Aldehyde dehydrogenase inhibitors: a comprehensive review of the pharmacology, mechanism of action, substrate specificity, and clinical application.

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of enzymes that play a key role in the metabolism of aldehydes of both endogenous and exogenous derivation. The human ALDH superfamily comprises 19 isozymes that possess important physiological and toxicological functions. The ALDH1A subfamily plays a pivotal role in embryogenesis and development by mediating retinoic acid signaling. ALDH2, as a key enzyme that oxidizes acetaldehyde, is crucial for alcohol metabolism. ALDH1A1 and ALDH3A1 are lens and corneal crystallins, which are essential elements of the cellular defense mechanism against ultraviolet radiation-induced damage in ocular tissues. Many ALDH isozymes are important in oxidizing reactive aldehydes derived from lipid peroxidation and thereby help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes have been reported in various human cancers and are associated with cancer relapse. As a direct consequence of their significant physiological and toxicological roles, inhibitors of the ALDH enzymes have been developed to treat human diseases. This review summarizes known ALDH inhibitors, their mechanisms of action, isozyme selectivity, potency, and clinical uses. The purpose of this review is to 1) establish the current status of pharmacological inhibition of the ALDHs, 2) provide a rationale for the continued development of ALDH isozyme-selective inhibitors, and 3) identify the challenges and potential therapeutic rewards associated with the creation of such agents.

The role of CYP2E1 in alcohol metabolism and sensitivity in the central nervous system.

Ethanol consumption has effects on the central nervous system (CNS), manifesting as motor incoordination, sleep induction (hypnosis), anxiety, amnesia, and the reinforcement or aversion of alcohol consumption. Acetaldehyde (the direct metabolite of ethanol oxidation) contributes to many aspects of the behavioral effects of ethanol. Given acetaldehyde cannot pass through the blood brain barrier, its concentration in the CNS is primarily determined by local production from ethanol. Catalase and cytochrome P450 2E1 (CYP2E1) represent the major enzymes in the CNS that catalyze ethanol oxidation. CYP2E1 is expressed abundantly within the microsomes of certain brain cells and is localized to particular brain regions. This chapter focuses on the discussion of CYP2E1 in ethanol metabolism in the CNS, covering topics including how it is regulated, where it is expressed and how it influences sensitivity to ethanol in the brain.

Ethanol as a prodrug: brain metabolism of ethanol mediates its reinforcing effects--a commentary.

BACKGROUND: This commentary discusses a study by Karahanian and colleagues (2011) on the role of central nervous system acetaldehyde in the reinforcing effects of ethanol. The goal is to emphasize the importance of the study and to discuss future directions. RESULTS: This important paper solidifies the idea that the levels of acetaldehyde in the central nervous system have profound effects in mediating the reinforcing actions of ethanol. This is accomplished by manipulating the brain levels of acetaldehyde produced from ethanol by the injection of lentivirus containing either an anti-catalase shRNA construct or a rat liver alcohol dehydrogenase into the central nervous system and observing the effects on alcohol preference by high ethanol-consuming rats. A factor not directly considered is that acetaldehyde is further metabolized to acetate, which also has some behavioral actions. CONCLUSIONS: The efficacy of lentivirus injections of enzyme inhibitors or enzymes themselves to alter a behavioral response to ethanol is clearly demonstrated here. The many other actions of ethanol that are postulated to be a result of the production of acetaldehyde in the brain remain to be investigated by similar techniques. Possible "therapeutic avenues to reduce chronic alcohol use" are envisioned.

Acetate-dependent mechanisms of inborn tolerance to ethanol.

