Aldehyde Dehydrogenases and Their Role in Carcinogenesis

Abstract

Aldehydes are highly reactive molecules that may have a variety of effects on biological systems. They can be generated from a virtually limitless number of endogenous and exogenous sources. Although some aldehyde-mediated effects such as vision are beneficial, many effects are deleterious, including cytotoxicity, mutagenicity, and carcinogenicity. A variety of enzymes have evolved to metabolize aldehydes to less reactive forms. Among the most effective pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs).

ALDHs are a family of NADP-dependent enzymes with common structural and functional features that catalyze the oxidation of a broad spectrum of aliphatic and aromatic aldehydes. Based on primary sequence analysis, three major classes of mammalian ALDHs — 1, 2, and 3 — have been identified. Classes 1 and 3 contain both constitutively expressed and inducible cytosolic forms. Class 2 consists of constitutive mitochondrial enzymes. Each class appears to oxidize a variety of substrates that may be derived either from endogenous sources such as amino acid, biogenic amine, or lipid metabolism or from exogenous sources, including aldehydes derived from xenobiotic metabolism.

Changes in ALDH activity have been observed during experimental liver and urinary bladder carcinogenesis and in a number of human tumors, including some liver, colon, and mammary cancers. Changes in ALDH define at least one population of preneoplastic cells having a high probability of progressing to overt neoplasms. The most common change is the appearance of class 3 ALDH dehydrogenase activity in tumors arising in tissues that normally do not express this form. The changes in enzyme activity occur early in tumorigenesis and are the result of permanent changes in ALDH gene expression.

This review discusses several aspects of ALDH expression during carcinogenesis. A brief introduction examines the variety of sources of aldehydes. This is followed by a discussion of the mammalian ALDHs. Because the ALDHs are a relatively understudied family of enzymes, this section presents what is currently known about the general structural and functional properties of the enzymes and the interrelationships of the various forms.

The remainder of the review discusses various aspects of the ALDHs in relation to tumorigenesis. The expression of ALDH during experimental carcinogenesis and what is known about the molecular mechanisms underlying those changes are discussed. This is followed by an extended discussion of the potential roles for ALDH in tumorigenesis. The role of ALDH in the metabolism of cyclophosphamidelike chemotherapeutic agents is described. This work suggests that modulation of ALDH activity may be an important determinant of the effectiveness of certain chemotherapeutic agents. The evidence that changes in ALDH are part of an adaptive response of preneoplastic and neoplastic cells to altered cell physiology or stress is then considered. Roles in the metabolism of aldehydes generated from lipid peroxidation and as part of the Ah gene-mediated response to xenobiotic exposure are both discussed. The data are consistent with a role for certain ALDHs in lipid aldehyde metabolism. Biochemical and genetic data also imply that changes in ALDH may be linked, in part, to cellular adaptation to oxidative stress.

Finally, a model of inducible ALDH gene regulation is proposed. The model incorporates current information about ALDH gene expression with the regulation of other genes known to be part of the adaptive responses occurring in neoplastic cells. The model suggests that regulation of class 1 and 3 ALDH gene activity may be complex, involving the tissue-specific ability to respond to a variety of physiological cues. The model also suggests several avenues for future research that should provide a clearer understanding of the regulation of this important gene family in response to a variety of factors.     

Human aldehyde dehydrogenase-catalyzed oxidation of ethylene glycol ether aldehydes

Ethylene glycol ethers (EGEs) are primary alcohols commonly used as solvents in numerous household and industrial products. Exposure to EGEs has been correlated with delayed encephalopathy, metabolic acidosis, sub-fertility and spermatotoxicity in humans. In addition, they also cause teratogenesis, carcinogenesis, hemolysis, etc., in various animal models. Metabolism EGEs parallels ethanol metabolism, i.e., EGEs are first converted to 2-alkoxy acetaldehydes (EGE aldehydes) by alcohol dehydrogenases, and then to alkoxyacetic acids by aldehyde dehydrogenases (ALDHs). The acid metabolite of EGEs is considered responsible for toxicities associated with EGEs. The role of human ALDHs in EGE metabolism is not clear; accordingly, we have investigated the ability of five different human ALDHs (ALDH1A1, ALDH2, ALDH3A1, ALDH5A1 and ALDH9A1) to catalyze the oxidation of various EGE aldehydes. The EGE aldehydes used in this study were synthesized via Swern oxidation. All of the human ALDHs were purified from human cDNA clones over-expressing these enzymes in E. coli. The ALDHs tested, so far, differentially catalyze the oxidation of EGE aldehydes to their corresponding acids (K(m) values range from approximately 10 microM to approximately 20.0mM). As judged by V(max)/K(m) ratios, short-chain alkyl-group containing EGE aldehydes are oxidized to their acids more efficiently by ALDH2, whereas aryl- and long-chain alkyl-group containing EGE aldehydes are oxidized to their acid more efficiently by ALDH3A1. Given the product of ALDH-catalyzed reaction is toxic, this process should be considered as a bio-activation (toxification) process.     

