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1.  Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCε) knockout mice 
Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCε). We recently identified aldehyde dehydrogenase 2 (ALDH2) as an PKCε substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCε knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCε and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCε, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCε knockout hearts. Our data suggest that ALDH2 is downstream of PKCε in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCε to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.
PMCID: PMC2837767  PMID: 19913552
2.  Isoflurane Preconditioning Confers Cardioprotection by Activation of ALDH2 
PLoS ONE  2013;8(2):e52469.
The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.
PMCID: PMC3585331  PMID: 23468836
3.  Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde 
European Heart Journal  2010;32(8):1025-1038.
The present study was designed to examine the mechanism involved in mitochondrial aldehyde dehydrogenase (ALDH2)-induced cardioprotection against ischaemia/reperfusion (I/R) injury with a focus on autophagy.
Wild-type (WT), ALDH2 overexpression, and knockout (KO) mice (n = 4–6 for each index measured) were subjected to I/R, and myocardial function was assessed using echocardiographic, Langendroff, and edge-detection systems. Western blotting was used to evaluate AMP-dependent protein kinase (AMPK), Akt, autophagy, and the AMPK/Akt upstream signalling LKB1 and PTEN.
ALDH2 overexpression and KO significantly attenuated and accentuated, respectively, infarct size, factional shortening, and recovery of post-ischaemic left ventricular function following I/R as well as hypoxia/reoxygenation-induced cardiomyocyte contractile dysfunction. Autophagy was induced during ischaemia and remained elevated during reperfusion. ALDH2 significantly promoted autophagy during ischaemia, which was accompanied by AMPK activation and mammalian target of rapamycin (mTOR) inhibition. On the contrary, ALDH2 overtly inhibited autophagy during reperfusion accompanied by the activation of Akt and mTOR. Inhibition and induction of autophagy mitigated ALDH2-induced protection against cell death in hypoxia and reoxygenation, respectively. In addition, levels of the endogenous toxic aldehyde 4-hydroxy-2-nonenal (4-HNE) were elevated by ischaemia and reperfusion, which was abrogated by ALDH2. Furthermore, ALDH2 ablated 4-HNE-induced cardiomyocyte dysfunction and protein damage, whereas 4-HNE directly decreased pan and phosphorylated LKB1 and PTEN expression.
Our data suggest a myocardial protective effect of ALDH2 against I/R injury possibly through detoxification of toxic aldehyde and a differential regulation of autophagy through AMPK- and Akt-mTOR signalling during ischaemia and reperfusion, respectively.
PMCID: PMC3076664  PMID: 20705694
ALDH2; Myocardial ischaemia/reperfusion; Akt; AMPK; Autophagy; 4-HNE
4.  Characterization of the East Asian Variant of Aldehyde Dehydrogenase-2 
The Journal of Biological Chemistry  2009;285(2):943-952.
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at ≥5 mm. In the presence of 1 mm NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.
PMCID: PMC2801295  PMID: 19906643
Cyclic GMP (cGMP); Enzyme Catalysis; Nitric Oxide; Oxidase; Superoxide Dismutase (SOD); Superoxide Ion; Bioactivation; Nitroglycerin
5.  Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant 
In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant, acrolein. ALDH2 also bioactivates nitroglycerin, but it is best known for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian Alcohol-induced Flushing Syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semi-dominant and heterozygotic individuals exhibit a similar, but not as severe phenotype. We recently identified a small molecule, Alda-1, which activates wild-type ALDH2 and restores near wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.
PMCID: PMC2857674  PMID: 20062057
6.  Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases* 
The Journal of Biological Chemistry  2008;283(26):17873-17880.
Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.
PMCID: PMC2440601  PMID: 18450747
7.  Aldehyde Dehydrogenase-2 Deficiency Aggravates Cardiac Dysfunction Elicited by Endoplasmic Reticulum Stress Induction 
Molecular Medicine  2012;18(1):785-793.
Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been characterized as an important mediator of endogenous cytoprotection in the heart. This study was designed to examine the role of ALDH2 knockout (KO) in the regulation of cardiac function after endoplasmic reticulum (ER) stress. Wild-type (WT) and ALDH2 KO mice were subjected to a tunicamycin challenge, and the echocardiographic property was examined. Protein levels of six items—78 kDa glucose-regulated protein (GRP78), phosphorylation of eukaryotic initiation factor 2 subunit α (p-eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP), phosphorylation of Akt, p47phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 4-hydroxynonenal—were determined by using Western blot analysis. Cytotoxicity and apoptosis were estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and caspase-3 activity, respectively. ALDH2 deficiency exacerbated cardiac contractile dysfunction and promoted ER stress after ER stress induction, manifested by the changes of ejection fraction and fractional shortening. In vitro study revealed that tunicamycin significantly upregulated the levels of GRP78, p-eIF2α, CHOP, p47phox NADPH oxidase and 4-hydroxynonenal, which was exacerbated by ALDH2 knockdown and abolished by ALDH2 overexpression, respectively. Overexpression of ALDH2 abrogated tunicamycin-induced dephosphorylation Akt. Inhibition of phosphatidylinositol 3-kinase using LY294002 did not affect ALDH2-conferred protection against ER stress, although LY294002 reversed the antiapoptotic action of ALDH2 associated with p47phox NADPH oxidase. These results suggest a pivotal role of ALDH2 in the regulation of ER stress and ER stress–induced apoptosis. The protective role of ALDH2 against ER stress–induced cell death was probably mediated by Akt via a p47phox NADPH oxidase-dependent manner. These findings indicate the critical role of ALDH2 in the pathogenesis of ER stress in heart disease.
PMCID: PMC3409283  PMID: 22430940
8.  Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of εPKC and activation of aldehyde dehydrogenase 2 
The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (εPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of εPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 minutes prior to (but not 15 minutes prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of εPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and εPKC-mediated protection from cardiac ischemia, in vivo.
PMCID: PMC2675554  PMID: 18983847
9.  Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance 
Journal of Clinical Investigation  2004;113(3):482-489.
Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (ρ0 cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.
PMCID: PMC324536  PMID: 14755345
10.  Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels 
Biochemical Pharmacology  2015;93(4):440-448.
Graphical abstract
The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.
PMCID: PMC4321882  PMID: 25576686
ALDH, aldehyde dehydrogenase; CB25, 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-1H-indole-2,3-dione; cGMP, 3′,5′-cyclic guanosine monophosphate; DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine; DMSO, dimethyl sulfoxide; DPI, diphenyleneiodonium; DTPA, diethylenetriamine pentaacetic acid; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; GDN, glycerol dinitrate; GTN, glycerol trinitrate (nitroglycerin); NAD, nicotinamide adenine dinucleotide; l-NNA, NG-nitro-l-arginine; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one; sGC, soluble guanylate cyclase.; Aldehyde dehydrogenase-2; Denitration; Nitroglycerin; Protein expression; Vascular relaxation
11.  ALDH2 protects against stroke by clearing 4-HNE 
Cell Research  2013;23(7):915-930.
Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke.
PMCID: PMC3698638  PMID: 23689279
ALDH2; 4-HNE; stroke; ethanol
12.  Impaired Cardiac SIRT1 Activity by Carbonyl Stress Contributes to Aging-Related Ischemic Intolerance 
PLoS ONE  2013;8(9):e74050.
Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1+/− knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1+/− hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.
PMCID: PMC3769351  PMID: 24040162
13.  Neurodegeneration and Motor Dysfunction in Mice Lacking Cytosolic and Mitochondrial Aldehyde Dehydrogenases: Implications for Parkinson's Disease 
PLoS ONE  2012;7(2):e31522.
Previous studies have reported elevated levels of biogenic aldehydes in the brains of patients with Parkinson's disease (PD). In the brain, aldehydes are primarily detoxified by aldehyde dehydrogenases (ALDH). Reduced ALDH1 expression in surviving midbrain dopamine neurons has been reported in brains of patients who died with PD. In addition, impaired complex I activity, which is well documented in PD, reduces the availability of the NAD+ co-factor required by multiple ALDH isoforms to catalyze the removal of biogenic aldehydes. We hypothesized that chronically decreased function of multiple aldehyde dehydrogenases consequent to exposure to environmental toxins and/or reduced ALDH expression, plays an important role in the pathophysiology of PD. To address this hypothesis, we generated mice null for Aldh1a1 and Aldh2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Aldh1a1−/−×Aldh2−/− mice exhibited age-dependent deficits in motor performance assessed by gait analysis and by performance on an accelerating rotarod. Intraperitoneal administration of L-DOPA plus benserazide alleviated the deficits in motor performance. We observed a significant loss of neurons immunoreactive for tyrosine hydroxylase (TH) in the substantia nigra and a reduction of dopamine and metabolites in the striatum of Aldh1a1−/−×Aldh2−/− mice. We also observed significant increases in biogenic aldehydes reported to be neurotoxic, including 4-hydroxynonenal (4-HNE) and the aldehyde intermediate of dopamine metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL). These results support the hypothesis that impaired detoxification of biogenic aldehydes may be important in the pathophysiology of PD and suggest that Aldh1a1−/−×Aldh2−/− mice may be a useful animal model of PD.