AIMS: To clarify the role of acetate in neurochemical mechanisms of the initial (inborn) tolerance to ethanol. METHODS: Rats with low and high inborn tolerance to hypnotic effect of ethanol were used. In the brain region homogenates (frontal and parietal cortex, hypothalamus, striatum, medulla oblongata) and brain cortex synaptosomes, the levels of acetate, acetyl-CoA, acetylcholine (AcH), the activity of pyruvate dehydrogenase (PDG) and acetyl-CoA synthetase were examined. RESULTS: It has been found that brain cortex of rats with high tolerance to hypnotic effect of ethanol have higher level of acetate and activity of acetyl-CoA synthetase, but lower level of acetyl-СCoA and activity of PDG. In brain cortex synaptosomes of tolerant rats, the pyruvate oxidation rate as well as the content of acetyl-CoA and AcH synthesis were lower when compared with intolerant animals. The addition of acetate into the medium significantly increased the AcH synthesis in synaptosomes of tolerant, but not of intolerant animals. Calcium ions stimulated the AcH release from synaptosomes twice as high in tolerant as in intolerant animals. Acetate eliminated the stimulating effect of calcium ions upon the release of AcH in synaptosomes of intolerant rats, but not in tolerant animals. As a result, the quantum release of AcH from synaptosomes in the presence of acetate was 6.5 times higher in tolerant when compared with intolerant rats. CONCLUSION: The brain cortex of rats with high inborn tolerance to hypnotic effect of ethanol can better utilize acetate for the acetyl-CoA and AcH synthesis, as well as being resistant to inhibitory effect of acetate to calcium-stimulated release of AcH. It indicates the metabolic and cholinergic mechanisms of the initial tolerance to ethanol.

Quantitative trait loci for sensitivity to acute ethanol and ethanol consummatory behaviors in rats.

Individuals with a low initial response to alcohol (i.e., ethanol) are at greater risk of developing alcohol abuse or dependence later in life. Similar to humans, individual differences in ethanol sensitivity also can be seen in rats, and several laboratories have used these individual differences to generate selectively bred rats that differ in acute ethanol sensitivity. We have worked with two sets of such rats (Inbred High or Low Alcohol Sensitivity strains, IHAS or ILAS, respectively; Inbred Alcohol Tolerant or Non-Tolerant strains, IAT and IANT, respectively) and have confirmed previously mapped quantitative trait loci (QTL) for these acute differences with the use of recombinant congenic lines; however, the relationship between acute sensitivity and ethanol drinking in these rats has yet to be determined. Thus, here we tested the hypothesis that QTLs underlying variation in initial low sensitivity to ethanol also will modulate variation in ethanol drinking behaviors. Separate groups of selectively inbred parent and congenic rats were tested for the loss of righting response (LORR) and also assessed for ethanol consummatory behavior using either operant self-administration or an intermittent-access two-bottle choice procedure. LORR testing confirmed the presence of a LORR duration QTL in all of the congenics; however, the lack of a corresponding difference in blood ethanol concentration at the regaining of the righting response suggests that these QTLs may be mediating a difference in ethanol metabolism rather than in neuronal sensitivity. IHAS/ILAS-derived congenic rats did not differ from parent rats at any point during operant self-administration. IAT/IANT-derived congenic rats showed small, but significant, increases in ethanol consumption relative to the parent strains only during the initial stages of operant self-administration. In contrast to operant testing, IHAS/ILAS-derived congenic rats showed significantly greater ethanol consumption and preference than parent rats during intermittent-access testing. There were not differences, however, between IAT/IANT congenic and parent rats during intermittent access. These data support the hypothesis that there is a genetic relationship between initial ethanol sensitivity and ethanol consumption, at least for the IHAS/ILAS-derived congenic rats. Our current studies, however, cannot eliminate pharmacokinetic or taste preference factors as contributing to the rats' responses, nor can we eliminate the possibility of a linkage effect because of the fairly large size of the QTL intervals; i.e., distinct genes may be mediating the acute sensitivity and drinking responses.

Removal of acetaldehyde from the body.

The reduction of acetaldehyde back to ethanol via NAD-linked alcohol dehydrogenase is an important mechanism for keeping acetaldehyde levels low following ethanol ingestion. However, this does not remove acetaldehyde from the body, but just delays its eventual removal. Acetaldehyde is removed from the body primarily by oxidation to acetate via a number of NAD-linked aldehyde dehydrogenase (ALDH) enzymes. There are nineteen known ALDHs in humans, but only a few of them appear to be involved in acetaldehyde oxidation. There are many analogous enzymes in other organisms. Genetic polymorphisms of several ALDHs have been identified in humans that are responsible for several hereditary defects in the metabolism of normal endogenous substrates. The best known ALDH genetic polymorphism is in ALDH2 gene, which encodes a mitochondrial enzyme primarily responsible for the oxidation of the ethanol-derived acetaldehyde. This common polymorphism is known to dominantly inhibit its enzymatic activity resulting in reduced ability to clear acetaldehyde in both homozygote and heterozygote individuals. These individuals are generally protected against alcohol abuse but are susceptible to oesophageal cancer. For those enzymes that are capable of reacting with acetaldehyde, they may do so at the expense of their normal substrates, resulting in abnormal accumulation of these substrates. Examples of this are the aldehydes of the biogenic amines, dopamine, noradrenaline, adrenaline, serotonin and long chain lipid aldehydes such as nonenal. Not all of these enzymes are capable of efficient oxidation of acetaldehyde; however, it is possible that acetaldehyde may function as an inhibitor of these enzymes as well. The aldehydes whose metabolism is interfered with may also serve as inhibitors of ALDHs as well. However, this aspect of aldehyde function has not been extensively studied. A number of other mechanisms for the removal of acetaldehyde exist. For example, reaction of acetaldehyde with protein or nucleic acids is responsible for the disappearance of a small amount of acetaldehyde, but may be responsible for some pathological effects of acetaldehyde. There are a few other enzymes such as aldehyde oxidase, xanthine oxidase, cytochrome P450 oxidase and glyceraldehyde-3-phosphate dehydrogenase that are capable of oxidizing acetaldehyde. However, these enzymes account for only a small fraction of the total activity.

Short-term selection for acute ethanol tolerance and sensitization from an F2 population derived from the high and low alcohol-sensitive selectively bred rat lines.

Previous studies have identified quantitative trait loci (QTL) in the inbred high and low alcohol-sensitive rat (IHAS1 and ILAS1) strains. The original development of the strains involved selection for ethanol sensitivity based on duration of the loss of the righting reflex (LORR) after a standard dose of ethanol. This paper confirms some of these QTL using a short-term selection procedure based on the difference between the blood ethanol level at LORR and regain of the righting response. An F(2) population of rats was developed by a reciprocal cross of IHAS1 and ILAS1 rats. Selection for five generations was carried out using delta-blood ethanol concentration (dBEC) as the selection trait, where dBEC=BECLR (BEC at loss of righting reflex)-BECRR (BEC at regain of righting reflex). The lines were labeled tolerant (TOL) or sensitive (SENS). Approximately one-third of the offspring for each generation in each line were genotyped using DNA markers that had been previously found to be linked to QTL on chromosomes 1, 2, 5, 12, and 13. By the fifth generation of selection, the lines showed a very large difference in dBEC, BECRR, and duration of LORR; BECLR showed little segregation during the selection, and latency to lose the righting reflex showed none. IHAS allele frequency increased in the SENS line for markers on chromosomes 1, 5, 12, and 13 while ILAS allele frequency increased in the TOL line. These results were in good agreement with the two previous QTL studies. On chromosome 2, the selection resulted in an accumulation of ILAS alleles in both lines. This study provides independent confirmation of the location of QTL on chromosomes 1, 5, 12, and 13 for ethanol sensitivity. It also suggests that genetic differences in duration of LORR are mediated primarily by the dBEC phenotype.

Modulation of GABAA receptors in cerebellar granule neurons by ethanol: a review of genetic and electrophysiological studies.

