-Rad Protein Assay kit.
Expression of rADH-47His and rADH-47Arg cDNAs in UChB rats
Animals
Rats of the UChB (University of Chile “bibulous”) lineage were used; this line, derived from the Wistar strain, has been bred selectively for high alcohol preference over several decades (21, 22). Fifteen alcohol-naive female UChB rats weighing between 150 and 200 g (~16 wk old) were housed in individual cages in a temperature- and humidity-controlled room under a 12-h light-dark cycle and had free access to water and food. Rats were not controlled for the estrous cycle; in separate cages, the estrous cycle is expected to be randomly distributed. Animal experimentation procedures were approved by the Institutional Animal Experimentation Ethics Board. Each animal received an intravenous injection of one of the adenoviral vectors AdV-rADH-47His, AdV-rADH-47Arg, or AdV-noncoding (5×1012 vp/kg, 5 animals/group) via the tail vein.
Voluntary ethanol intake by animals
Ninety-six hours after the adenoviral vector administration (designated as d 1), rats were allowed access to a 10% ethanol solution and water for only 1 h each day (2–3 PM in the normal light cycle), during which the consumption of both fluids was recorded. The voluntary ethanol intake was determined for 13 d and expressed as grams of ethanol per kilogram per hour. Although a water bottle was also available during the 1 h of access to 10% ethanol, water consumption was too small to be measured accurately during that hour, and therefore water intake was recorded for the total 24 h. On d 21 (25 d after administration of the adenoviral vectors) the rats were given a standard dose of ethanol, and arterial acetaldehyde levels were determined (see below). Thereafter, animals were decapitated, and tissues (brain, heart, kidneys, liver, lungs, and spleen) were removed immediately, weighed, and stored at −80°C for analysis of ADH activity. Serum alanine aminotransferase (ALT) levels were determined with a commercial kit (Valtek, Santiago, Chile).
Arterial acetaldehyde determination
To determine arterial acetaldehyde levels, ethanol was administered intraperitoneally (as a 20% solution in saline) at a dose of 1 g/kg. Blood samples for acetaldehyde measurement were drawn from the carotid artery of the anesthetized rats (ketamine hydrochloride, 60 mg/kg, plus acetopromazine, 2 mg/kg) at 1 min (when possible) and at 2.5, 5, 10, 15, and 30 min after ethanol administration. The blood samples (0.1 ml) were diluted 10-fold in distilled water, and acetaldehyde was measured by gas chromatography of headspace gas (27), a method that does not yield artifactual formation of acetaldehyde (27).
Alcohol dehydrogenase activity in different tissues
Small samples (~0.5 g each) of the various tissues obtained from the decapitated rats (see above) were weighed, immediately cut into small pieces, washed twice with ice-cold phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4), and homogenized in 5 vol of 0.25 M sucrose containing 5 mM Tris, 0.5 mM EDTA, and 0.5 mM DTT; each sample received five 30-s rounds of homogenization at 4°C with an Ultra Turrax T25 homogenizer (Janke & Kinkel, IKA Labortechnik, Staufen, Germany). Cell debris was removed by centrifugation at 20,800 g for 20 min at 4°C, and the supernatant was collected. The ADH activity in the samples (100 μl of supernatant) was determined in duplicate as described previously. ADH activity of each organ was normalized to body weight and expressed as micromoles of NADH per minute per kilogram of body weight. Given the differences in the weight of different organs, expression of this activity per kilogram of body weight reflects more accurately its relative contribution to acetaldehyde generation.
Statistical analyses
Data are expressed as means ± se. Statistical differences were analyzed by Student’s t test or ANOVA for repeated measures for the time factor, with a post hoc test (Student-Newman-Keuls) when required. A level of P < 0.05 was considered statistically significant.
RESULTS
Site-directed mutagenesis and kinetic characteristics of rat ADH-47His and ADH-47Arg
Rat ADH cDNA was subjected to site-directed mutagenesis to encode rADH-47His rather than rADH-47Arg, and both cDNAs were incorporated into plasmids downstream of a CMV promoter. HEK-293 cells, which lack ADH activity, were transfected with these plasmids, and the synthesis of the ADH proteins was confirmed by Western blot using a polyclonal antibody. As seen in Fig. 1, plasmids carrying either the cDNA coding for the wild-type rADH-47Arg or the mutated rADH-47His were expressed (protein levels) to the same degree. Both ADHs were further characterized by determining their Km for NAD+, as it is known that while the Arg47His substitution increases 100-fold the Vmax of the human enzyme, it also markedly reduces the affinity for nicotinamide adenine dinucleotides (36). The Km for NAD+ of rat ADH in supernatants of cells transfected with the plasmid encoding rADH-47His was nearly 10-fold higher (P<0.001) than that for rADH-47Arg (0.93±0.05 and 0.10±0.01 mM, respectively). Consistent with studies on human ADH1B*2 (36), the affinity for NADH of the rat ADH-47His was significantly lower than that of ADH-47Arg, as seen by the increase in the apparent Ki for NADH (90±4.8 vs. 49±1.9 μM; P<0.001).
