Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of the amino acid L-tryptophan (Trp) combined with the kynureninase inhibitor benserazide (BSZ) to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo and to induce aversion to alcohol. Methods: Trp, BSZ or both were administered to male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring acetaldehyde accumulation in blood after ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: Combined administration of Trp + BSZ, but neither compound alone, produced a strong inhibition of ALDH activity and an increase in blood acetaldehyde concentration after ethanol, and induced aversion to alcohol in rats and decreased preference in mice. Another kynureninase inhibitor, carbidopa, induced aversion to alcohol by itself, which was reversed by Trp co-administration. Conclusions: The present results establish a prior art for the use of a combination of Trp plus BSZ in the treatment of alcoholism by aversion, which merits rapid clinical development.
A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2–2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2–2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (−61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2–2 phenotype.
alcoholism; disulfiram; ALDH2-2; acetaldehyde; treatment
Ethanol is metabolized into acetaldehyde mainly by the action of alcohol dehydrogenase in the liver, while mainly by the action of catalase in the brain. Aldehyde dehydrogenase-2 metabolizes acetaldehyde into acetate in both organs. Gene specific modifications reviewed here show that an increased liver generation of acetaldehyde (by transduction of a gene coding for a high-activity liver alcohol dehydrogenase ADH1*B2) leads to increased blood acetaldehyde levels and aversion to ethanol in animals. Similarly aversive is an increased acetaldehyde level resulting from the inhibition of liver aldehyde dehydrogenase-2 (ALDH2) synthesis (by an antisense coding gene against aldh2 mRNA). The situation is diametrically different when acetaldehyde is generated in the brain. When the brain ventral tegmental area (VTA) is endowed with an increased ability to generate acetaldehyde (by transfection of liver rADH) the reinforcing effects of ethanol are increased, while a highly specific inhibition of catalase synthesis (by transduction of a shRNA anti catalase mRNA) virtually abolishes the reinforcing effects of ethanol as seen by a complete abolition of ethanol intake in rats bred for generations as high ethanol drinkers. Data shows two divergent effects of increases in acetaldehyde generation: aversive in the periphery but reinforcing in the brain.
alcohol dehydrogenase; catalase; aldehyde dehydrogenase; reinforcement; aversion
Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.
Aldehyde dehydrogenases; post-translational modifications; cellular defense; drug toxicity; disease states; translational research
Aldehyde dehydrogenase (ALDH) isozymes are critically important in the metabolism of acetaldehyde, thus preventing its accumulation after ethanol exposure. We previously reported that mitochondrial ALDH2 could be inactivated via -nitrosylation in ethanol-exposed rats. This study was aimed at investigating whether cytosolic ALDH1, with a relatively low- value (11–18 μM) for acetaldehyde, could be also inhibited in ethanol-exposed rats. Chronic or binge ethanol exposure significantly decreased ALDH1 activity, which was restored by addition of dithiothreitol. Immunoblot analysis with the anti--nitroso-Cys antibody showed one immunoreactive band in the immunoprecipiated ALDH1 only from ethanol-exposed rats, but not from pair-fed controls, suggesting -nitrosylation of ALDH1. Therefore inactivation of ALDH1 via -nitrosylation can result in accumulation of acetaldehyde upon ethanol exposure.
Cytosolic aldehyde dehydrogenase (ALDH1); Ethanol; S-nitrosylation; reversible inhibition; acetaldehyde metabolism
Alcohol and MDMA (ecstasy) are frequently co-abused, but recent findings indicate a harmful drug interaction between these two agents. In our previous study, we showed that MDMA exposure inhibits the activity of the acetaldehyde (ACH) metabolizing enzyme, aldehyde dehydrogenase2 (ALDH2). Based on this finding, we hypothesized that the co-administration of MDMA and ethanol would reduce the metabolism of ACH and result in increased accumulation of ACH. Rats were treated with MDMA or vehicle and then administered a single dose of ethanol. Liver ALDH2 activity decreased by 35% in the MDMA-treated rats compared to control rats. The peak concentration and the area under the concentration versus time curve of plasma ACH was 31% and 59% higher, respectively, in the MDMA-ethanol group compared to the ethanol-only group. In addition, the MDMA-ethanol group had 80% higher plasma transaminase levels than the ethanol-only group, indicating greater hepatocellular damage. Our results not only support a drug interaction between MDMA and ethanol but a novel underlying mechanism for the interaction.