Expression and initial characterization of human ALDH3B1

Aldehyde dehydrogenases (ALDHs) are critical enzymes in the metabolism of endogenous and exogenous aldehydes. The human genome contains 19 putatively functional ALDH genes; ALDH3B1 belongs to the ALDH3 family. While recent studies have linked the ALDH3B1 locus to schizophrenia, nothing was known, until now, about the properties and significance of the ALDH3B1 protein. The aim of this study was to characterize the ALDH3B1 protein. Human ALDH3B1 was baculovirus-expressed and found to be catalytically active towards medium- and long-chain aliphatic aldehydes and the aromatic aldehyde benzaldehyde. Western blot analyses indicate that ALDH3B1 is highly expressed in kidney and liver and moderately expressed in various brain regions. ALDH3B1-transfected HEK293 cells were significantly protected against cytotoxicity induced by the lipid peroxidation product octanal when compared to vector-transfected cells. This study shows for the first time the functionality, expression and protective role of ALDH3B1 and indicates a potential physiological role of ALDH3B1 against oxidative stress.     

Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells

Aldehyde dehydrogenases (ALDHs) are a superfamily of several isoenzymes widely expressed in bacteria, yeast, plant and animals. Three major classes of ALDHs have been traditionally identified, classes 1, 2 and 3. Both exogenous and endogenous aldehydes, including aldehydes derived from lipid peroxidation, are oxidized by the ALDH superfamily. Several changes in ALDH isoenzyme expression take place in hepatoma cells, in particular cytosolic class 3 ALDH (ALDH3), not expressed in normal hepatocytes, appears and increases with the degree of deviation. It has been demonstrated that cytosolic ALDH3 is important in determining the resistance of tumor cells to antitumor drugs, such as cyclophosphamide. Moreover, hepatoma-associated ALDH3 seems to be important in metabolizing aldehydes derived from lipid peroxidation, and in particular the cytostatic aldehyde 4-hydroxynonenal (4-HNE). We demonstrated previously that restoring endogenous lipid peroxidation in hepatoma cells by enriching them with arachidonic acid causes a decrease of mRNA, protein and enzyme activity of ALDH3 and that this decrease reduces cell growth and/or causes cell death, depending on basal class 3 ALDH activity. To confirm the correlation between inhibition of class 3 ALDH and reduction of cell proliferation, we exposed hepatoma cells to antisense oligonucleotides (ODNs) against ALDH3. In JM2 hepatoma cell line, with high ALDH3 activity, the exposure to antisense ODNs significantly decreases mRNA and enzyme activity (90%). At the same time, cell growth was reduced by about 70%. The results confirm that in hepatoma cells ALDH3 expression is closely related with cell growth, and that its inhibition is important in reducing the proliferation of hepatoma cells overexpressing ALDH3.     

Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells

Aldehyde dehydrogenases (ALDHs) are a superfamily of several isoenzymes widely expressed in bacteria, yeast, plant and animals. Three major classes of ALDHs have been traditionally identified, classes 1, 2 and 3. Both exogenous and endogenous aldehydes, including aldehydes derived from lipid peroxidation, are oxidized by the ALDH superfamily. Several changes in ALDH isoenzyme expression take place in hepatoma cells, in particular cytosolic class 3 ALDH (ALDH3), not expressed in normal hepatocytes, appears and increases with the degree of deviation. It has been demonstrated that cytosolic ALDH3 is important in determining the resistance of tumor cells to antitumor drugs, such as cyclophosphamide. Moreover, hepatoma-associated ALDH3 seems to be important in metabolizing aldehydes derived from lipid peroxidation, and in particular the cytostatic aldehyde 4-hydroxynonenal (4-HNE). We demonstrated previously that restoring endogenous lipid peroxidation in hepatoma cells by enriching them with arachidonic acid causes a decrease of mRNA, protein and enzyme activity of ALDH3 and that this decrease reduces cell growth and/or causes cell death, depending on basal class 3 ALDH activity. To confirm the correlation between inhibition of class 3 ALDH and reduction of cell proliferation, we exposed hepatoma cells to antisense oligonucleotides (ODNs) against ALDH3. In JM2 hepatoma cell line, with high ALDH3 activity, the exposure to antisense ODNs significantly decreases mRNA and enzyme activity (90%). At the same time, cell growth was reduced by about 70%. The results confirm that in hepatoma cells ALDH3 expression is closely related with cell growth, and that its inhibition is important in reducing the proliferation of hepatoma cells overexpressing ALDH3.     

Aldehyde dehydrogenases and cell proliferation

Aldehyde dehydrogenases (ALDHs) oxidize aldehydes to the corresponding carboxylic acids using either NAD or NADP as a coenzyme. Aldehydes are highly reactive aliphatic or aromatic molecules that play an important role in numerous physiological, pathological, and pharmacological processes. ALDHs have been discovered in practically all organisms and there are multiple isoforms, with multiple subcellular localizations. More than 160 ALDH cDNAs or genes have been isolated and sequenced to date from various sources, including bacteria, yeast, fungi, plants, and animals. The eukaryote ALDH genes can be subdivided into several families; the human genome contains 19 known ALDH genes, as well as many pseudogenes. Noteworthy is the fact that elevated activity of various ALDHs, namely ALDH1A2, ALDH1A3, ALDH1A7, ALDH2*2, ALDH3A1, ALDH4A1, ALDH5A1, ALDH6, and ALDH9A1, has been observed in normal and cancer stem cells. Consequently, ALDHs not only may be considered markers of these cells, but also may well play a functional role in terms of self-protection, differentiation, and/or expansion of stem cell populations. The ALDH3 family includes enzymes able to oxidize medium-chain aliphatic and aromatic aldehydes, such as peroxidic and fatty aldehydes. Moreover, these enzymes also have noncatalytic functions, including antioxidant functions and some structural roles. The gene of the cytosolic form, ALDH3A1, is localized on chromosome 17 in human beings and on the 11th and 10th chromosome in the mouse and rat, respectively. ALDH3A1 belongs to the phase II group of drug-metabolizing enzymes and is highly expressed in the stomach, lung, keratinocytes, and cornea, but poorly, if at all, in normal liver. Cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons or chlorinated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, in rat liver cells and increases during carcinogenesis. It has been observed that this increased activity is directly correlated with the degree of deviation in hepatoma and lung cancer cell lines, as is the case in chemically induced hepatoma in rats. High ALDH3A1 expression and activity have been correlated with cell proliferation, resistance against aldehydes derived from lipid peroxidation, and resistance against drug toxicity, such as oxazaphosphorines. Indeed, cells with a high ALDH3A1 content are more resistant to the cytostatic and cytotoxic effects of lipidic aldehydes than are those with a low content. A reduction in cell proliferation can be observed when the enzyme is directly inhibited by the administration of synthetic specific inhibitors, antisense oligonucleotides, or siRNA or indirectly inhibited by the induction of peroxisome proliferator-activated receptor γ (PPARγ) with polyunsaturated fatty acids or PPARγ transfection. Conversely, cell proliferation is stimulated by the activation of ALDH3A1, whether by inhibiting PPARγ with a specific antagonist, antisense oligonucleotides, siRNA, or a medical device (i.e., composite polypropylene prosthesis for hernia repair) used to induce cell proliferation. To date, the mechanisms underlying the effects of ALDHs on cell proliferation are not yet fully clear. A likely hypothesis is that the regulatory effect is mediated by the catabolism of some endogenous substrates deriving from normal cell metabolism, such as 4-hydroxynonenal, which have the capacity to either stimulate or inhibit the expression of genes involved in regulating proliferation.     

Piperlonguminine a new mitochondrial aldehyde dehydrogenase activator protects the heart from ischemia/reperfusion injury

BackgroundDetoxification of aldehydes by aldehyde dehydrogenases (ALDHs) is crucial to maintain cell function. In cardiovascular diseases, reactive oxygen species generated during ischemia/reperfusion events trigger lipoperoxidation, promoting cell accumulation of highly toxic lipid aldehydes compromising cardiac function. In this context, activation of ALDH2, may contribute to preservation of cell integrity by diminishing aldehydes content more efficiently.MethodsThe theoretic interaction of piperlonguminine (PPLG) with ALDH2 was evaluated by docking analysis. Recombinant human ALDH2 was used to evaluate the effects of PPLG on the kinetics of the enzyme. The effects of PPLG were further investigated in a myocardial infarction model in rats, evaluating ALDHs activity, antioxidant enzymes, oxidative stress markers and mitochondrial function.ResultsPPLG increased the activity of recombinant human ALDH2 and protected the enzyme from inactivation by lipid aldehydes. Additionally, administration of this drug prevented the damage induced by ischemia/reperfusion in rats, restoring heart rate and blood pressure, which correlated with protection of ALDHs activity in the tissue, a lower content of lipid aldehydes, and the preservation of mitochondrial function.ConclusionActivation of ALDH2 by piperlonguminine ameliorates cell damage generated in heart ischemia/reperfusion events, by decreasing lipid aldehydes concentration promoting cardioprotection.     

Engineering the nucleotide coenzyme specificity and sulfhydryl redox sensitivity of two stress-responsive aldehyde dehydrogenase isoenzymes of Arabidopsis thaliana

Lipid peroxidation is one of the consequences of environmental stress in plants and leads to the accumulation of highly toxic, reactive aldehydes. One of the processes to detoxify these aldehydes is their oxidation into carboxylic acids catalyzed by NAD(P)+-dependent ALDHs (aldehyde dehydrogenases). We investigated kinetic parameters of two Arabidopsis thaliana family 3 ALDHs, the cytosolic ALDH3H1 and the chloroplastic isoform ALDH3I1. Both enzymes had similar substrate specificity and oxidized saturated aliphatic aldehydes. Catalytic efficiencies improved with the increase of carbon chain length. Both enzymes were also able to oxidize α,β-unsaturated aldehydes, but not aromatic aldehydes. Activity of ALDH3H1 was NAD+-dependent, whereas ALDH3I1 was able to use NAD+ and NADP+. An unusual isoleucine residue within the coenzyme-binding cleft was responsible for the NAD+-dependence of ALDH3H1. Engineering the coenzyme-binding environment of ALDH3I1 elucidated the influence of the surrounding amino acids. Enzyme activities of both ALDHs were redox-sensitive. Inhibition was correlated with oxidation of both catalytic and non-catalytic cysteine residues in addition to homodimer formation. Dimerization and inactivation could be reversed by reducing agents. Mutant analysis showed that cysteine residues mediating homodimerization are located in the N-terminal region. Modelling of the protein structures revealed that the redox-sensitive cysteine residues are located at the surfaces of the subunits.     

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

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 dehydrogenase 1B1 (ALDH1B1) is a potential biomarker for human colon cancer

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P)⁺-dependent enzymes, which catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding acids. Increased expression and/or activity of ALDHs, particularly ALDH1A1, have been reported to occur in human cancers. It is proposed that the metabolic function of ALDH1A1 confers the “stemness” properties to normal and cancer stem cells. Nevertheless, the identity of ALDH isozymes that contribute to the enhanced ALDH activity in specific types of human cancers remains to be elucidated. ALDH1B1 is a mitochondrial ALDH that metabolizes a wide range of aldehyde substrates including acetaldehyde and products of lipid peroxidation (LPO). In this study, we immunohistochemically examined the expression profile of ALDH1A1 and ALDH1B1 in human adenocarcinomas of colon (N = 40), lung (N = 30), breast (N = 33) and ovary (N = 33) using an NIH tissue array. The immunohistochemical expression of ALDH1A1 or ALDH1B1 in tumor tissues was scored by their intensity (scale = 1–3) and extensiveness (% of total cancer cells). Herein we report a 5.6-fold higher expression score for ALDH1B1 in cancerous tissues than that for ALDH1A1. Remarkably, 39 out of 40 colonic cancer specimens were positive for ALDH1B1 with a staining intensity of 2.8 ± 0.5. Our study demonstrates that ALDH1B1 is more profoundly expressed in the adenocarcinomas examined in this study relative to ALDH1A1 and that ALDH1B1 is dramatically upregulated in human colonic adenocarcinoma, making it a potential biomarker for human colon cancer.     

Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases

Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.     

The ALDH gene superfamily of Arabidopsis

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily of NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. The Arabidopsis genome contains 14 unique ALDH sequences encoding members of nine ALDH families, including eight known families and one novel family (ALDH22) that is currently known only in plants. Here, we identify members of the ALDH gene superfamily in Arabidopsis; provide a revised, unified nomenclature for these ALDH genes; analyze the molecular relationship among Arabidopsis ALDH genes and compare them to ALDH genes from other species, including prokaryotes and mammals; and describe the role of ALDHs in cytoplasmic male sterility, plant defense and abiotic stress tolerance.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.     

Substrate specificity,plasma membrane localization,and lipid modification of the aldehyde dehydrogenase ALDH3B1

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum.     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

Substrate specificity of human and yeast aldehyde dehydrogenases

Human aldehyde dehydrogenase (ALDH) family may contribute to metabolism of hydrocarbons, biogenic amines, retinoids, steroids, and lipid peroxidation. We previously reported kinetic properties of human cytosolic ALDH1 and mitochondrial ALDH2 towards oxidation of the straight-chain and branched-chain aliphatic aldehydes with various chain lengths [S.J. Yin, M.F. Wang, C.L. Han, S.L. Wang, Substrate binding pocket structure of human aldehyde dehydrogenases: a substrate specificity approach, Adv. Exp. Med. Biol. 372 (1995) 9-16]. We present here substrate specificities for aromatic and heterocyclic aldehydes with purified human liver ALDH1 and ALDH2, and also with yeast mitochondrial ALDH2 for comparison. Kinetic assay for human ALDHs was performed in 50mM HEPES, pH 7.5 and 25 degrees C, containing 0.5mM NAD(+), 1.7% (v/v) acetonitrile (as a solvent carrier for aldehydes) and varied concentrations of substrate, and for yeast ALDH2 the assay was determined in the same reaction mixture except containing 3mM NAD(+) and addition of 200 mM KCl. With respect to phenylacetaldehyde, 2-phenylpropionaldehyde, benzaldehyde, p-nitrobenzaldehyde, cinnamaldehyde, 2-furaldehyde and indole-3-acetaldehyde, human liver ALDH1 exhibited K(M) ranging from 0.25 to 4.8 microM, V(max) of 0.34-2.4U/mg, and catalytic efficiency, V(max)/K(M), 0.070-3.9U/(mg microM); human ALDH2 exhibited K(M) ranging from less than 0.15-0.74 microM, V(max) of 0.039-0.51 U/mg, and V(max)/K(M), 0.15-1.0U/(mg microM). Human ALDH1 and ALDH2 exhibited substate inhibition constants (K(i)) for phenylacetaldehyde, 95 and 430 microM, respectively. Yeast ALDH2 exhibited K(M) for straight-chain aliphatic aldehydes (C1-C10), 2.3-210 microM, and substrate inhibition constants (C2-C10), 79-2900 microM, with a trend of being smaller K(M) and K(i) for longer chain lengths; and K(M) for cinnamaldehyde, benzaldehyde, and 2-furaldehyde, 5.0, 79, and 1000 microM, respectively. Therefore human ALDH1/ALDH2 and yeast ALDH2 can contribute to detoxification or metabolism of various exogenous/endogenous aliphatic and aromatic aldehydes. The systematic changes in kinetic parameters for oxidation of structurally related aldehydes may reflect subtle functional topographic distinctions of substrate pocket for human and yeast ALDHs.     