Alcohol dehydrogenase levels, Km for NAD+, and Ki for NADH in lysates of HEK-293 cells transfected with plasmids coding for the mutated rADH-47His or the wild-type rADH-47Arg enzymes. HEK-293 cells were transfected with 2 μg of one of the plasmids pAAV-rADH-47His, pAAV-rADH-47Arg, or the noncoding pAAV-MCS as transfection control plasmid. The detection of ADH by Western blot and the determination of kinetic constants for the ADH reaction were performed on cells harvested 48 h after transfection. Results are means ± se of 3 experiments performed in duplicate. N.A., not applicable. ***P < 0.001 for Km for NAD+ (t=14.2, df=4) and Ki for NADH (t=7.9, df=4); Student’s t test.
Transduction of rat hepatoma cells with adenoviral vectors encoding ADH-47His and ADH-47Arg
The mutant and the wild-type ADH cDNAs were incorporated into adenoviral vectors (AdV-rADH-47His and AdV-rADH-47Arg). An additional control AdV vector carrying only an intronic sequence instead of an ADH cDNA was also prepared (AdV-noncoding). The cDNA viral constructs were shown to be active by transduction of H4-II-E-C3 rat hepatoma cells. The ability of the viral vectors to generate active ADHs was assessed, and their respective Vmax (under saturating concentrations of NAD+ and ethanol) were determined. Data in Fig. 2 show that the transduction with AdV-rADH-47His, encoding the rat analog of human ADH1B*2, resulted in a 6-fold increase in ADH activity when compared to that in cells transduced with AdV-rADH-47Arg. Figure 2 (inset) also shows that both AdVs, coding for either the wild-type or the mutated ADH, increased the ADH protein by ~2.5-fold, indicating that the increase in enzymatic activity observed was due to the single amino acid difference rather than to different degrees of gene expression.
Alcohol dehydrogenase activity and levels in rat hepatoma cells transduced with adenoviral vectors coding for the mutated rADH-47His or the wild-type rADH-47Arg enzymes. H4-II-E-C3 cells were transduced with 1 × 103 vp/cell of one of the adenoviral vectors AdV-rADH-47His, AdV-rADH-47Arg, or AdV-noncoding as control. Cells were lysed 48 h after transduction to measure the ADH activity and determine enzyme levels by Western blot. Bars represent means ± se of 5 experiments performed in duplicate. ***P < 0.001 for AdV-rADH-47His vs. AdV-noncoding (t=26.6, df=8) and AdV-rADH-47Arg vs. AdV-noncoding (t=15.2, df=8); Student’s t test.
Administration of adenoviral vectors encoding ADH-47His and ADH-47Arg to rats: determination of ADH activity, arterial acetaldehyde, and ethanol intake
Rats of the UChB high alcohol drinker line were administered one of the 3 AdV vectors (i.e., AdV-rADH-47His, AdV-rADH-47Arg, or AdV-noncoding), and voluntary ethanol consumption was measured in a limited access paradigm in which 10% ethanol and water were offered to the animals for only 1 h each day. Figure 3 indicates that animals that received the AdV-rADH-47His vector showed a significantly lower voluntary ethanol intake (50% reduction; P<0.001) than animals that received the AdV-noncoding vector. Administration of AdV-rADH-47Arg also produced a reduction in ethanol intake, although it was less marked than the above (−30%; P<0.01) and faded quickly. The in vivo inhibitory effect on ethanol consumption of animals that received either vector was found to be consistent with the ADH activity measured in liver samples. Figure 4 shows that liver ADH activity in animals transduced with AdV-rADH-47His was 90% (P<0.01) higher than that in animals that received the AdV-noncoding control, while in animals that received AdV-rADH-47Arg, the activity was 32% (P<0.01) higher than that of controls. The administration of the adenoviral vectors did not increase ADH activity in other tissues; this finding is consistent with the preferential liver tropism reported for adenoviral vectors administered systemically (37). To rule out possible hepatotoxic effects of the adenoviral vectors, serum ALT levels were measured 3 wk after adenoviral vector administration. No increases in serum ALT levels were found in the animals treated with adenovirus (data not shown). The daily water consumption in animals receiving adenoviral vectors carrying ADH cDNAs was not different from that of animals that received the control adenoviral vector (data not shown).
Voluntary ethanol intake of UChB rats treated with adenoviral vectors coding for the mutated rADH-47His or the wild-type rADH-47Arg enzymes. Fifteen rats from the high-drinker UChB line were injected with 5 × 1012 vp/kg of one of the adenoviral vectors AdV-rADH-47His, AdV-rADH-47Arg, or AdV-noncoding as control. Ninety-six hours after administration of the adenoviral vector (d 1), the animals were allowed access to a 10% ethanol solution for only 1 h each day. Water was continuously available. Points represent means ± se of daily ethanol intake during the 1 h access to ethanol; 5 animals/group. Rats treated with AdV-rADH-47His (left panel) showed a 50% reduction in voluntary alcohol intake vs. control animals [ANOVA; F(1,25)=71.3, P<0.001], whereas in rats treated with AdV-rADH-47Arg (right panel), the reduction was 30% [F(1,25)=8.7, P<0.01].