alcohol; ethanol; 3,4-methylenedioxymethamphetamine (MDMA); ecstasy; metabolism; acetaldehyde; aldehyde dehydrogenase; drug interaction; liver toxicity; transaminase
The anti-alcoholism medication disulfiram (Antabuse) inhibits aldehyde dehydrogenase (ALDH), which results in the accumulation of acetaldehyde upon ethanol ingestion and produces the aversive “Antabuse reaction” that deters alcohol consumption. Disulfiram has also been shown to deter cocaine use, even in the absence of an interaction with alcohol, indicating the existence of an ALDH-independent therapeutic mechanism. We hypothesized that disulfiram’s inhibition of dopamine β-hydroxylase (DBH), the catecholamine biosynthetic enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons, underlies the drug’s ability to treat cocaine dependence. We tested the effects of disulfiram on cocaine and food self-administration behavior and drug-primed reinstatement of cocaine seeking in rats. We then compared the effects of disulfiram with those of the selective DBH inhibitor, nepicastat. Disulfiram, at a dose (100 mg/kg, i.p.) that reduced brain NE by ~40%, did not alter responding for food or cocaine on a fixed ratio 1 (FR1) schedule, whereas it completely blocked cocaine-primed (10 mg/kg, i.p.) reinstatement of drug seeking following extinction. A lower dose of disulfiram (10 mg/kg) that did not reduce NE had no effect on cocaine-primed reinstatement. Nepicastat recapitulated the behavioral effects of disulfiram (100 mg/kg) at a dose (50 mg/kg, i.p.) that produced a similar reduction in brain NE. Food-primed reinstatement of food seeking was not impaired by DBH inhibition. Our results suggest that disulfiram’s efficacy in the treatment of cocaine addiction is associated with the inhibition of DBH and interference with the ability of environmental stimuli to trigger relapse.
dopamine β-hydroxylase; disulfiram; nepicastat; norepinephrine; cocaine; reinstatement
Alcohol consumption leads to myocardial contractile dysfunction possibly due to the toxicity of ethanol and its major metabolite acetaldehyde. This study was designed to examine the influence of mitochondrial aldehyde dehydrogenase-2 (ALDH2) knockout (KO) on acute ethanol exposure-induced cardiomyocyte dysfunction. Wild-type (WT) and ALDH2 KO mice were subjected to acute ethanol (3 g/kg, i.p.) challenge and cardiomyocyte contractile function was assessed 24 hrs later using an IonOptix® edge-detection system. Western blot analysis was performed to evaluate ALDH2, protein phosphatase 2A (PP2A), phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β). ALDH2 KO accentuated ethanol-induced elevation in cardiac acetaldehyde levels. Ethanol exposure depressed cardiomyocyte contractile function including decreased cell shortening amplitude and maximal velocity of shortening/relengthening as well as prolonged relengthening duration and a greater decline in peak shortening in response to increasing stimulus frequency, the effect of which was significantly exaggerated by ALDH2 KO. ALDH2 KO also unmasked an ethanol-induced prolongation of shortening duration. In addition, short-term in vitro incubation of ethanol-induced cardiomyocyte mechanical defects were exacerbated by the ALDH inhibitor cyanamide. Ethanol treatment dampened phosphorylation of Akt and GSK-3β associated with up-regulated PP2A, which was accentuated by ALDH2 KO. ALDH2 KO aggravated ethanol-induced decrease in mitochondrial membrane potential. These results suggested that ALDH2 deficiency led to worsened ethanol-induced cardiomyocyte function, possibly due to upregulated expression of protein phosphatase, depressed Akt activation and subsequently impaired mitochondrial function. These findings depict a critical role of ALDH2 in the pathogenesis of alcoholic cardiomyopathy.