Cerebellar granule neurons (CGNs) receive inhibitory input from Golgi cells in the form of phasic and tonic currents that are mediated by postsynaptic and extrasynaptic gamma-aminobutyric acid type A (GABAA) receptors, respectively. Extrasynaptic receptors are thought to contain alpha6betaxdelta subunits. Here, we review studies on ethanol (EtOH) modulation of these receptors, which have yielded contradictory results. Although studies with recombinant receptors expressed in Xenopus oocytes indicate that alpha6beta3delta receptors are potently enhanced by acute exposure to low (>or=3 mM) EtOH concentrations, this effect was not observed when these receptors were expressed in Chinese hamster ovary cells. Slice recordings of CGNs have consistently shown that EtOH increases the frequency of phasic spontaneous inhibitory postsynaptic currents (sIPSCs), as well as the tonic current amplitude and noise. However, there is a lack of consensus as to whether EtOH directly acts on extrasynaptic receptors or modulates them indirectly; that is, via an increase in spillover of synaptically released GABA. It was recently demonstrated that an R to Q mutation of amino acid 100 of the alpha6 subunit increases the effect of EtOH on both sIPSCs and tonic current. These electrophysiological findings have not been reproducible in our hands. Moreover, it was shown the alpha6-R100Q mutation enhances sensitivity to the motor-impairing effects of EtOH in outbred Sprague-Dawley rats, but this was not observed in a line of rats selectively bred for high sensitivity to EtOH-induced motor alterations (Alcohol Non-Tolerant rats). We conclude that currently there is insufficient evidence conclusively supporting a direct potentiation of extrasynaptic GABAA receptors following acute EtOH exposure in CGNs.

Confirmation of quantitative trait loci for ethanol sensitivity and neurotensin receptor density in crosses derived from the inbred high and low alcohol sensitive selectively bred rat lines.

RATIONALE: Genetically influenced alcohol sensitivity is thought to be an important risk factor for the development of alcoholism. An effective first step for identifying genes that mediate variation in alcohol sensitivity is through quantitative trait loci (QTL) mapping in model organisms. OBJECTIVE: Fourteen provisional QTLs related to alcohol sensitivity were previously mapped in an F2 derived from the IHAS1 and ILAS1 rat lines. The objective of the current study was to confirm those QTLs in an independently derived F2 and in congenics that were bred for two of the loci. MATERIALS AND METHODS: IHAS1 X ILAS1 F2 (n=450) were tested for alcohol-induced loss of righting reflex (LORR), blood ethanol concentration at regain of righting reflex (BECRR), sensitivity and acute tolerance on the Rotarod, and neurotensin receptor density (NTR1). Rats were genotyped at the 14 candidate loci and QTL mapping was conducted. Reciprocal congenic strains were bred for loci on chromosomes 2 and 5 and tested for LORR and BECRR. RESULTS: Four LORR QTLs were mapped at the suggestive or significant level (chromosomes 2, 5, 12, and 13). BECRR was mapped to chromosomes 5, 12, and 13 either in the original or current experiment. Results of the congenic experiment also support QTLs for LORR and BECRR on chromosomes 2 and 5. QTLs for NTR1 density and behavior on the Rotarod were not confirmed. CONCLUSIONS: QTL mapping in crosses derived from the IHAS1 and ILAS1 has successfully identified loci related to alcohol sensitivity. Recombinant congenics are now being bred to more finely map the confirmed QTLs.

Cyclosporine-A discourages ethanol intake in C57bl/6j mice: a preliminary study.

OBJECTIVE: Remarkably high rates of abstinence occur among alcohol-dependent persons after a liver transplant. This was thought to be solely due to selection or other nonbiological factors. Observing sustained abstinence for many months to years after a liver transplant, however, we asked whether immunosuppressants might exert a biological effect that supported abstinence from ethanol. METHOD: We hypothesized that cyclosporine-A-treated mice would drink less ethanol given a choice between water and 10% ethyl alcohol. After a 3-day ethanol conditioning period, C57bl/6j mice (n = 20) medicated with cyclosporine-A (50 mg/kg) and a control group of unmedicated mice (n = 20) injected with saline were given free access to either water or the ethanol solution. RESULTS: The medicated mice drank significantly less ethanol throughout the 11-day choice protocol. This was significant whether measured by mean alcohol consumption per day (p = .003) or by alcohol preference as a percent of total liquid intake per day (p = .03). The two groups did not differ significantly in total daily fluid consumption. CONCLUSION: These data suggest that cyclosporine-A lowers alcohol preference in mice. Mechanism of action, similar effects by other centrally acting immunosuppressants and translation to humans all remain to be studied. If fruitful, elucidating the actions of cyclosporine and other immunosuppressants that activate central nervous system receptors may illuminate the pathophysiology of alcohol use and addiction.