PMCID: PMC3284575  PMID: 22384032
14.  Molecular Characterization, Expression Analysis and Role of ALDH3B1 in The Cellular Protection Against Oxidative Stress 
Free radical biology & medicine  2010;49(9):1432-1443.
Aldehyde dehydrogenase (ALDH) enzymes are critical in the detoxification of aldehydes. The human genome contains 19 ALDH genes, mutations in which are the basis of several diseases. The expression, subcellular localization, enzyme kinetics and role of ALDH3B1 against aldehyde- and oxidant-induced cytotoxicity were investigated. ALDH3B1 was purified from Sf9 cells using chromatographic methods and enzyme kinetics were determined spectrophotometrically. ALDH3B1 demonstrated high affinity for hexanal (Km 62 μM), octanal (Km 8 μM), 4-hydroxy-2-nonenal (4HNE) (Km 52 μM) and benzaldehyde (Km 46 μM). Low affinity was seen towards acetaldehyde (Km 23.3 mM), malondialdehyde (Km 152 mM) and the ester p-nitrophenylacetate (Km 3.6 mM). ALDH3B1 mRNA was abundant in testis, lung, kidney and ovary. ALDH3B1 protein was highly expressed in these tissues and the liver. Immunofluorescence microscopy of ALDH3B1-transfected human embryonic kidney (HEK293) cells and subcellular fractionation of mouse kidney and liver revealed a cytosolic protein localization. ALDH3B1-transfected HEK293 cells were significantly protected from the lipid peroxidation-derived aldehydes trans-2-octenal, 4HNE and hexanal, and the oxidants H2O2 and menadione. In addition, ALDH3B1 protein expression was up-regulated by 4HNE in ARPE-19 cells. The results detailed in this study support a pathophysiological role for ALDH3B1 in protecting cells from the damaging effects of oxidative stress.
PMCID: PMC3457645  PMID: 20699116
aldehyde dehydrogenase 3B1 (ALDH3B1); 4-hydroxy-2-nonenal; lipid peroxidation; mRNA; protein expression; enzyme kinetics; aldehyde toxicity
15.  Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications 
Journal of proteomics  2011;74(12):2691-2702.
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.
PMCID: PMC3177986  PMID: 21609791
Aldehyde dehydrogenases; post-translational modifications; cellular defense; drug toxicity; disease states; translational research
16.  Differential effects of organic nitrates on arterial diameter among healthy Japanese participants with different mitochondrial aldehyde dehydrogenase 2 genotypes: randomised crossover trial 
BMJ Open  2011;1(1):e000133.
To determine whether polymorphisms at codon 487 (*1, GAA=Glu; *2, AAA=Lys) of mitochondrial aldehyde dehydrogenase 2 (ALDH2) influence nitroglycerine (glyceryl trinitrate (GTN))-induced vasodilation, and whether GTN or isosorbide dinitrate (ISDN) is a more effective antianginal agent in each ALDH2 genotype.
A randomised, open-label, crossover trial with 117 healthy Japanese (20–39 years) whose genotypes were determined (*1/*1, n=47; *1/*2, n=48; *2/*2, n=22) was performed at Kyushu University Hospital, Fukuoka, Japan. Participants were randomly assigned to treatment: sublingual spray of GTN (0.3 mg) or ISDN (1.25 mg). After ≥1 week, measurements were repeated using the other drug. The main outcome measures were the maximal rate of increase in the brachial artery diameter determined by ultrasonography, the time required to attain maximal dilation (Tmax) and the time required to attain 90% maximal dilation (T0.9).