Ethanol; ALDH2; Cardiomyocyte; Contractile function; Akt; Protein phosphatase
The antialcoholism medication disulfiram (Antabuse) inhibits aldehyde dehydrogenase (ALDH), which results in the accumulation of acetaldehyde upon ethanol ingestion and produces the aversive ‘Antabuse reaction' that deters alcohol consumption. Disulfiram has also been shown to deter cocaine use, even in the absence of an interaction with alcohol, indicating the existence of an ALDH-independent therapeutic mechanism. We hypothesized that disulfiram's inhibition of dopamine β-hydroxylase (DBH), the catecholamine biosynthetic enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons, underlies the drug's ability to treat cocaine dependence. We tested the effects of disulfiram on cocaine and food self-administration behavior and drug-primed reinstatement of cocaine seeking in rats. We then compared the effects of disulfiram with those of the selective DBH inhibitor, nepicastat. Disulfiram, at a dose (100 mg/kg, i.p.) that reduced brain NE by ∼40%, did not alter the response for food or cocaine on a fixed ratio 1 schedule, whereas it completely blocked cocaine-primed (10 mg/kg, i.p.) reinstatement of drug seeking following extinction. A lower dose of disulfiram (10 mg/kg) that did not reduce NE had no effect on cocaine-primed reinstatement. Nepicastat recapitulated the behavioral effects of disulfiram (100 mg/kg) at a dose (50 mg/kg, i.p.) that produced a similar reduction in brain NE. Food-primed reinstatement of food seeking was not impaired by DBH inhibition. Our results suggest that disulfiram's efficacy in the treatment of cocaine addiction is associated with the inhibition of DBH and interference with the ability of environmental stimuli to trigger relapse.
dopamine β-hydroxylase; disulfiram; nepicastat; norepinephrine; cocaine; reinstatement; addiction & substance abuse; catecholamines; psychostimulants; animal models; norepinephrine; cocaine; self-administration; rat
It is well established that inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However not all of the anti-alcohol properties of diadzin are due to inhibition of ALDH-2. This is in agreement with our earlier work showing significant interaction effects of both pyrozole (ALDH-2 inhibitor) and methyl-pyrozole (non-inhibitor) and ethanol’s depressant effects. Moreover, it has been suggested that selective ALDH 2 inhibitors reduce craving for alcohol by increasing dopamine in the nucleus accumbens (NAc). In addition there is significant evidence related to the role of the genetics of bitter receptors (TAS2R) and its stimulation as an aversive mechanism against alcohol intake. The inclusion of bitters such as Gentian & Tangerine Peel in Declinol provides stimulation of gut TAS2R receptors which is potentially synergistic with the effects of Kudzu. Finally the addition of Radix Bupleuri in the Declinol formula may have some protective benefits not only in terms of ethanol induced liver toxicity but neurochemical actions involving endorphins, dopamine and epinephrine. With this information as a rationale, we report herein that this combination significantly reduced Alcohol Use Disorders Identification Test (AUDIT) scores administered to ten heavy drinkers (M=8, F=2; 43.2 ± 14.6 years) attending a recovery program. Specifically, from the pre-post comparison of the AUD scores, it was found that the score of every participant decreased after the intervention which ranged from 1 to 31. The decrease in the scores was found to be statistically significant with the p-value of 0.00298 (two-sided paired test; p-value = 0.00149 for one-sided test). Albeit this being a small pilot, we are encouraged about these significant results, and caution any interpretation until larger controlled studies are executed.
Declinol; Kudzu; Daidzin; ALDH 2 inhibitors; Dopamine; Gentian and tangerine peel; Radix burpleuri; Alcoholism and reward deficiency
Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.
Aldehyde dehydrogenase; Glutathione; Nitric oxide; NO donors; S-Nitrosylation; ALDH2, mitochondrial aldehyde dehydrogenase; BSO, l-buthionine-sulphoximine; DTT, dithiothreitol; GSH-EE, glutathioneethylester; GSNO, S-nitrosoglutathione; HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; NO, nitric oxide; S-NO-Cys, S-nitroso-cysteine; ROS/RNS, reactive oxygen/nitrogen species; SIN-1, 3-morpholinosydnonimine · HCl; SNAP, S-nitroso-N-acetyl-d, l-penicillamine
In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant, acrolein. ALDH2 also bioactivates nitroglycerin, but it is best known for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian Alcohol-induced Flushing Syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semi-dominant and heterozygotic individuals exhibit a similar, but not as severe phenotype. We recently identified a small molecule, Alda-1, which activates wild-type ALDH2 and restores near wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.
Many Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH2) activity responsible for the oxidation of acetaldehyde produced during ethanol metabolism. These individuals suffer the alcohol-flush reaction when they drink alcoholic beverages. The alcohol-flush reaction is the result of excessive acetaldehyde accumulation, and the unpleasant symptoms tend to reduce alcohol consumption. The subunit of this homotetrameric enzyme was sequenced and the abnormality in the inactive enzyme shown to be a substitution of lysine for glutamate at position 487. We have used the polymerase chain reaction to determine the genotypes of 24 livers from Japanese individuals. Correlating genotype with phenotype leads to the conclusion that the allele (ALDH2(2)) encoding the abnormal subunit is dominant.