Formation of ethyl nitrite in vivo after ethanol administration.

The purpose of the current study was to ascertain whether ethyl nitrite could be detected in vitro from the reaction of ethanol with peroxynitrite, as well as after administration of ethanol to mice. Ethyl nitrite analyte was determined by using gas chromatography--mass spectrometry with headspace analysis with the use of a solid-phase microextraction device. Peroxynitrite was allowed to react with ethanol under a variety of conditions in vitro. Ethyl nitrite was generated when peroxynitrite was allowed to react with ethanol. Male, inbred short-sleep mice were injected intraperitoneally with either ethanol [5.2 g/kg; 15.0% (weight/volume) ethanol in saline] or a 50:50 mixture of deuterium-labeled ethanol (D5-ethanol) and ethanol. Blood samples, as well as whole brain and liver sections, were obtained from mice 30 min later for determination of ethanol, D5-ethanol, ethyl nitrite, and deuterium-labeled ethyl nitrite (D5-ethyl nitrite). Time courses for the appearance of ethyl nitrite in blood samples, as well as in whole brain and liver sections, obtained from mice were carried out. After ethanol administration, ethyl nitrite was detected and quantitated in mouse blood, brain, and liver. A small fraction of ethyl nitrite was present. When a 50:50 mixture of ethanol and D5-ethanol was given to animals, both ethyl nitrite and D5-ethyl nitrite were found in blood and brain in approximately the same ratio as that of ethanol and D5-ethanol. The level of D5-ethyl nitrite in liver was more than twice that of ethyl nitrite, indicating a possible isotope effect in the metabolism of ethyl nitrite. Ethyl nitrite is a new metabolite of ethanol in vivo. The mechanism of ethyl nitrite formation is most likely the reaction of ethanol with peroxynitrite generated in vivo from nitric oxide.

Acetaldehyde: déjà vu du jour.

OBJECTIVE: The possibility that acetaldehyde is responsible for some of the central nervous system effects of ethanol has been a popular hypothesis for many years. This review examines the evidence of a role for acetaldehyde in the actions of ethanol in the brain. METHOD: The literature review was confined primarily to effects of acetaldehyde in the central nervous system in the realization that a great deal of information is also available on the actions of acetaldehyde in the periphery. The emphasis is on more recent findings, with only occasional references to older work. RESULTS: There are studies implicating acetaldehyde in nearly every central nervous system effect of ethanol that has been studied. With a few exceptions, the evidence for most of these effects is conflicting. For many years the dogma was that the brain did not metabolize ethanol. Any effects of acetaldehyde were therefore held to be due to acetaldehyde diffusing in from the blood. Recently, however, it has been established that ethanol is metabolized to acetaldehyde (primarily by catalase) and then to acetate (by aldehyde dehydrogenase) in the brain. These findings remove the problem that acetaldehyde does not penetrate the brain very well but leave questions as to what it does there. Almost invariably, the concentrations of acetaldehyde in the brain, under normal conditions of ethanol intoxication, are in the low micromolar range. Inhibition of aldehyde dehydrogenase will lead to increases of both peripheral and central acetaldehyde and usually to increases in the effects of ethanol or to behaviorally aversive effects. Stimulation of catalase should lead to increased levels of acetaldehyde in the brain, but this has not been directly demonstrated. Inhibition of catalase should lead to decreased acetaldehyde concentrations in vivo, but, again, this has not been directly demonstrated. Various effects of the direct application of acetaldehyde to the brain have been noted, but in most studies the concentration of acetaldehyde resulting from such manipulations has not been determined, and it is probably higher than that occurring during ethanol intoxication. These experiments tell us what acetaldehyde is capable of doing, not what it does after administration of ethanol. Still, this is a first step. CONCLUSIONS: Acetaldehyde is a product of ethanol metabolism in the brain. It clearly has central nervous system effects in its own right. The jury is still out as to whether it has effects under normal conditions of ethanol intoxication. This will remain the case until direct measurement of acetaldehyde concentrations in the brain is routinely accomplished under conditions in which behavioral effects of ethanol are also measured.

Putative role of brain acetaldehyde in ethanol addiction.

The putative contribution of brain acetaldehyde (AcH) to ethanol (EtOH) tolerance and dependence (addiction) is reviewed. Although the role of AcH in EtOH addiction has been controversial, there are data showing a relationship. AcH can be formed in the brain tissues through the peroxidatic activity of catalase and by oxidation via other oxidizing enzymes such as cytochrome P-4502E1. Significant formation of AcH occurs in vitro in brain tissue at concentrations of EtOH that can be achieved by voluntary consumption of EtOH by rodents. AcH itself possesses reinforcing properties, which suggests that some of the behavioral pharmacological effects attributed to EtOH may be a result of the formation of AcH, and supports the involvement of AcH in EtOH addiction. Modulation of aldehyde dehydrogenase (ALDH) and brain catalase activity can change EtOH-related addictive behaviors presumably by changing AcH levels. Moreover, some condensation reaction products of AcH may promote some actions of EtOH and its consumption. On the basis of the findings, it can be concluded that AcH may mediate some of the CNS actions of EtOH including tolerance and dependence, although further exploration the involvement of AcH in EtOH addiction is warranted.

Ethanol metabolism and effects: nitric oxide and its interaction.

Ethanol (EtOH) in alcoholic beverages is consumed by a large number of individuals and its elimination is primarily by oxidation. The role of nitric oxide (NO) in EtOH's effects is important since NO is one of the most prominent biological factors in mammals. NO is constantly formed endogenously from L-arginine. Dose and length of EtOH exposure, and cell type are the main factors affecting EtOH effects on NO production. Either acute or chronic EtOH ingestion affects inducible NO synthase (iNOS) activity. However it seems that EtOH suppresses induced-NO production by inhibition of iNOS in different cells. On the other hand, it is clear that acute low doses of EtOH increase both the release of NO and endothelial NOS (eNOS) expression, and augment endothelium-mediated vasodilatation, whereas higher doses impair endothelial functions. EtOH selectively affects neuronal NOS (nNOS) activity in different brain cells, which may relate to various behavioral interactions. Therefore, there is an excellent chance for EtOH and NO to react with each other. Effects of EtOH on NO production and NOS activity may be important to EtOH modification of cell or organ function. Nitrosated compounds (alkyl nitrites) are often found as the interaction products, which might be one of the minor pathways of EtOH metabolism. NO also inhibits EtOH metabolizing enzymes. Furthermore, NO is involved in EtOH induced liver damage and has a role in fetal development during EtOH exposure in pregnancy. The mechanisms underlying these effects are only partially understood. Hence, the current discussion of the interaction of EtOH and NO is presented.

Neurotoxicity and metabolism of the catecholamine-derived 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde: the role of aldehyde dehydrogenase.

Aldehydes are highly reactive molecules formed during the biotransformation of numerous endogenous and exogenous compounds, including biogenic amines. 3,4-Dihydroxyphenylacetaldehyde is the aldehyde metabolite of dopamine, and 3,4-dihydroxyphenylglycolaldehyde is the aldehyde metabolite of both norepinephrine and epinephrine. There is an increasing body of evidence suggesting that these compounds are neurotoxic, and it has been recently hypothesized that neurodegenerative disorders may be associated with increased levels of these biogenic aldehydes. Aldehyde dehydrogenases are a group of NAD(P)+ -dependent enzymes that catalyze the oxidation of aldehydes, such as those derived from catecholamines, to their corresponding carboxylic acids. To date, 19 aldehyde dehydrogenase genes have been identified in the human genome. Mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sjögren-Larsson syndrome, type II hyperprolinemia, gamma-hydroxybutyric aciduria, and pyridoxine-dependent seizures, most of which are characterized by neurological abnormalities. Several pharmaceutical agents and environmental toxins are also known to disrupt or inhibit aldehyde dehydrogenase function. It is, therefore, possible to speculate that reduced detoxification of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde from impaired or deficient aldehyde dehydrogenase function may be a contributing factor in the suggested neurotoxicity of these compounds. This article presents a comprehensive review of what is currently known of both the neurotoxicity and respective metabolism pathways of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde with an emphasis on the role that aldehyde dehydrogenase enzymes play in the detoxification of these two aldehydes.

Oxidation of ethanol in the brain and its consequences.

Acetaldehyde, a toxic byproduct of alcohol (i.e., ethanol) metabolism, has long been suspected of causing at least some of the central nervous system actions of ethanol. However, the data to support such a hypothesis have been difficult to obtain. One roadblock is the very low blood levels of acetaldehyde following ethanol intake and the finding that even elevated acetaldehyde levels in the blood do not easily gain access to the brain. The recent discovery of the oxidation of ethanol to acetaldehyde in the adult brain may help explain the acute effects of ethanol.

CYP2E1 and catalase influence ethanol sensitivity in the central nervous system.

OBJECTIVES: Genetic factors are known to influence the sensitivity and tolerance to ethanol in humans and laboratory animals. Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway (ADHs) and, to a lesser extent, by microsomal oxidization (CYP2E1) and the catalase-H2O2 system. METHODS: In this study, we examined the role of CYP2E1 and catalase in ethanol metabolism and sensitivity, using transgenic knockout Cyp2e1(-/-) mice, acatalasemic (Cs/Cs) mice, double mutant Cyp2e1(-/-)/Cs/Cs mice and their respective wild-type counterparts 129/sv, C3H/HeJ, 129/sv X C3H/HeJ mice. Ethanol was administered to the mouse lines and ethanol pharmacokinetics and sleep times were evaluated. RESULTS: Although the rates of whole blood ethanol elimination following i.p. administration were found to be similar regardless of dose or genetic stock, Cs/Cs, Cyp2e1(-/-) and Cyp2e1(-/-)/Cs/Cs mice exhibited longer ethanol-induced sleep times, especially at higher ethanol doses. This infers that there is less acetaldehyde produced in the brains of these animals and is in opposition to the idea that increased acetaldehyde increases the actions of ethanol. The Cyp2e1(-/-) animals produced lower whole blood levels of acetaldehyde than wild-type controls; however, this difference was seen only at higher doses of ethanol. The amount of acetaldehyde produced following the incubation of ethanol with liver and brain microsomes was greater in tissues derived from 129/sv than in those from Cyp2e1(-/-) mice. CONCLUSIONS: Although the contribution of CYP2E1 and catalase in ethanol oxidation may be of little significance, these enzymes appear to play a significant role in ethanol sensitivity in the brain.

Enzymatic mechanisms of ethanol oxidation in the brain.

BACKGROUND: The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450-dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats. METHODS: Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography. RESULTS: The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively. CONCLUSIONS: Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates.

Gene array profiles of alcohol and aldehyde metabolizing enzymes in brains of C57BL/6 and DBA/2 mice.

BACKGROUND: Differences in ethanol metabolizing enzymes expressed in brain have been suggested to contribute to the significant differences in ethanol (alcohol) preference between inbred C57BL/6 and DBA/2 mouse strains. METHODS: We have utilized 2 different platforms of oligonucleotide microarray technology (CodeLink UniSet I BioArray from G.E. Healthcare and MG U74A v2.0 from Affymetrix) to simultaneously assess expression of alcohol and acetaldehyde metabolizing enzymes in the whole brain of naïve (no exposure to alcohol) C57BL/6 and DBA/2 mice. RESULTS: There were no significant differences between the 2 strains of mice in gene expression intensity for alcohol dehydrogenases (ADH), catalase, and a number of the cytochrome P450 family of genes, which can be involved in ethanol catabolism. However, significantly higher expression of mRNA for aldehyde dehydrogenase 2 (ALDH2), an isoform mainly responsible for the catabolism of acetaldehyde, was observed in whole brains of DBA/2 mice with both platforms. Aldehyde dehydrogenase 2 protein was also higher in DBA/2 brain. Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA was found to be higher in brains of DBA/2 mice, when measured with the CodeLink platform, but not when measured with Affymetrix arrays or quantitative reverse transcriptase-real-time polymerase chain reaction (qRT-PCR). The ALDH1A1 protein, however, reflected the results obtained with the CodeLink arrays and was higher in DBA/2 brain, compared with brains of C57BL/6 mice. In contrast, the expression intensity for the aldehyde dehydrogenase 7A1 (ALDH7A1) mRNA and protein was significantly higher in C57BL/6 mice than DBA/2 mice. These expression differences are consistent with more rapid metabolism of acetaldehyde in brains of DBA/2 mice. CONCLUSIONS: The use of 2 different microarray platforms provides important cross-validation of many results, and some discrepancies can be resolved with qRT-PCR and immunoblotting. The expression differences that were validated may affect alcohol/aldehyde metabolism in brain and/or alcohol preference in the 2 strains of mice.

Genetic dissociation between ethanol sensitivity and rapid tolerance in mouse and rat strains selectively bred for differential ethanol sensitivity.

BACKGROUND: The Inbred Long- and Short-Sleep mice (ILS and ISS) and the Inbred High- and Low-Alcohol-Sensitive rats (IHAS and ILAS) were selectively bred for differential alcohol sensitivity with use of the duration of loss-of-righting-reflex test (LORR), with the IHAS and ILS animals being much more sensitive than the ILAS and ISS animals, respectively. The current study was undertaken to determine whether acute sensitivity in these strains is genetically correlated to a rapid tolerance to alcohol, a form of tolerance that is evident 24 hr after a single alcohol dose. METHODS: Separate groups of animals were administered a single pretreatment dose of alcohol (0-6 g/kg for the mice; 0-4 g/kg for the rats). Alcohol sensitivity was tested 24 hr later with the LORR test, and blood ethanol concentration was tested at regain of righting (BECRR). Alcohol-induced hypothermia also was determined in the mice. Independently derived replicate rat strains were used for all experiments (IHAS1, ILAS1; IHAS2, ILAS2); no such replicates exist for the ILS and ISS strains. RESULTS: Alcohol pretreatment caused a dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the ILS strain, but LORR increased in the ISS strain with no effect on BECRR. Both strains became hypothermic during the LORR test on day two, but the only significant effect of alcohol pretreatment was in the ISS strain, in which alcohol-induced hypothermia was enhanced. Alcohol pretreatment caused a significant dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the IHAS1 but not in the IHAS2 strain. In contrast, ILAS1 and ILAS2 strains both showed a significant increase in LORR duration and also a significant increase in BECRR. CONCLUSIONS: Alcohol pretreatment caused a dose-dependent decrease in LORR duration and an increase in BECRR in the IHAS1 and ILS strain, suggesting the development of functional rapid tolerance. In contrast, LORR duration increased in the ILAS1, ILAS2, and ISS groups, but BECRR either increased (ILAS1, ILAS2) or did not change (ISS). These observations suggest that central nervous system sensitivity was decreased in the ILAS1 and ILAS2 groups (i.e., rapid functional tolerance) or unchanged in the ISS strain, but that some pharmacokinetic property also was altered in these strains. Overall, the results do not support a genetic relation between alcohol sensitivity and the development of rapid tolerance.