The maximal artery diameter increase in response to GTN or ISDN did not differ among genotypes. However, GTN Tmax was significantly longer for *2/*2 (299.7 s, 269.0–330.4) than *1/*1 (254.7 s, 238.6–273.4; p=0.0190). GTN T0.9 was significantly longer in the *1/*2 (206.1 s, 191.7–219.3) and *2/*2 (231.4 s, 211.8–251.0) genotypes than *1/*1 (174.9 s, 161.5–188.3; p=0.0068, p<0.0001, respectively). In contrast, the time-course of ISDN-induced vasodilation did not differ among genotypes. GTN Tmax and T0.9 among *1 allele carriers (*1/*1 and *1/*2) were significantly shorter than those of ISDN, whereas the time course of GTN and ISDN vasodilation did not differ among participants carrying *2/*2.
The amplitude of GTN-induced vasodilation was not influenced by the ALDH2 genotype, but the response was significantly delayed in *2 allele carriers, especially *2/*2. GTN dilated the artery more quickly than ISDN in *1/*1 and *1/*2, but not in *2/*2.
Trial registration number
UMIN000001492 (UMIN-CTR database).
Article summary
Article focus
There were few reliable human studies on the influence of aldehyde dehydrogenase 2 (ALDH2) polymorphisms at codon 487 on the response to nitroglycerine (GTN). In particular, there was no information about the response to GTN in *2/*2 homozygotes.
It was unclear whether GTN or isosorbide dinitrate (ISDN) is a more effective antianginal agent in each ALDH2 genotype.
We aimed at comparing vasodilation induced by GTN and ISDN in individuals with different ALDH2 genotypes.
Key messages
The maximal rate of increase in arterial diameter after GTN treatment did not differ among ALDH2 genotype groups.
The time required to attain 90% maximal vasodilation was longer in the *1/*2 and *2/*2 groups than in the *1/*1 group.
GTN (0.3 mg) induced vasodilation more rapidly than ISDN (1.25 mg) in the *1/*1 and *1/*2 groups, but there was no difference between GTN and ISDN in the *2/*2 group.
Strengths and limitations of this study
The choice of healthy young individuals as participants may have strengthened the validity of the study; the participants' backgrounds were likely to be more homogeneous by excluding the influence of multiple concomitant factors that could not be controlled when studying patients or older subjects. However, the results could have been different if the study had been performed with patients with angina, because haemodynamic changes in patients might alter the response to nitrates.
Sublingual spray formulations of nitrates were used instead of tablets to minimise the fluctuation in absorption rate. However, this may have made the dosage slightly unreliable.
A randomised crossover design was used, and arterial diameter was determined by a single measurer blinded to genotype. However, the participants and investigators were not blinded to treatment.
Arterial diameter was accurately measured using a semiautomatic ultrasonography system. However, the brachial artery diameter was measured instead of the coronary artery diameter, and moreover, we did not evaluate venous dilation.
PMCID: PMC3191425  PMID: 22021773
Clinical pharmacology; therapeutics; hypertension; ischaemic heart disease; heart failure
17.  Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2 
The Journal of Biological Chemistry  2012;287(45):38124-38134.
Background: Aldehyde dehydrogenase-2 (ALDH2) catalyzes bioactivation of glyceryl trinitrate (GTN) resulting in vasodilation. The exact mechanism is still unclear.
Results: Structures of ALDH2 in complex with GTN and of a thionitrate intermediate were obtained.
Conclusion: The structures represent snapshots of the first reaction step of GTN bioactivation by ALDH2.
Significance: The results provide new insight into the mechanism of vascular GTN bioactivation by ALDH2.
Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.
PMCID: PMC3488082  PMID: 22988236
Crystal Structure; Enzyme Inhibitors; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Mutation; Enzyme Structure; Aldehyde Dehydrogenase; Glyceryl Trinitrate; Sulfinic Acid; Thionitrate
18.  Tolerance to nitroglycerin through proteasomal down-regulation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency 
British Journal of Pharmacology  2013;168(8):1868-1877.
Background and Purpose
L-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation.
Experimental Approach
Ascorbate deficiency was induced in Gulo(-/-) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo(-/-), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined.
Key Results
Like GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo(-/-) mice with the 26S proteasome inhibitor bortezomib.
Conclusions and Implications
Our data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.
PMCID: PMC3623057  PMID: 23194305
aldehyde dehydrogenase-2; ascorbate deficiency; bortezomib; genetic mouse model; nitrate tolerance; nitroglycerin; proteasomal degradation; vascular relaxation; vitamin C
19.  Tolerance to nitroglycerin through proteasomal down-regulation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency 
British Journal of Pharmacology  2013;168(8):1868-1877.
Background and Purpose
L-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation.
Experimental Approach
Ascorbate deficiency was induced in Gulo(-/-) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo(-/-), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined.
Key Results
Like GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo(-/-) mice with the 26S proteasome inhibitor bortezomib.
Conclusions and Implications
Our data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.
PMCID: PMC3623057  PMID: 23194305
aldehyde dehydrogenase-2; ascorbate deficiency; bortezomib; genetic mouse model; nitrate tolerance; nitroglycerin; proteasomal degradation; vascular relaxation; vitamin C
20.  Multiple and Additive Functions of ALDH3A1 and ALDH1A1: CATARACT PHENOTYPE AND OCULAR OXIDATIVE DAMAGE IN Aldh3a1(−/−)/Aldh1a1(−/−) KNOCK-OUT MICE*S 
The Journal of biological chemistry  2007;282(35):25668-25676.
ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(−/−)/Aldh3a1(−/−) double knock-out and Aldh1a1(−/−) and Aldh3a1(−/−) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6–9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(−/−) and Aldh3a1(−/−)/Aldh1a1(−/−) mice compared with Aldh1a1(−/−) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.
PMCID: PMC2253645  PMID: 17567582
21.  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 the present 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.
PMCID: PMC3112362  PMID: 21216231
ALDH1B1; epithelial cancer; colon cancer; cancer stem cell; biomarker
22.  Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system 
Science translational medicine  2014;6(255):255ra130.
Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.
PMCID: PMC4215699  PMID: 25253673
23.  ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance 
Science translational medicine  2011;3(107):107ra111.
Nitroglycerin, which helps impaired cardiac function as it is converted to nitric oxide, is used worldwide to treat patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction (MI). Here we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent MI. In a rat model of MI, 16 hours of prior, sustained nitroglycerin treatment (7.2 mg/kg/day) resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the MI. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 (16 mg/kg/day) with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after MI in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect MI patients from this nitroglycerin-induced increase in cardiac injury, while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin.
PMCID: PMC3547591  PMID: 22049071
24.  Partially Irreversible Inactivation of Mitochondrial Aldehyde Dehydrogenase by Nitroglycerin*S⃞ 
The Journal of Biological Chemistry  2008;283(45):30735-30744.
Mitochondrial aldehyde dehydrogenase (ALDH2) may be involved in the biotransformation of glyceryl trinitrate (GTN), and the inactivation of ALDH2 by GTN may contribute to the phenomenon of nitrate tolerance. We studied the GTN-induced inactivation of ALDH2 by UV/visible absorption spectroscopy. Dehydrogenation of acetaldehyde and hydrolysis of p-nitrophenylacetate (p-NPA) were both inhibited by GTN. The rate of inhibition increased with the GTN concentration and decreased with the substrate concentration, indicative of competition between GTN and the substrates. Inactivation of p-NPA hydrolysis was greatly enhanced in the presence of NAD+, and, to a lesser extent, in the presence of NADH. In the presence of dithiothreitol (DTT) inactivation of ALDH2 was much slower. Dihydrolipoic acid (LPA-H2) was less effective than DTT, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. When DTT was added after complete inactivation, dehydrogenase reactivation was quite modest (≤16%). The restored dehydrogenase activity correlated inversely with the GTN concentration but was hardly affected by the concentrations of acetaldehyde or DTT. Partial reactivation of dehydrogenation was also accomplished by LPA-H2 but not by GSH. We conclude that, in addition to the previously documented reversible inhibition by GTN that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to ALDH inactivation. Importantly, ALDH2-catalyzed GTN reduction was partly inactivated by preincubation with GTN, suggesting that the inactivation of GTN reduction is also partly irreversible. These observations are consistent with a significant role for irreversible inactivation of ALDH2 in the development of nitrate tolerance.
PMCID: PMC2576553  PMID: 18786921
25.  Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity 
Chemico-Biological Interactions  2011;191(1-3):269-277.
Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H2O2) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation.
PMCID: PMC3387551  PMID: 21338592
Aldehyde dehydrogenase 7A1; ALDH7A1; Antiquitin; Oxidative stress; 4HNE; 4-Hydroxynonenal

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