Alcohol dehydrogenase activity in different tissues of rats treated with adenoviral vectors coding for the mutated rADH-47His or the wild type rADH-47Arg enzymes. Three weeks after administration of the adenoviral vectors, the animals were killed, and ADH activity was determined in brain, heart, kidney, liver, lung, and spleen. Bars represent mean ± se ADH activity in tissues (μmol NADH/min/kg body wt); 5 animals/group. Rats treated with AdV-rADH-47His showed an 88% increase in liver ADH activity vs. control animals; rats treated with AdV-rADH-47Arg showed an increase of 32%. Inset: liver ADH levels of rats treated with AdV-rADH-47His or AdV-rADH-47Arg were similar, and both were higher than levels in rats treated with the AdV-noncoding control vector. **P < 0.01 for AdV-rADH-47His vs. AdV-noncoding (t=3.4, df=8) and AdV-rADH-47Arg vs. AdV-noncoding (t=3.3, df=8); Student’s t test.
The present study in animals allowed the determination of arterial acetaldehyde levels immediately following the administration of ethanol. Figure 5 shows that a marked surge (burst) in the arterial acetaldehyde level occurs virtually immediately on the administration of ethanol in animals that received either AdV-rADH-47His or AdV-rADH-47Arg; the maximal levels were 5- and 3.5-fold higher (P<0.001), respectively, than those of control animals that received AdV-noncoding.
Arterial blood acetaldehyde concentrations after administration of ethanol to UChB rats treated with adenoviral vectors coding for the mutated rADH-47His or the wild-type rADH-47Arg enzymes. Three weeks after administration of the adenoviral vectors, animals were injected with ethanol intraperitoneally at a dose of 1 g/kg, and acetaldehyde levels were measured in arterial blood (carotid artery) by gas chromatography. Points represent mean ± se blood acetaldehyde concentration; 5 animals/group. At 2.5 min after ethanol administration, maximal concentrations of acetaldehyde in animals that received AdV-rADH-47His and AdV-rADH-47Arg were 5-fold (t=7.2, df=8) and 3.5-fold (t=8.2, df=8) higher than those of control animals that received AdV-noncoding, respectively. ***P < 0.001. ANOVA for repeated measures revealed significant differences between treatments [F(2,14)=11.6, P<0.01]. Post hoc comparisons revealed significant differences (P<0.05) between AdV-rADH-47His and AdV-noncoding and also between AdV-rADH-47Arg and AdV-noncoding. No statistically significant differences were found between AdV-rADH-47His and AdV-rADH-47Arg.
DISCUSSION
As indicated above, early studies showed that marked elevations in blood acetaldehyde levels following ethanol intake in humans who carry an inactive aldehyde dehydrogenase (ALDH2*2) lead to marked protection (66 to 99%) against alcohol abuse and alcoholism (3, 4, 6,7,8). Subsequent studies showed that a polymorphism in the gene that codes for a highly active human alcohol dehydrogenase (Arg47His; ADH1B*2) also leads to marked protection against alcoholism (4, 5, 10,11,12,13). The protection against alcoholism afforded by ADH1B*2 is of similar magnitude to that afforded by ALDH2*2 (18, 38).
It is noteworthy that in humans, although the Arg47His polymorphism results in a variant enzyme (ADH1B*2) that is 100-fold more active than ADH1B*1 (36), an accumulation of acetaldehyde following ethanol intake has not been reported in individuals who carry the ADH1B*2 allele, even if they are homozygous for this allele (16, 17). The aim of the present study was to investigate, by a gene delivery method, the protective mechanism of the 47His-ADH1B polymorphism in an animal model. Several rat strains/lines have been bred for their high voluntary consumption of ethanol (39,40,41), which would have been valuable for the present work. One of these, the UChB rat line, derived from the Wistar strain, was used in the present study (21, 22).
To mimic the human condition, the cDNA of wild-type rat ADH coding for rADH-47Arg was mutated to encode rADH-47His. Such cDNA was incorporated into a gene construct that was administered to UChB rats in an adenoviral vector. In the human ADH1B*1, Arg47 is part of the active site for NAD+, and a change to His47 (ADH1B*2) markedly reduces the affinity of the enzyme for nicotinamide adenine dinucleotides (36). An effect of this type was observed in rat ADH when Arg47 was replaced by His; the Km for NAD+ was shown to increase nearly 10-fold (from 100 to 910 μM). Since in mammalian cells the concentration of cytosolic NAD+ is in the range of 350–400 μM (42, 43), the initial in vivo velocity of rADH-47His, according to the Michaelis-Menten equation, is expected to be 90% greater than that of rADH-47Arg. This would occur for rADH-47His considering both its higher Km for NAD+ and 6-fold higher Vmax. In the human ADH1B, the Arg47His mutation also lowers the affinity for NADH, the product generated at the same active site (36). Since the release of NADH from the enzyme constitutes the rate-limiting step of ADHs (23, 24) the Vmax is markedly increased in the 47His isoform. It is noted that rat ADH-47His displayed an NADH affinity that was about half that of the wild-type ADH-47Arg, as shown by a significant increase in the apparent Ki for NADH, an effect that could further contribute to an increased initial velocity of rADH-47His in vivo, since NADH levels are increased on ethanol oxidation.
Given both the increase in the Km for NAD+ and the reduction in the Ki for NADH of rADH-47His vs. rADH-47Arg, the burst of acetaldehyde observed in animals injected with AdV-ADH-47His may not only stem from an increased initial velocity but also from the existence of relatively high NAD+ and low NADH levels at the time that ethanol starts being oxidized. These levels are reversed on ethanol oxidation, reaching a new redox steady state shown as 100–400% increases in the lactate/pyruvate ratio during ethanol metabolism (25, 26), which reflects relative increases in NADH vs. NAD+. It has been postulated (27) that before achieving the new redox steady state, the initially high systemic pyruvate concentration available to the liver via the circulation allows a time-limited reoxidation of NADH by the action of lactate dehydrogenase, with regeneration of NAD+. This reaction allows ADH to generate acetaldehyde at its near-maximal capacity only for a limited time; thus, the acetaldehyde surge will occur before the final lactate/pyruvate ratio is established.
In animals transduced with AdV-rADH-47His, ethanol metabolism led to a time-limited surge in arterial acetaldehyde that reached maximal levels 5-fold higher than those observed in animals that received the noncoding viral vector. Such an increase is likely to be the basis of the 50% reduction (P<0.001) in voluntary ethanol consumption observed in rats that received AdV-rADH-47His relative to that in rats that received the control viral vector. This reduction is virtually identical to the overall protection against alcoholism observed in humans who carry the ADH1B*2 allele (see meta-analysis in ref. 5). Hence, the mechanism by which ADH1B*2 confers protection against alcoholism in humans is likely to be, as suggested in this animal study, a marked initial surge in arterial acetaldehyde.
Since acetaldehyde is metabolized by an aldehyde dehydrogenase, ALDH2, that also requires NAD+, it is reasonable to ask why, in AdV-rADH-47His-treated animals, more acetaldehyde is generated at early times than is removed. This most likely occurs because the rate-limiting step of ALDH2, unlike that of ADH, is not the release of NADH from the enzyme, but rather the release of the acyl group (acetate) generated in this enzymatic reaction (44). The fact that a rapid NADH reoxidation allows acetaldehyde generation to exceed its removal was demonstrated by studies in which the administration of sodium pyruvate together with ethanol generated an acetaldehyde burst that led to a marked aversion to ethanol in rats (27). In AdV-rADH-47His-treated animals, the burst levels of acetaldehyde (80–90 μM) are in the range of those that markedly reduce alcohol intake both in animals treated with disulfiram (45), an inhibitor of aldehyde dehydrogenase, and in humans who carry a dominant negative mutation in ALDH2 (16, 17).
An additional point to be made is that in humans, ADH activity is not a main rate-limiting factor in the elimination of ethanol (19, 20). In conducting this study, it was necessary to reduce the possibility that the animals would have an ADH activity low enough to become a major rate-limiting factor. The liver of female rats has twice the ADH protein and ADH activity of the male liver (46, 47); therefore, female rats were used in the present study.
The marked liver tropism of adenoviruses deserves a comment. While adenoviral vectors (70 nm) have the potential of infecting all types of mammalian cells in vitro, their size is too large to allow these vectors to cross the pores of the capillaries of most tissues (~20 nm or less) when delivered intravenously. However, the pores (fenestra) of liver sinusoids that carry blood to hepatocytes are 3–5 times larger than the adenoviral diameter (35, 37). It is noted that when primates or rodents receive therapeutic genes by the single intravenous administration of “gutless” (helper-dependent) adenoviral vectors, gene expression is detected in the liver for 2 yr (48, 49). Attaining a time-limited burst of acetaldehyde by increasing ADH activity in the liver, as seen in the present study, may be therapeutically advantageous over the use of drugs such as disulfiram, which on ethanol ingestion elevate acetaldehyde levels by inhibiting ALDHs in all tissues. In alcoholics who continue abusing alcohol, disulfiram exposes all tissues to sustained high levels of acetaldehyde, including those in the upper respiratory tract and the esophagus, which are susceptible to acetaldehyde toxicity (14, 15). Alcoholics who carry one ALDH2*2 allele, who are deficient in this enzyme activity in all tissues, and who are exposed to sustained high levels of acetaldehyde show a 5- to 20-fold greater risk of upper respiratory tract and esophageal cancer than alcoholics who are homozygous for the ALDH2*1 allele (14, 15).