Aldehyde dehydrogenase gene superfamily: the 2000 update

Aldehyde dehydrogenase (ALDH) superfamily represents a group of NAD(P)(+)-dependent enzymes that catalyze the oxidation of a wide spectrum of endogenous and exogenous aldehydes. With the advent of megabase genome sequencing, the ALDH superfamily is expanding rapidly on many fronts. As expected, ALDH genes are found in virtually all genomes analyzed to date, indicating the importance of these enzymes in biological functions. Complete genome sequences of various species have revealed additional ALDH genes. As of July 2000, the ALDH superfamily consists of 331 distinct genes, of which eight are found in archaea, 165 in eubacteria, and 158 in eukaryota. The number of ALDH genes in some species with their genomes completely sequenced and annotated, Escherichia coli and Caenorhabditis elegans, ranges from 10 to 17. In the human genome, 17 functional genes and three pseudogenes have been identified to date. Divergent evolution, based on multiple alignment analysis of 86 eukaryotic ALDH amino-acid sequences, was the basis of the standardized ALDH gene nomenclature system (Pharmacogenetics 9: 421-434, 1999). Thus far, the eukaryotic ALDHs comprise 20 gene families. A complete list of all ALDH sequences known to date is presented here along with the evolution analysis of the eukaryotic ALDHs.     

Phenobarbital inducibility and differences in protein expression of an animal model

Aldehyde dehydrogenases (ALDHs) are a group of enzymes which catalyze the conversion of aldehydes to the corresponding carboxylic acids in a NAD(P)(+)-dependent reaction. In mammals, different ALDHs are constitutively expressed in liver, stomach, eye and skin. In addition, inducible ALDH-isoenzymes are detectable in many tissues; apart from other physico- and immuno-chemical differences, two cytosolic ALDHs (ALDH1A3 and ALDH3A1) are known to be activated in rat liver, by different types of inducers of drug metabolism. Phenobarbital-type inducers increase the ALDH1A3, while polycyclic hydrocarbons (such as BaP and TCDD) increase the expression of the two members of ALDH3A subfamily (3A1 and 3A2). In this study, we used two Wistar rat substrains which have been well-characterized for different inducibility of ALDH1A3 enzyme activity after treatment with phenobarbital. Animals that respond (RR) or do not respond (rr) to treatment have been inbred for almost 25 years, offering a useful experimental model. Apart from the level of ALDH1A3 induced enzyme expression after phenobarbital treatment, no other differences between the two substrains have been noticed, as far as drug metabolizing enzyme activities (like the pentoxy- and ethoxy-O-dealkylation rate) are concerned. According to the present results, the ALDH1A3 expression is still the only difference between the two substrains. Immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or/and CYP1A1/1A2 showed no differences between the two substrains. Additionally, data concerning time- and dose-response induction of ALDH1A3 after phenobarbital and griseofulvin treatment are presented. It is concluded that these two Wistar rat substrains represent a unique animal model for studying what seems to be the only difference between these substrains - the genetic basis of the phenobarbital induction.     

Phenobarbital inducibility and differences in protein expression of an animal model

Aldehyde dehydrogenases (ALDHs) are a group of enzymes which catalyze the conversion of aldehydes to the corresponding carboxylic acids in a NAD(P)⁺-dependent reaction. In mammals, different ALDHs are constitutively expressed in liver, stomach, eye and skin. In addition, inducible ALDH-isoenzymes are detectable in many tissues; apart from other physico- and immuno-chemical differences, two cytosolic ALDHs (ALDH1A3 and ALDH3A1) are known to be activated in rat liver, by different types of inducers of drug metabolism. Phenobarbital-type inducers increase the ALDH1A3, while polycyclic hydrocarbons (such as BaP and TCDD) increase the expression of the two members of ALDH3A subfamily (3A1 and 3A2). In this study, we used two Wistar rat substrains which have been well-characterized for different inducibility of ALDH1A3 enzyme activity after treatment with phenobarbital. Animals that respond (RR) or do not respond (rr) to treatment have been inbred for almost 25 years, offering a useful experimental model. Apart from the level of ALDH1A3 induced enzyme expression after phenobarbital treatment, no other differences between the two substrains have been noticed, as far as drug metabolizing enzyme activities (like the pentoxy- and ethoxy-O-dealkylation rate) are concerned. According to the present results, the ALDH1A3 expression is still the only difference between the two substrains. Immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or/and CYP1A1/1A2 showed no differences between the two substrains. Additionally, data concerning time- and dose-response induction of ALDH1A3 after phenobarbital and griseofulvin treatment are presented. It is concluded that these two Wistar rat substrains represent a unique animal model for studying what seems to be the only difference between these substrains — the genetic basis of the phenobarbital induction.