Chronic alcohol consumption increases the risk for cancer of the organs and tissues of the respiratory tract and the upper digestive tract (i.e., upper aerodigestive tract), liver, colon, rectum, and breast. Various factors may contribute to the development (i.e., pathogenesis) of alcohol-associated cancer, including the actions of acetaldehyde, the first and most toxic metabolite of alcohol metabolism. The main enzymes involved in alcohol and acetaldehyde metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are encoded by multiple genes. Because some of these genes exist in several variants (i.e., are polymorphic), and the enzymes encoded by certain variants may result in elevated acetaldehyde levels, the presence of these variants may predispose to certain cancers. Several mechanisms may contribute to alcohol-related cancer development. Acetaldehyde itself is a cancer-causing substance in experimental animals and reacts with DNA to form cancer-promoting compounds. In addition, highly reactive, oxygen-containing molecules that are generated during certain pathways of alcohol metabolism can damage the DNA, thus also inducing tumor development. Together with other factors related to chronic alcohol consumption, these metabolism-related factors may increase tumor risk in chronic heavy drinkers.
Alcohol and other drug (AOD) consumption; heavy drinking; chronic AOD use; ethanol metabolism; carcinogenesis; cancer; upper respiratory system cancer; oropharyngeal cancer; laryngeal cancer; aerodigestive tract cancer; esophageal cancer; liver cancer; colon cancer; colorectal cancer; breast cancer; acetaldehyde; alcohol dehydrogenase (ADH); aldehyde dehydrogenase (ALDH); genetic factors; cytochrome P450 2E1 (CYP2E1); reactive oxygen species
Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at ≥5 mm. In the presence of 1 mm NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.
Cyclic GMP (cGMP); Enzyme Catalysis; Nitric Oxide; Oxidase; Superoxide Dismutase (SOD); Superoxide Ion; Bioactivation; Nitroglycerin
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal (4-HNE) and the bioactivation of nitroglycerin (GTN) to nitric oxide (NO). In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our lab recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon (PKCε) and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator (Alda-1) which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2, and discuss potential clinical implications of these findings.
Alcoholism has been associated with folate deficiency in humans and laboratory animals. Previous study showed that ethanol feeding reduces the dehydrogenase and hydrolase activity of 10-formyltetrahydrofolate dehydrogenase (FDH) in rat liver. Hepatic ethanol metabolism generates acetaldehyde and acetate. The mechanisms by which ethanol and its metabolites produce toxicity within the liver cells are unknown. We purified FDH from rat liver and investigated the effect of ethanol, acetaldehyde and acetate on the enzyme in vitro. Hepatic FDH activity was not reduced by ethanol or acetate directly. However, acetaldehyde was observed to reduce the dehydrogenase activity of FDH in a dose- and time-dependent manner with an apparent IC50 of 4 mM, while the hydrolase activity of FDH was not affected by acetaldehyde in vitro. These results suggest that the inhibition of hepatic FDH dehydrogenase activity induced by acetadehyde may play a role in ethanol toxicity.
Ethanol toxicity; acetaldehyde; 10-formyltetrahydrofolate dehydrogenase/hydrolase; folate
Acetaldehyde associated with alcoholic beverages was recently classified as carcinogenic (Group 1) to humans based on uniform epidemiological and biochemical evidence. ALDH2 (aldehyde dehydrogenase 2) deficient alcohol consumers are exposed to high concentrations of salivary acetaldehyde and have an increased risk of upper digestive tract cancer. However, this interaction is not seen among ALDH2 deficient non-drinkers or rare drinkers, regardless of their smoking status or consumption of edibles containing ethanol or acetaldehyde. Therefore, the aim of this study was to examine the effect of the ALDH2 genotype on the exposure to locally formed acetaldehyde via the saliva without ethanol ingestion.
The ALDH2 genotypes of 17 subjects were determined by PCR-RFLP. The subjects rinsed out their mouths with 5 ml of 40 vol% alcohol for 5 seconds. Salivary ethanol and acetaldehyde levels were measured by gas chromatography.
Acetaldehyde reached mutagenic levels rapidly and the exposure continued for up to 20 minutes. The mean salivary acetaldehyde concentrations did not differ between ALDH2 genotypes.
