Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse

A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype.     

ALDH3A1 Plays a Functional Role in Maintenance of Corneal Epithelial Homeostasis

Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1^-/-/3a1^-/- double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.     

Omega -crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.     

Defining a link between gap junction communication, proteolysis, and cataract formation

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.     

Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian—South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.     

Lipid abnormalities in succinate semialdehyde dehydrogenase (Aldh5a1-/-) deficient mouse brain provide additional evidence for myelin alterations

Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1(-/-) mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143-156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1(-/-) brain. In comparison to wild-type littermates (Aldh5a1(+/+)), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1(-/-)mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1(-/-) brain tissue (one-tailed t test, p=0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology.     

Mechanisms involved in the protection of UV-induced protein inactivation by the corneal crystallin ALDH3A1

Various lines of evidence have shown that ALDH3A1 (aldehyde dehydrogenase 3A1) plays a critical and multifaceted role in protecting the cornea from UV-induced oxidative stress. ALDH3A1 is a corneal crystallin, which is defined as a protein recruited into the cornea for structural purposes without losing its primary function (i.e. metabolism). Although the primary role of ALDH3A1 in the metabolism of toxic aldehydes has been clearly demonstrated, including the detoxification of aldehydes produced during UV-induced lipid peroxidation, the structural role of ALDH3A1 in the cornea remains elusive. We therefore examined the potential contribution of ALDH3A1 in maintaining the optical integrity of the cornea by suppressing the aggregation and/or inactivation of other proteins through chaperone-like activity and other protective mechanisms. We found that ALDH3A1 underwent a structural transition near physiological temperatures to form a partially unfolded conformation that is suggestive of chaperone activity. Although this structural transition alone did not correlate with any protection, ALDH3A1 substantially reduced the inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal and malondialdehyde when co-incubated with NADP(+), reinforcing the importance of the metabolic function of this corneal enzyme in the detoxification of toxic aldehydes. A large excess of ALDH3A1 also protected glucose-6-phosphate dehydrogenase from inactivation because of direct exposure to UVB light, which suggests that ALDH3A1 may shield other proteins from damaging UV rays. Collectively, these data demonstrate that ALDH3A1 can reduce protein inactivation and/or aggregation not only by detoxification of reactive aldehydes but also by directly absorbing UV energy. This study provides for the first time mechanistic evidence supporting the structural role of the corneal crystallin ALDH3A1 as a UV-absorbing constituent of the cornea.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Mutation in the betaA3/A1-crystallin encoding gene Cryba1 causes a dominant cataract in the mouse

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.     

Scallop lens Omega-crystallin (ALDH1A9): a novel tetrameric aldehyde dehydrogenase

Scallop eye lens Omega-crystallin is an inactive aldehyde dehydrogenase (ALDH1A9) related to cytoplasmic ALDH1A1 and mitochondrial ALDH2 that migrates by gel filtration chromatography as a homodimer. Because mammalian ALDH1A1 and ALDH2 are homotetramers, we investigated the native molecular mass of scallop Omega-crystallin by multi-angle laser light scattering. The results indicate that the scallop Omega-crystallin is a tetrameric, not a dimeric protein. Moreover, phylogenetic tree analysis shows that scallop Omega-crystallin clusters with the mitochondrial ALDH2 and ALDH1B1 rather than the cytoplasmic ALDH1A, yet it lacks the mitochondrial N-terminal leader sequence characteristic of the mitochondrial ALDHs. The mitochondrial grouping, enzymatic inactivity, and anomalous gel filtration behavior make scallop cytoplasmic Omega-crystallin an interesting protein for structural studies of evolutionary adaptations to become an enzyme-crystallin.     

Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys.     