Paradoxically, heavy drinkers and alcoholics who are homozygous for the ADH1B*2 allele display a 60–90% protection against the development of upper respiratory tract and esophageal cancer vs. heavy drinkers and alcoholics who are homozygous for the ADH1B*1 allele (14, 15). It is well known that some toxins or stressors are beneficial at low doses while damaging at higher doses, an effect known as hormesis (50). Although not all mechanisms of hormesis are understood, conditions such as short physiological challenges or low doses of ionizing radiation are known to elicit general protective and reparative mechanisms, including an enhanced DNA repair (see refs. 51,52,53,54). On the basis of the present study, it may be suggested that in ADH1B*2 drinkers, protection against cancers might result from an activation of repair mechanisms induced by only a limited exposure to acetaldehyde. In Drosophila, exposure to low concentrations of acetaldehyde increases longevity at lethal doses, providing a clear example of hormesis (55). Also, extended longevity has been reported in mice that consume moderate, but not high, amounts of alcohol throughout their adult life (56).
Overall, the present study in animals transduced with the rat equivalent of human ADH1B*2 shows that high levels of blood acetaldehyde occur only during the first few minutes of ethanol metabolism, which could be responsible for the reduction in voluntary ethanol intake seen in these animals. These observations may constitute the basis of the protection against alcoholism seen in humans who carry the ADH1B*2 allele. The mechanism may have eluded clinical investigators heretofore since blood acetaldehyde levels were not measured shortly after ethanol intake, and acetaldehyde levels were not determined in arterial blood, but rather in venous blood. In the present animal study, hepatic gene delivery and the design used were helpful in overcoming the limitations inherent to human studies.
Acknowledgments
This work was supported by grants from the U.S. National Institutes of Health (R01-AA 015421) and the Millennium Scientific Initiative (ICM P05-001). M.R. held a doctoral fellowship from CONICYT-Chile. We thank Dr. Fernando Ezquer for pAAV-rADH-47Arg direction reversal, Juan Santibáñez for skillful technical assistance, and Prof. Harold Kalant for his critical review of the manuscript.
References
- Rehm J, Taylor B, Room R. Global burden of disease from alcohol, illicit drugs and tobacco. Drug Alcohol Rev. 2006;25:503–513. [Abstract] [Google Scholar]
- World Health Organization Geneva, Switzerland: Second report of WHO Expert Committee, WHO Technical Report Series, no. 944; Problems related to alcohol consumption. 2007 [Abstract] [Google Scholar]
- Thomasson H R, Edenberg H J, Crabb D W, Mai X L, Jerome R E, Li T K, Wang S P, Lin Y T, Lu R B, Yin S J. Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men. Am J Hum Genet. 1991;48:677–681. [Europe PMC free article] [Abstract] [Google Scholar]
- Chen C C, Lu R B, Chen Y C, Wang M F, Chang Y C, Li T K, Yin S J. Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism. Am J Hum Genet. 1999;65:795–807. [Europe PMC free article] [Abstract] [Google Scholar]
- Zintzaras E, Stefanidis I, Santos M, Vidal F. Do alcohol-metabolizing enzyme gene polymorphisms increase the risk of alcoholism and alcoholic liver disease? Hepatology. 2006;43:352–361. [Abstract] [Google Scholar]
- Harada S, Agarwal D P, Goedde H W, Tagaki S, Ishikawa B. Possible protective role against alcoholism for aldehyde dehydrogenase isozyme deficiency in Japan. Lancet. 1982;2:827. [Abstract] [Google Scholar]
- Higuchi S. Polymorphisms of ethanol metabolizing enzyme genes and alcoholism. Alcohol Alcohol Suppl. 1994;2:29–34. [Abstract] [Google Scholar]
- Tu G C, Israel Y. Alcohol consumption by Orientals in North America is predicted largely by a single gene. Behav Genet. 1995;25:59–65. [Abstract] [Google Scholar]
- Mizoi Y, Tatsuno Y, Adachi J, Kogame M, Fukunaga T, Fujiwara S, Hishida S, Ijiri I. Alcohol sensitivity related to polymorphism of alcohol-metabolizing enzymes in Japanese. Pharmacol Biochem Behav. 1983;18:127–133. [Abstract] [Google Scholar]
- Borràs E, Coutelle C, Rosell A, Fernández-Muixi F, Broch M, Crosas B, Hjelmqvist L, Lorenzo A, Gutiérrez C, Santos M, Szczepanek M, Heilig M, Quattrocchi P, Farrés J, Vidal F, Richart C, Mach T, Bogdal J, Jörnvall H, Seitz H, Couzigou P, Parés X. Genetic polymorphism of alcohol dehydrogenase in Europeans: the ADH2*2 allele decreases the risk for alcoholism and is associated with ADH3*1. Hepatology. 2000;31:984–989. [Abstract] [Google Scholar]
- Chambers G K, Marshall S J, Robinson G M, Maguire S, Newton-Howes J, Chong N. The genetics of alcoholism in Polynesians: alcohol and aldehyde dehydrogenase genotypes in young men. Alcohol Clin Exp Res. 2002;26:949–955. [Abstract] [Google Scholar]
- Neumark Y D, Friedlander Y, Thomasson H R, Li T K. Association of the ADH2*2 allele with reduced ethanol consumption in Jewish men in Israel: a pilot study. J Stud Alcohol. 1998;59:133–139. [Abstract] [Google Scholar]
- Wall T L, Shea S H, Luczak S E, Cook T A, Carr L G. Genetic associations of alcohol dehydrogenase with alcohol use disorders and endophenotypes in white college students. J Abnorm Psychol. 2005;114:456–465. [Abstract] [Google Scholar]
- Yokoyama A, Muramatsu T, Omori T, Yokoyama T, Matsushita S, Higuchi S, Maruyama K, Ishii H. Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics. Carcinogenesis. 2001;22:433–439. [Abstract] [Google Scholar]
- Chen Y J, Chen C, Wu D C, Lee C H, Wu C I, Lee J M, Goan Y G, Huang S P, Lin C C, Li T C, Chou Y P, Wu M T. Interactive effects of lifetime alcohol consumption and alcohol and aldehyde dehydrogenase polymorphisms on esophageal cancer risks. Int J Cancer. 2006;119:2827–2831. [Abstract] [Google Scholar]
- Mizoi Y, Yamamoto K, Ueno Y, Fukunaga T, Harada S. Involvement of genetic polymorphism of alcohol and aldehyde dehydrogenases in individual variation of alcohol metabolism. Alcohol Alcohol. 1994;29:707–710. [Abstract] [Google Scholar]
- Peng G S, Chen Y C, Tsao T P, Wang M F, Yin S J. Pharmacokinetic and pharmacodynamic basis for partial protection against alcoholism in Asians, heterozygous for the variant ALDH2*2 gene allele. Pharmacogenet Genomics. 2007;17:845–855. [Abstract] [Google Scholar]
- Chen Y C, Peng G S, Wang M F, Tsao T P, Yin S J. Polymorphism of ethanol-metabolism genes and alcoholism: correlation of allelic variations with the pharmacokinetic and pharmacodynamic consequences. Chem Biol Interact. 2009;178:2–7. [Abstract] [Google Scholar]
- Duranceaux N C, Schuckit M A, Eng M Y, Robinson S K, Carr L G, Wall T L. Associations of variations in alcohol dehydrogenase genes with the level of response to alcohol in non-Asians. Alcohol Clin Exp Res. 2006;30:1470–1478. [Abstract] [Google Scholar]
- Neumark Y D, Friedlander Y, Durst R, Leitersdorf E, Jaffe D, Ramchandani V A, O'Connor S, Carr L G, Li T K. Alcohol dehydrogenase polymorphisms influence alcohol-elimination rates in a male Jewish population. Alcohol Clin Exp Res. 2004;28:10–14. [Abstract] [Google Scholar]
- Mardones J, Segovia-Riquelme N. Thirty-two years of selection of rats by ethanol preference: UChA and UChB strains. Neurobehav Toxicol Teratol. 1983;5:171–178. [Abstract] [Google Scholar]
- Quintanilla M E, Israel Y, Sapag A, Tampier L. The UChA and UChB rat lines: metabolic and genetic differences influencing ethanol intake. Addict Biol. 2006;11:310–323. [Abstract] [Google Scholar]
- Cronholm T. Hydrogen transfer between ethanol molecules during oxidoreduction in vivo. Biochem J. 1985;229:315–322. [Europe PMC free article] [Abstract] [Google Scholar]
- Stone C L, Bosron W F, Dunn M F. Amino acid substitutions at position 47 of human β1β1 and β2β2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants. J Biol Chem. 1993;268:892–899. [Abstract] [Google Scholar]
- Wahid S, Khanna J M, Carmichael F J, Lindros K O, Rachamin G, Israel Y. Alcohol-induced redox changes in the liver of the spontaneously hypertensive rat: effect of chronic ethanol treatment. Biochem Pharmacol. 1981;30:1277–1282. [Abstract] [Google Scholar]
- Lecky F E, Little R A, Maycock P F, Rainey T, Barton R N, Yates D W, Knottenbelt J D, Evans A. Effect of alcohol on the lactate/pyruvate ratio of recently injured adults. Crit Care Med. 2002;30:981–985. [Abstract] [Google Scholar]
- Quintanilla M E, Tampier L, Sapag A, Gerdtzen Z, Israel Y. Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective. Am J Physiol Endocrinol Metab. 2007;293:531–537. [Abstract] [Google Scholar]
- Nuutinen H U, Salaspuro M P, Valle M, Lindros K O. Blood acetaldehyde concentration gradient between hepatic and antecubital venous blood in ethanol-intoxicated alcoholics and controls. Eur J Clin Invest. 1984;14:306–311. [Abstract] [Google Scholar]
- Garver E, Tu G C, Cao Q N, Aini M, Zhou F, Israel Y. Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol. J Exp Med. 2001;5:571–580. [Europe PMC free article] [Abstract] [Google Scholar]
- Crabb D W, Edenberg H J. Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence. Gene. 1986;48:287–291. [Abstract] [Google Scholar]
- Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77:51–59. [Abstract] [Google Scholar]
- Lineweaver H, Burk D. The determination of enzyme dissociation constants. J Am Chem Soc. 1934;56:658–666. [Google Scholar]
- Dixon M. The determination of enzyme inhibitor constants. Biochem J. 1953;55:170–171. [Europe PMC free article] [Abstract] [Google Scholar]
- He T C, Zhou S, Da Costa L T, Yu J, Kinzler K W, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci. 1998;95:2509–2514. [Europe PMC free article] [Abstract] [Google Scholar]
- Mittereder N, March K L, Trapnell B C. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol. 1996;70:7498–7509. [Europe PMC free article] [Abstract] [Google Scholar]
- Hurley T D, Edenberg H J, Bosron W F. Expression and kinetic characterization of variants of human β1β1 alcohol dehydrogenase containing substitutions at amino acid 47. J Biol Chem. 1990;265:16366–16372. [Abstract] [Google Scholar]
- Fechner H, Haack A, Wang H, Wang X, Eizema K, Pauschinger M, Schoemaker R G, Van Veghel R, Houtsmuller A B, Schultheiss H P, Lamers J M, Poller W. Expression of Coxsackie adenovirus receptor and alphav-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers. Gene Ther. 1999;6:1520–1535. [Abstract] [Google Scholar]
- Kim D J, Choi I G, Park B L, Lee B C, Ham B J, Yoon S, Bae J S, Cheong H S, Shin H D. Major genetic components underlying alcoholism in Korean population. Hum Mol Genet. 2008;6:854–858. [Abstract] [Google Scholar]
- Hilakivi L, Eriksson C J, Sarviharju M, Sinclair J D. Revitalization of the AA and ANA rat lines: effects on some line characteristics. Alcohol. 1984;1:71–75. [Abstract] [Google Scholar]
- Lê A D, Israel Y, Juzytsch W, Quan B, Harding S. Genetic selection for high and low alcohol consumption in a limited-access paradigm. Alcohol Clin Exp Res. 2001;25:1613–1620. [Abstract] [Google Scholar]
- Li T K, McBride W J. Pharmacogenetic models of alcoholism. Clin Neurosci. 1995;3:182–188. [Abstract] [Google Scholar]
- Yamada K, Hara N, Shibata T, Osago H, Tsuchiya M. The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry. Anal Biochem. 2006;352:282–285. [Abstract] [Google Scholar]
- Yang H, Yang T, Baur J, Pérez E, Matsui T, Carmona J J, Lamming D W, Souza-Pinto N C, Bohr V A, Rosenzweig A, De Cabo R, Sauve A A, Sinclair D A. Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell. 2007;130:1095–1107. [Europe PMC free article] [Abstract] [Google Scholar]
- Ni L, Sheikh S, Weiner H. Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase. J Biol Chem. 1997;272:18823–18826. [Abstract] [Google Scholar]
- Tampier L, Quintanilla M E, Israel Y. Tolerance to disulfiram by chronic alcohol intake in the rat. Alcohol Clin Exp Res. 2008;32:937–941. [Abstract] [Google Scholar]
- Rachamin G, Macdonald J A, Wahid S, Clapp J J, Khanna J M, Israel Y. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem J. 1980;186:483–490. [Europe PMC free article] [Abstract] [Google Scholar]
- Simon F R, Fortune J, Iwahashi M, Sutherland E. Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis. Am J Physiol Gastrointest Liver Physiol. 2002;283:646–655. [Abstract] [Google Scholar]
- Morral N, O'Neal W, Rice K, Leland M, Kaplan J, Piedra P A, Zhou H, Parks R J, Velji R, Aguilar-Córdova E, Wadsworth S, Graham F L, Kochanek S, Dee Carey K, Beaudet A L. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc Natl Acad Sci. 1999;96:12816–12821. [Europe PMC free article] [Abstract] [Google Scholar]
- Toietta G, Mane V P, Norona W S, Finegold M J, Ng P, McDonagh A F, Beaudet A L, Lee B. Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector. Proc Natl Acad Sci. 2005;102:3930–3935. [Europe PMC free article] [Abstract] [Google Scholar]
- Calabrese E J, Baldwin L A. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol. 2003;43:175–197. [Abstract] [Google Scholar]
- Schwartz D R, Sack M N. Targeting the mitochondria to augment myocardial protection. Curr Opin Pharmacol. 2008;8:160–165. [Europe PMC free article] [Abstract] [Google Scholar]
- Li N, Wu H, Yang S, Chen D. Ischemic preconditioning induces XRCC1, DNA polymerase-beta, and ligase III and correlates with enhanced base excision repair. DNA Repair. 2007;6:1297–1306. [Abstract] [Google Scholar]
- Kang M J, Jung S M, Kim M J, Bae J H, Kim H B, Kim J Y, Park S J, Song H S, Kim D W, Kang C D, Kim S H. DNA-dependent protein kinase is involved in heat shock protein-mediated accumulation of hypoxia-inducible factor-1alpha in hypoxic preconditioned HepG2 cells. FEBS J. 2008;275:5969–5981. [Abstract] [Google Scholar]
- Feinendegen L E. Evidence for beneficial low level radiation effects and radiation hormesis. Br J Radiol. 2005;78:3–7. [Abstract] [Google Scholar]
- Parsons P A. Acetaldehyde utilization in Drosophila: an example of hormesis. Biol J Linn Soc. 1989;37:183–189. [Google Scholar]
- Schmidt W, Popham R E, Israel Y. Dose-specific effects of alcohol on the lifespan of mice and the possible relevance to man. Br J Addict. 1987;82:775–788. [Abstract] [Google Scholar]
Articles from The FASEB Journal are provided here courtesy of The Federation of American Societies for Experimental Biology
Full text links
Read article at publisher's site: https://doi.org/10.1096/fj.09-132563
Read article for free, from open access legal sources, via Unpaywall:
https://europepmc.org/articles/pmc2797030
Free to read at www.fasebj.org
http://www.fasebj.org/cgi/content/abstract/24/1/266
Free after 12 months at www.fasebj.org
http://www.fasebj.org/cgi/content/full/24/1/266
Citations & impact
Impact metrics
Citations of article over time
Smart citations by scite.ai
Explore citation contexts and check if this article has been
supported or disputed.
https://scite.ai/reports/10.1096/fj.09-132563
Article citations
Aldehyde dehydrogenase 2 polymorphism is associated with chemotherapy-related cognitive impairment in patients with breast cancer who receive chemotherapy.
Cancer Med, 12(5):5209-5221, 06 Oct 2022
Cited by: 1 article | PMID: 36200595 | PMCID: PMC10028021
Effects of Gene Polymorphisms, Metabolic Activity, and Content of Alcohol Dehydrogenase and Acetaldehyde Dehydrogenases on Prognosis of Hepatocellular Carcinoma Patients.
Turk J Gastroenterol, 33(7):606-614, 01 Jul 2022
Cited by: 2 articles | PMID: 35879918 | PMCID: PMC9404678
FACTORS CONTRIBUTING TO THE ESCALATION OF ALCOHOL CONSUMPTION.
Neurosci Biobehav Rev, 132:730-756, 25 Nov 2021
Cited by: 6 articles | PMID: 34839930 | PMCID: PMC8892842
Review Free full text in Europe PMC
The C-terminal extension of Mycobacterium tuberculosis Hsp16.3 regulates its oligomerization, subunit exchange dynamics and chaperone function.
FEBS J, 284(2):277-300, 12 Jan 2017
Cited by: 9 articles | PMID: 27885799
Rat animal models for screening medications to treat alcohol use disorders.
Neuropharmacology, 122:201-243, 16 Feb 2017
Cited by: 38 articles | PMID: 28215999 | PMCID: PMC5659204
Review Free full text in Europe PMC
Go to all (29) article citations
Data
Data behind the article
This data has been text mined from the article, or deposited into data resources.
BioStudies: supplemental material and supporting data
RefSeq - NCBI Reference Sequence Database
- (1 citation) RefSeq - NM_019286.3
Similar Articles
To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
Reduction of ethanol consumption in alcohol-preferring rats by dual expression gene transfer.
Alcohol Alcohol, 47(2):102-108, 02 Jan 2012
Cited by: 10 articles | PMID: 22214999 | PMCID: PMC3284687
Acetaldehyde burst protection of ADH1B*2 against alcoholism: an additional hormesis protection against esophageal cancers following alcohol consumption?
Alcohol Clin Exp Res, 35(5):806-810, 01 Feb 2011
Cited by: 2 articles | PMID: 21284671 | PMCID: PMC3142558
Review Free full text in Europe PMC
Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective.
Am J Physiol Endocrinol Metab, 293(2):E531-7, 08 May 2007
Cited by: 33 articles | PMID: 17488809
Effect of the allelic variants of aldehyde dehydrogenase ALDH2*2 and alcohol dehydrogenase ADH1B*2 on blood acetaldehyde concentrations.
Hum Genomics, 3(2):121-127, 01 Jan 2009
Cited by: 75 articles | PMID: 19164089 | PMCID: PMC3525274
Review
Free full text in Europe PMCEffect of the allelic variant of alcohol dehydrogenase ADH1B*2 on ethanol metabolism.
Alcohol Clin Exp Res, 38(6):1502-1509, 05 May 2014
Cited by: 9 articles | PMID: 24797321
Funding
Funders who supported this work.
NIAAA NIH HHS (2)
Grant ID: R01-AA 015421
Grant ID: R01 AA015421