For ALDH2 deficient subjects, an elevated exposure to endogenously formed acetaldehyde requires the presence of ethanol in the systemic circulation.
Our findings provide a logical explanation for how there is an increased incidence of upper digestive tract cancers among ALDH2 deficient alcohol drinkers, but not among those ALDH2 deficient subjects who are locally exposed to acetaldehyde without bloodborne ethanol being delivered to the saliva. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands.
Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke.
ALDH2; 4-HNE; stroke; ethanol
Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 Å crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid-body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant likely reflects the energetic penalty for reestablishing this site for productive coenzyme binding, while the structural alterations near the active site are consistent with the lowered Vmax.
In order to determine the specificity of abnormalities of alcohol metabolism in patients with alcoholic liver disease, blood acetaldehyde concentrations after oral ethanol challenge and the activities of alcohol metabolising enzymes in liver biopsy samples have been determined in patients with alcoholic liver disease and a wide variety of non-alcoholic liver disorders. Significant decreases in hepatic cytosolic aldehyde dehydrogenase activity were associated with significant increases in acetaldehyde concentrations after ethanol in both patient groups compared with control subjects. There was a significant correlation between hepatic cytosolic aldehyde dehydrogenase and mean blood acetaldehyde concentration 30-180 min after ethanol ingestion (y = 17.4-0.45x; r = -0.56; p less than 0.01) confirming the importance of this enzyme in controlling blood acetaldehyde concentrations. These findings suggest that disturbances in alcohol metabolism in patients with alcoholic liver disease are the consequence of liver damage rather than a specific abnormality predisposing to alcohol induced liver injury.
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.
Molinate is a thiocarbamate herbicide used as a pre-emergent in rice patty fields. It has two predominant sulfoxidation metabolites, molinate sulfoxide and molinate sulfone. Previous work demonstrated an in vivo decrease in liver aldehyde dehydrogenase (ALDH) activity in rats treated with molinate and motor function deficits in dogs dosed chronically with this compound. ALDH is an enzyme important in the catabolism of many neurotransmitters, such as dopamine. Inhibition of this enzyme may lead to the accumulation of endogenous neurotoxic metabolites such as 3,4-dihydroxyphenylacetaldehyde (DOPAL), a dopamine metabolite, which may account for the observed neurotoxicity. In this study, the relative reactivity of molinate and both of its sulfoxidation metabolites towards ALDH were investigated, as well as the mechanism of inhibition. ALDH activity was monitored in two different model systems, human recombinant ALDH (hALDH2) and mouse striatal synaptosomes. Molinate sulfone was found to be the most potent ALDH inhibitor, compared to molinate and molinate sulfoxide. The reactivity of these three compounds was also assessed, using N-acetyl Cys, model peptides, and hALDH2. It was determined that molinate sulfone is capable of covalently modifying Cys residues, including catalytic Cys302 of ALDH, accounting for the observed enzyme inhibition.
Ethanol is primarily metabolized in the liver by 2 rate-limiting reactions: conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) and subsequent conversion of acetaldehyde to acetate by aldehyde dehydrogenase (ALDH). ADH and ALDH exist in multiple isozymes that differ in their kinetic properties. Notably, polymorphisms within the genes that encode for these isozymes vary in their allele frequencies between ethnic groups, and thus, they have been considered as candidate genes that may differentially influence risk for the development of alcohol dependence across ethnic groups.
Objectives and Methods
Associations between alcohol dependence and polymorphisms in ADH1B, ADH1C, and ALDH2, were compared in a community sample of Native Americans living on reservations (n=791) and Mexican Americans (n=391) living within the same county.
Two Mexican Americans and no Native Americans possessed one ALDH2*2 allele. Presence of at least one ADH1B*2 allele was found in 7% of the Native Americans and 13% of the Mexican Americans, but was only associated with protection against alcohol dependence in the Mexican Americans. Presence of at least one ADH1B*3 allele was found in 4% if the Native Americans and 2% of the Mexican Americans, but was associated with protection against alcohol dependence only in the Native Americans. No associations between alcohol dependence and polymorphisms in ADH1C were found.
Conclusions and Scientific Significance
Polymorphisms in ADH1B are protective against alcoholism in these two populations; however, these findings do not explain the high prevalence of alcoholism in these populations.
ADH; ALDH; Alcohol dependence; candidate genes; Mexican Americans; Native Americans; population genetics