Comparative studies of vertebrate aldehyde dehydrogenase 3: sequences, structures, phylogeny and evolution. Evidence for a mammalian origin for the ALDH3A1 gene

Mammalian ALDH3 genes (ALDH3A1, ALDH3A2, ALDH3B1 and ALDH3B2) encode enzymes of peroxidic and fatty aldehyde metabolism. ALDH3A1 also plays a major role in anterior eye tissue UV-filtration. BLAT and BLAST analyses were undertaken of several vertebrate genomes using rat, chicken and zebrafish ALDH3-like amino acid sequences. Predicted vertebrate ALDH3 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure as reported by Liu et al. [27] for rat ALDH3A1. Phylogeny studies of human, rat, opossum, platypus, chicken, xenopus and zebrafish ALDH3-like sequences supported three hypotheses: (1) the mammalian ALDH3A1 gene was generated by a tandem duplication event of an ancestral vertebrate ALDH3A2 gene; (2) multiple mammalian and chicken ALDH3B-like genes were generated by tandem duplication events within genomes of related species; and (3) vertebrate ALDH3A and ALDH3B genes were generated prior to the appearance of bony fish more than 500 million years ago.     

Extracellular matrix and integrin signaling in lens development and cataract

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.     

Enzymatic and metabolic evidence for a region specific mitochondrial dysfunction in brains of murine succinic semialdehyde dehydrogenase deficiency (Aldh5a1-/- mice)

Succinic semialdehyde dehydrogenase deficiency, a rare inherited defect of gamma-aminobutyrate (GABA) catabolism, presents with characteristic biochemical abnormalities in the central nervous system (CNS). These include elevated concentrations of GABA, gamma-hydroxybutyrate (GHB), succinic semialdehyde (SSA), 4,5-dihydroxyhexanoic acid (DHHA) and alanine as well as decreased concentrations of glutamine. GABA degradation is coupled to Krebs cycle function in mammalian CNS ("GABA shunt") through succinate and alpha-ketoglutarate. Accordingly, we hypothesized that disruption of Krebs cycle and respiratory chain function in the CNS is involved in the neuropathogenesis of this disease. For this purpose, we investigated cerebral activities of Krebs cycle and respiratory chain enzymes as well as the glutathione content in Aldh5a1(-/-) mice, a recently generated mouse model for this disease. In CNS tissue of Aldh5a1(-/-) mice, we found a significantly decreased glutathione content (hippocampus, cortex) and decreased activities of complexes I-IV (hippocampus) suggesting increased oxidative stress and mitochondrial dysfunction. However, specific activities of Krebs cycle and respiratory chain were not affected by GABA, GHB, SSA, or DHHA (up to 1 mmol/L). Although our results suggest hippocampal and cortical dysfunction in Aldh5a1(-/-) brain, we found no evidence that accumulating key metabolites of SSADH deficiency directly induce impairment of energy metabolism.     

Orally active multi-functional antioxidants delay cataract formation in streptozotocin (type 1) diabetic and gamma-irradiated rats

Background

Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation.

Methods/Principal Findings

Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic “sugar” cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic “sugar” cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress.

Conclusions/Significance

Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed.     

Corneal and stomach expression of aldehyde dehydrogenases: from fish to mammals

We have studied the distribution of the ALDH3A1, ALDH1A1 and ALDH2 proteins in the cornea and stomach of several animal species, including mammals (C57BL/6J and SWR/J mice, rat and pig), birds (chicken and turkey), amphibians (frog) and fish (trout and zebrafish). High ALDH3A1 protein levels and catalytic activities were detected in C57BL/6J mouse, rat and pig. We found complete absence of the ALDH3A1 protein in SWR/J mice, which carry the Aldh3a1(c) allele characterized by four amino acid substitutions (G88R, I154N, H305R and I352V) and lack of enzymatic activity. This indicates that the SWR/J mouse strain is a natural gene knockout model for ALDH3A1. Traces of ALDH3A1 were detected in rabbit, whereas expression was absent from chicken, turkey, frog, trout, and zebrafish. Interestingly, significant levels of the cytosolic ALDH1A1 and mitochondrial ALDH2 proteins were detected by immunoblot analysis in all examined species that are deficient in ALDH3A1 expression. In contrast, no ALDH1A1 or ALDH2 protein was detected in the species expressing ALDH3A1. It can, therefore, be concluded that corneal expression of ALDH3A1 or ALDH1A1/ALDH2 occurs in a taxon-specific manner, supporting the protective role of these ALDHs in cornea against the UV-induced oxidative damage.     

Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation

Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer's and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (V(max)/K(m)=23). However, Aldh1a1 exhibits far higher affinity for acrolein (K(m)=23.2 μM) compared to Aldh3a1 (K(m)=464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 ml/min/mg vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein-protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈3-fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1mM NAD(+) was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies.