ALDH2 Activity Research Articles

Abstract 699: Mitochondrial Aldehyde Dehydrogenase (aldh-2) Protects Against Lipopolysaccharide-induced Myocardial Contractile Dysfunction by Suppression of ER Stress via Regulation of Autophagy

Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria, is the principal culprit factor responsible for the development of multi-organ failure including septic heart failure - a major contributor to increased mortality in patients with sepsis. However, there is still no efficacious treatment for septic cardiomyophathy. ALDH2 is well-known for its distinct beneficial role in cardiac pathologies including heart failure, ischemia and reperfusion injury. Little is known with regards to its effect on septic cardiomyopathy and the underlying mechanism. This study was designed to examine whether ALDH2 affects LPS-induced myocardial dysfunction and the underlying mechanism involved with a focus on ER stress and autophagy. WT and ALDH2 transgenic mice were subjected to LPS (4 mg/kg, 4 h). Myocardial mechanical and intracellular Ca2+ properties were examined in FVB wild-type and ALDH2 transgenic mice using echocardiography and IonOptix SoftEdge techniques. Protein markers for ER stress, autophagy and related signaling molecules were evaluated. LPS compromised cardiac contractile function shown as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca2+ homeostasis, associated with overt ER stress, upregulated autophagy, suppressed phosphorylation of Akt and its downstream signal molecule mTOR, the effects of which were attenuated by ALDH2. In vitro study revealed that the ER stress inducer tunicamycin exacerbated LPS-induced myocardial dysfunction, which was abrogated by the ALDH2 activator Alda-1 and the autophagy inhibitor 3-MA. Interestingly, the beneficial effect of Alda-1 was obliterated by the autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. We conclude that ALDH2 protects against LPS-induced myocardial dysfunction possibly through suppression of ER stress and inhibition of autophagy.

652 Prevalence and Significance of Elevated Serum IgG4 Levels in Primary Sclerosing Cholangitis

A S L D A b st ra ct s further investigated whether activation of ALDH2 using the novel small molecule, Alda-1, attenuates mitochondrial dysfunction and liver injury after chronic ethanol exposure. Methods: Male C57BL/6 mice were fed a liquid diet containing 27% of calories from ethanol and injected with Alda-1 (50 mg/kg, i.p. daily) or vehicle for 3 weeks. Results: Adducts of malondialdehyde-acetaldehyde (MAA) formed from acetaldehyde increased in liver tissue after ethanol feeding, which Alda-1 decreased, consistent with enhancement of acetaldehyde degradation by Alda-1. Chronic ethanol consumption caused mitochondrial depolarization in ~40% of hepatocytes as revealed by intravital confocal/multiphoton microscopy, which Alda-1 partially prevented. Ethanol increased serum ALT from 30 U/L to ~110 U/L, which Alda-1 treatment decreased to 53 U/L. Chronic ethanol exposure caused hepatic steatosis, as indicated by wide-spread Oil-Red-O-stained fat droplets and increased hepatic triglycerides. Alda-1 treatment decreased fat droplets and triglycerides in the liver after ethanol consumption. By H&E histology, hepatocyte ballooning, accumulation of macroand microvesicular fat droplets, necrosis, apoptosis and white blood cell infiltration developed after chronic ethanol exposure. TUNEL-positive cells also increased in association with caspase3 activation, indicating apoptosis. Myeloperoxidase and F4/80 increased markedly in liver tissue, suggesting neutrophil and monocyte/macrophage infiltration. Alda-1 decreased these markers of cell death and inflammation. Conclusion: Chronic ethanol consumption causes mitochondrial dysfunction and steatohepatitis in vivo, at least in part, due to production of toxic acetaldehydes. Activation ofmitochondrial ALDH2 decreasesmitochondrial dysfunction and steatohepatitis and is a promising strategy to prevent/minimize liver injury from chronic ethanol exposure (NIAAA, NIDDK & Charleston Alcohol Research Center).

Effects of ALDH2 genotype, PPI treatment and L-cysteine on carcinogenic acetaldehyde in gastric juice and saliva after intragastric alcohol administration.

Acetaldehyde (ACH) associated with alcoholic beverages is Group 1 carcinogen to humans (IARC/WHO). Aldehyde dehydrogenase (ALDH2), a major ACH eliminating enzyme, is genetically deficient in 30–50% of Eastern Asians. In alcohol drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive tract cancers, i.e., head and neck cancer and esophageal cancer. However, there is only a limited evidence for stomach cancer. In this study we demonstrated for the first time that ALDH2 deficiency results in markedly increased exposure of the gastric mucosa to acetaldehyde after intragastric administration of alcohol. Our finding provides concrete evidence for a causal relationship between acetaldehyde and gastric carcinogenesis. A plausible explanation is the gastric first pass metabolism of ethanol. The gastric mucosa expresses alcohol dehydrogenase (ADH) enzymes catalyzing the oxidation of ethanol to acetaldehyde, especially at the high ethanol concentrations prevailing in the stomach after the consumption of alcoholic beverages. The gastric mucosa also possesses the acetaldehyde-eliminating ALDH2 enzyme. Due to decreased mucosal ALDH2 activity, the elimination of ethanol-derived acetaldehyde is decreased, which results in its accumulation in the gastric juice. We also demonstrate that ALDH2 deficiency, proton pump inhibitor (PPI) treatment, and L-cysteine cause independent changes in gastric juice and salivary acetaldehyde levels, indicating that intragastric acetaldehyde is locally regulated by gastric mucosal ADH and ALDH2 enzymes, and by oral microbes colonizing an achlorhydric stomach. Markedly elevated acetaldehyde levels were also found at low intragastric ethanol concentrations corresponding to the ethanol levels of many foodstuffs, beverages, and dairy products produced by fermentation. A capsule that slowly releases L-cysteine effectively eliminated acetaldehyde from the gastric juice of PPI-treated ALDH2-active and ALDH2-deficient subjects. These results provide entirely novel perspectives for the prevention of gastric cancer, especially in established risk groups.

Discovery of a series of aromatic lactones as ALDH1/2-directed inhibitors

In humans, the aldehyde dehydrogenase superfamily consists of 19 isoenzymes which mostly catalyze the NAD(P)+-dependent oxidation of aldehydes. Many of these isoenzymes have overlapping substrate specificities and therefore their potential physiological functions may overlap. Thus the development of new isoenzyme-selective probes would be able to better delineate the function of a single isoenzyme and its individual contribution to the metabolism of a particular substrate. This specific study was designed to find a novel modulator of ALDH2, a mitochondrial ALDH isoenzyme most well-known for its role in acetaldehyde oxidation. 53 compounds were initially identified to modulate the activity of ALDH2 by a high-throughput esterase screen from a library of 63,000 compounds. Of these initial 53 compounds, 12 were found to also modulate the oxidation of propionaldehyde by ALDH2. Single concentration measurements at 10μM compound were performed using ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and/or ALDH5A1 to determine the selectivity of these 12 compounds toward ALDH2. Four of the twelve compounds shared an aromatic lactone structure and were found to be potent inhibitors of the ALDH1/2 isoenzymes, but have no inhibitory effect on ALDH3A1, ALDH4A1 or ALDH5A1. Two of the aromatic lactones show selectivity within the ALDH1/2 class, and one appears to be selective for ALDH2 compared to all other isoenzymes tested.

Genetic Polymorphisms in ADH1B and ALDH2 Are Associated with Colorectal Tumors in Japan: A Case-Control Study

Background: Unlike with esophageal cancer, acetaldehyde levels and genetic polymorphisms in alcohol dehydrogenase have not yet been shown to be contributing factors for colorectal cancer (CRC). This study aimed to clarify the mechanism of CRC development related to alcohol consumption and to the presence of genetic polymorphisms in the alcohol dehydrogenase, ADH1B and aldehyde dehydrogenase, ALDH2. Methods: This was a case-control study (221 cases and 179 controls) in patients with adenomas and intramucosal tumors who underwent endoscopic removal of all tumors. The amount of alcohol consumption was determined using a self-recorded questionnaire, and the tumor information was obtained from colonoscopy results. Blood samples were taken to analyze the following polymorphisms: ALDH2 Glu504Lys and ADH1B His48Arg. Results: The polymorphisms in ADH1B and ALDH2 had little influence on the development of colorectal adenoma or intramucosal cancer. Patients with ALDH2 (Glu/Glu) were more tolerant of alcohol than those with ALDH2 (Glu/Lys and Lys/Lys). Next, we examined certain combinations of the ADH1B genotypes. In the ALDH2 (Glu/Glu) group, an increased risk (OR = 3.4; 95% CI 1.4 - 8.4; P = 0.009) was observed among moderate/heavy drinkers with ADH1B (His/His). In the ALDH2 (Glu/Lys and Lys/Lys) group, an increased risk (OR = 4.2; 95% CI 1.1 - 16.7; P = 0.041) was found among moderate/heavy drinkers with ADH1B (Arg/His and Arg/Arg). Conclusions: ADH1B and ALDH2 activity may be involved in the development of CRC.

Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases.

Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP) of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer's disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations.

Bioavailability, plasma protein binding and metabolic stability studies of a ALDH2 activator, alda-1, using a validated LC-ESI-MS/MS method in rat plasma

Alda-1 was found to be a poorly bioavailable, 82–86% protein bound, high extraction compound.

Mitochondrial aldehyde dehydrogenase activation by Alda-1 inhibits atherosclerosis and attenuates hepatic steatosis in apolipoprotein E-knockout mice.

BackgroundMitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice.Methods and ResultsAlda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids.ConclusionsCollectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation.

Mitochondrial aldehyde dehydrogenase activity protects against lipopolysaccharide‑induced cardiac dysfunction in rats.

Myocardial dysfunction in sepsis is associated with an increased risk of mortality. The mitochondrial aldehyde dehydrogenase (ALDH2) enzyme is crucial for protecting the heart from ischemic injury. The aim of the present study was to determine the role of ALDH2 in cardiac dysfunction induced by lipopolysaccharide (LPS). Male rats were treated intraperitoneally with LPS, and their stroke volume and cardiac output were evaluated using an M‑mode echocardiograph. The expression levels and activity of ALDH2, nitric oxide content and inducible nitric oxide synthase (iNOS) activity, and the opening of the mitochondrial permeability transition pore (MPTP) were also evaluated. Treatment with LPS (5, 10, or 20 mg/kg) resulted in a steady decrease in cardiac output and stroke volume. The ALDH2 activity was decreased in a dose‑dependent manner; however, the ALDH2 protein expression levels remained unchanged. Alda‑1, a specific activator of ALDH2, increased the activity of ALDH2 and lessened LPS‑induced cardiac dysfunction. A marked opening of the MPTP was observed 12 h following treatment with LPS, which was prevented by Alda‑1. The improvement in cardiac function in response to treatment with Alda‑1, was partially prevented by treatment with the MPTP inhibitor atractyloside. LPS treatment induced an increase in iNOS activation and inhibition of ALDH2 activity. The iNOS selective inhibitor, aminoguanidine, partially reversed the LPS‑induced ALDH2 activity decrease and MPTP opening. These results indicate that ALDH2 activity may have a role in protecting against LPS‑induced cardiac dysfunction. The potential mechanism may involve inhibition of MPTP opening and iNOS expression.

Mitochondrial aldehyde dehydrogenase 2 plays protective roles in heart failure after myocardial infarction via suppression of the cytosolic JNK/p53 pathway in mice.

BackgroundIncreasing evidence suggests a critical role for mitochondrial aldehyde dehydrogenase 2 (ALDH2) in protection against cardiac injuries; however, the downstream cytosolic actions of this enzyme are largely undefined.Methods and ResultsProteomic analysis identified a significant downregulation of mitochondrial ALDH2 in the heart of a rat heart failure model after myocardial infarction. The mechanistic insights underlying ALDH2 action were elucidated using murine models overexpressing ALDH2 or its mutant or with the ablation of the ALDH2 gene (ALDH2 knockout) and neonatal cardiomyocytes undergoing altered expression and activity of ALDH2. Left ventricle dilation and dysfunction and cardiomyocyte death after myocardial infarction were exacerbated in ALDH2‐knockout or ALDH2 mutant‐overexpressing mice but were significantly attenuated in ALDH2‐overexpressing mice. Using an anoxia model of cardiomyocytes with deficiency in ALDH2 activities, we observed prominent cardiomyocyte apoptosis and increased accumulation of the reactive aldehyde 4‐hydroxy‐2‐nonenal (4‐HNE). We subsequently examined the impacts of mitochondrial ALDH2 and 4‐HNE on the relevant cytosolic protective pathways. Our data documented 4‐HNE‐stimulated p53 upregulation via the phosphorylation of JNK, accompanying increased cardiomyocyte apoptosis that was attenuated by inhibition of p53. Importantly, elevation of 4‐HNE also triggered a reduction of the cytosolic HSP70, further corroborating cytosolic action of the 4‐HNE instigated by downregulation of mitochondrial ALDH2.ConclusionsDownregulation of ALDH2 in the mitochondria induced an elevation of 4‐HNE, leading to cardiomyocyte apoptosis by subsequent inhibition of HSP70, phosphorylation of JNK, and activation of p53. This chain of molecular events took place in both the mitochondria and the cytosol, contributing to the mechanism underlying heart failure.

P-24 * GENE-GENE INTERACTION OF ALCOHOL METABOLIZING HEPATIC ENZYME GENE AND MU-OPIOID RECEPTOR GENE IN KOREAN PATIENTS WITH ALCOHOL DEPENDENCE

Introduction. Many previous studies tried to find etiologic genes of alcohol dependence. Among these genes, alcohol metabolizing hepatic enzyme genes (ADH2 and ALDH2) and mu-opioid receptor gene (OPRM1) have become a great interest recently. Accordingly, our purpose is to investigate gene-gene interaction of alcohol metabolizing hepatic enzyme gene and mu-opioid receptor gene in the Korean male and female patients with alcohol dependence. Methods. The subjects were 241 male alcohol-dependent patients, 60 female alcohol-dependent patients, 79 male normal controls, and 59 female normal controls. The frequencies of the ADH2 (1/1 and 2+), ALDH2 (1/1 and 2+), and OPRM1 All8G (A/A and G+) genotypes were investigated in male and female alcohol-dependent patients and the normal control group. Results. In female alcohol-dependent patients, who has ADH2 1/1 genotype, OPRM1 G+ genotype frequencies were significantly higher than the female normal control group. Furthermore, OPRM1 G+ genotype frequencies were significantly higher in the female alcohol-dependent patients who have ALDH2 2+ genotype, compared to the female normal control group. Conclusion. G+ genotype frequency in OPRM1 was higher only in female alcoholics, who had low activity of ADH2 or ALDH2. These results suggest that the cause of drinking in women is strongly related to inducing reward.

Abstract 57: Impaired Aldehyde Dehydrogenase 2 Activity Contributes To Inhibition Of Mitochondrial Respiration In Hyperglycemic Rats

Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial isozyme of the heart involved in the metabolism of toxic aldehydes produced from oxidative stress. A decrease in ALDH2 levels and activity was reported in the hearts of experimental hyperglycemia along with an increase in 4-hydroxy-2-nonenal (4HNE). 4HNE is produced in the mitochondria upon lipid peroxidation and known to form adduct with mitochondrial proteins. We hypothesize that reduced ALDH2 activity impairs mitochondrial respiration due to 4HNE adducts formation, ultimately resulting in cardiac damage in hyperglycemia. A single dose (65 mg/kg; i.p.) of streptozotocin in rats resulted in hyperglycemia with a blood glucose level of 443 ± 9 mg/dl versus 121 ± 7 mg/dl in control animals, p<0.0001. N=7-11 in both groups. After 6 months of hyperglycemia, heart function was recorded and the rats were sacrificed. Increases in cardiomyocyte cross sectional area (446 ± 32 μm 2 Vs 221 ± 10 μm 2 ; p<0.0001) indicated cardiac hypertrophy in diabetic rats. Both diastolic and systolic dysfunctions were recorded with diabetic rats compared to controls. For instance, % fractional shortening was lower in diabetic group versus control; (p<0.0001). Most importantly, myocardial ALDH2 activity and levels were reduced and immunostaining for 4HNE protein adducts was increased in diabetic hearts compared to controls. The mitochondrial oxygen consumption rate (OCR), an index of mitochondrial respiration, was decreased in mitochondria isolated from diabetic hearts compared to controls (p<0.0001). To check the direct effect of 4HNE and ALDH2 inhibition, we treated H9C2 cardiomyocytes with 4HNE and disufiram, an ALDH inhibitor, respectively, which reduced mitochondrial respiration (p<0.0001) and cell viability (p<0.0001) along with reduced ALDH2 activity and increased 4HNE adducts formation. We conclude that chronic hyperglycemia-induced 4HNE accumulation due to low ALDH2 activity in the heart leads to defective mitochondrial respiration and cardiac damage and dysfunction.

A novel protective mechanism for mitochondrial aldehyde dehydrogenase (ALDH2) in type i diabetes-induced cardiac dysfunction: Role of AMPK-regulated autophagy

Mitochondrial aldehyde dehydrogenase (ALDH2) is known to offer myocardial protection against stress conditions including ischemia-reperfusion injury, alcoholism and diabetes mellitus although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on diabetes-induced myocardial injury with a focus on autophagy. Wild-type FVB and ALDH2 transgenic mice were challenged with streptozotozin (STZ, 200mg/kg, i.p.) for 3months to induce experimental diabetic cardiomyopathy. Diabetes triggered cardiac remodeling and contractile dysfunction as evidenced by cardiac hypertrophy, decreased cell shortening and prolonged relengthening duration, the effects of which were mitigated by ALDH2. Lectin staining displayed that diabetes promoted cardiac hypertrophy, the effect of which was alleviated by ALDH2. Western blot analysis revealed dampened autophagy protein markers including LC3B ratio and Atg7 along with upregulated p62 following experimental diabetes, the effect of which was reconciled by ALDH2. Phosphorylation level of AMPK was decreased and its downstream signaling molecule FOXO3a was upregulated in both diabetic cardiac tissue and in H9C2 cells with high glucose exposure. All these effect were partly abolished by ALDH2 overexpression and ALDH2 agonist Alda1. High glucose challenge dampened autophagy in H9C2 cells as evidenced by enhanced p62 levels and decreased levels of Atg7 and LC3B, the effect of which was alleviated by the ALDH2 activator Alda-1. High glucose-induced cell death and apoptosis were reversed by Alda-1. The autophagy inhibitor 3-MA and the AMPK inhibitor compound C mitigated Alda-1-offered beneficial effect whereas the autophagy inducer rapamycin mimicked or exacerbated high glucose-induced cell injury. Moreover, compound C nullified Alda-1-induced protection against STZ-induced changes in autophagy and function. Our results suggested that ALDH2 protects against diabetes-induced myocardial dysfunction possibly through an AMPK -dependent regulation of autophagy. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

(13) C magnetic resonance spectroscopic imaging of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation can be used to assess aldehyde dehydrogenase activity in vivo.

Aldehyde dehydrogenase (ALDH2) is an emerging drug target for the treatment of heart disease, cocaine and alcohol dependence, and conditions caused by genetic polymorphisms in ALDH2. Noninvasive measurement of ALDH2 activity in vivo could inform the development of these drugs and accelerate their translation to the clinic. [1-(13) C, U-(2) H5 ] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using (13) C MR spectroscopy and spectroscopic imaging. Oxidation of [1-(13) C, U-(2) H5 ] ethanol to [1-(13) C] acetate was observed. Saturation of the acetaldehyde resonance, which was below the level of detection in vivo, demonstrated that acetate was produced via acetaldehyde. Irreversible inhibition of ALDH2 activity with disulfiram resulted in a proportional decrease in the amplitude of the acetate resonance. (13) C magnetic resonance spectroscopy measurements of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation allow real-time assessment of ALDH2 activity in liver in vivo.

Oxidative stress and autonomic dysregulation contribute to the acute time-dependent myocardial depressant effect of ethanol in conscious female rats.

The molecular mechanisms of the acute hypotensive and indirectly assessed cardiac depressant effect of ethanol (EtOH)-evoked myocardial depression and hypotension in female rats are not known. We tested the hypothesis that a time-dependent myocardial depression caused by EtOH is initiated by its direct and indirect (cardiac vagal dominance) effects and is exacerbated by gradual development of oxidative stress. In conscious female rats, we directly measured left ventricular developed pressure (LVDP), the maximal rise of ventricular pressure over time (dP/dtmax ), blood pressure (BP), heart rate (HR), and sympathovagal activity following intragastric EtOH (1 g/kg) or water over 90 minutes. Catalytic activity of acetaldehyde (ACA)-generating (alcohol dehydrogenase [ADH] and catalase) and eliminating aldehyde dehydrogenase [ALDH2] enzymes along with mediators of oxidative stress were measured in myocardial tissues collected at 30, 60, or 90 minutes after EtOH or water. EtOH reduced myocardial function (LVDP and dP/dtmax ) within 5 to 10 minutes before the steady fall in BP in conscious proestrus rats. Further, EtOH shifted the sympathovagal balance, analyzed by spectral analysis of high frequency and low frequency of interbeat intervals, toward vagal dominance. Prior vagal blockade (atropine) or antioxidant (tempol) treatment attenuated EtOH-evoked myocardial depression and hypotension. Ex vivo studies revealed time-dependent: (i) enhancement of ADH, but not ALDH2 activity (indicative of elevated ACA levels), (ii) increases in phosphorylated Akt and ERK1/2, NADPH-oxidase activity, reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal-modified proteins. These molecular responses along with reduced myocardial catalase activity were most evident at 90 minutes post-EtOH when the reductions in cardiac function and BP reached their nadir. Vagal dominance and time-dependent myocardial oxidative stress along with the accumulation of cardiotoxic aldehydes mediate EtOH-evoked myocardial dysfunction and hypotension in conscious proestrus female rats.

Impairment of aldehyde dehydrogenase-2 by 4-hydroxy-2-nonenal adduct formation and cardiomyocyte hypertrophy in mice fed a high-fat diet and injected with low-dose streptozotocin

Reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are generated in the myocardium in cardiac disease. 4HNE and other toxic aldehydes form adducts with proteins, leading to cell damage and organ dysfunction. Aldehyde dehydrogenases (ALDHs) metabolize toxic aldehydes such as 4HNE into nontoxic metabolites. Both ALDH levels and activity are reduced in cardiac disease. We examined whether reduced ALDH2 activity contributes to cardiomyocyte hypertrophy in mice fed a high-fat diet and injected with low-dose streptozotocin (STZ). These mice exhibited most of the characteristics of metabolic syndrome/type-2 diabetes mellitus (DM): increased blood glucose levels depicting hyperglycemia (415.2 ± 18.7 mg/dL vs. 265.2 ± 7.6 mg/dL; P < 0.05), glucose intolerance with normal plasma insulin levels, suggesting insulin resistance and obesity as evident from increased weight (44 ± 3.1 vs. 34.50 ± 1.32 g; P < 0.05) and body fat. Myocardial ALDH2 activity was 60% lower in these mice (0.1 ± 0.012 vs. 0.04 ± 0.015 µmol/min/mg protein; P < 0.05). Myocardial 4HNE levels were also elevated in the hyperglycemic hearts. Co-immunoprecipitation study showed that 4HNE formed adducts on myocardial ALDH2 protein in the mice exhibiting metabolic syndrome/type-2 DM, and they had obvious cardiac hypertrophy compared with controls as evident from increased heart weight (HW), HW to tibial length ratio, left ventricular (LV) mass and cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy was correlated inversely with ALDH2 activity (R (2 )= 0.7; P < 0.05). Finally, cardiac dysfunction was observed in mice with metabolic syndrome/type-2 DM. Therefore, we conclude that reduced ALDH2 activity may contribute to cardiac hypertrophy and dysfunction in mice presenting with some of the characteristics of metabolic syndrome/type-2 DM when on a high-fat diet and low-dose STZ injection.

The Thiocarbamate Disulphide Drug, Disulfiram Induces Osteopenia in Rats by Inhibition of Osteoblast Function Due to Suppression of Acetaldehyde Dehydrogenase Activity

Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function.

Aldehyde dehydrogenase 2 ameliorates doxorubicin-induced myocardial dysfunction through detoxification of 4-HNE and suppression of autophagy

Mitochondrial aldehyde dehydrogenase (ALDH2) protects against cardiac injury via reducing production of 4-hydroxynonenal (4-HNE) and ROS. This study was designed to examine the impact of ALDH2 on doxorubicin (DOX)-induced cardiomyopathy and mechanisms involved with a focus on autophagy. 4-HNE and autophagic markers were detected by Western blotting in ventricular tissues from normal donors and patients with idiopathic dilated cardiomyopathy. Cardiac function, 4-HNE and levels of autophagic markers were detected in WT, ALDH2 knockout or ALDH2 transfected mice treated with or without DOX. Autophagy regulatory signaling including PI-3K, AMPK and Akt was examined in DOX-treated cardiomyocytes incubated with or without ALDH2 activator Alda-1. DOX-induced myocardial dysfunction, upregulation of 4-HNE and autophagic proteins were further aggravated in ALDH2 knockout mice while they were ameliorated in ALDH2 transfected mice. DOX downregulated Class I and upregulated Class III PI3-kinase, the effect of which was augmented by ALDH2 deletion. Accumulation of 4-HNE and autophagic protein markers in DOX-induced cardiomyocytes was significantly reduced by Alda-1. DOX depressed phosphorylated Akt but not AMPK, the effect was augmented by ALDH2 knockout. The autophagy inhibitor 3-MA attenuated, whereas autophagy inducer rapamycin mimicked DOX-induced cardiomyocyte contractile defects. In addition, rapamycin effectively mitigated Alda-1-offered protective action against DOX-induced cardiomyocyte dysfunction. Our data further revealed downregulated ALDH2 and upregulated autophagy levels in the hearts from patients with dilated cardiomyopathy. Taken together, our findings suggest that inhibition of 4-HNE and autophagy may be a plausible mechanism underscoring ALDH2-offered protection against DOX-induced cardiac defect. This article is part of a Special Issue entitled “Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy”.

Selective ALDH3A1 Inhibition by Benzimidazole Analogues Increase Mafosfamide Sensitivity in Cancer Cells

Aldehyde dehydrogenase enzymes irreversibly oxidize aldehydes generated from metabolism of amino acids, fatty acids, food, smoke, additives, and xenobiotic drugs. Cyclophosphamide is one such xenobiotic used in cancer therapies. Upon activation, cyclophosphamide forms an intermediate, aldophosphamide, which can be detoxified to carboxyphosphamide by aldehyde dehydrogenases (ALDH), especially ALDH1A1 and ALDH3A1. Consequently, selective inhibition of ALDH3A1 could increase chemosensitivity toward cyclophosphamide in ALDH3A1 expressing tumors. Here, we report detailed kinetics and structural characterization of a highly selective submicromolar inhibitor of ALDH3A1, 1-[(4-fluorophenyl)sulfonyl]-2-methyl-1H-benzimidazole (CB7, IC50 of 0.2 μM). CB7 does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 activity. Structural, kinetics, and mutagenesis studies show that CB7 binds to the aldehyde binding pocket of ALDH3A1. ALDH3A1-expressing lung adenocarcinoma and glioblastoma cell lines are sensitized toward mafosfamide (MF) treatment in the presence analogues of CB7, whereas primary lung fibroblasts lacking ALDH3A1 expression, are not.

A patient with rapid growth of superficial-type poorly differentiated hypopharyngeal squamous cell carcinoma

An 81-year-old man was diagnosed as having cStage IVa esophageal cancer and treated by chemoradiation therapy. His treatment response to the CRT was classified as CR. Then, 34 months after the CRT, he was detected to have a small elevated lesion with abnormal blood vessels coursing over it in the rt pyriform sinus, with the lesion showing rapid increase in size to a clearly elevated lesion over the next 6 months. The surface of the lesion was covered by normal mucosa and blood vessels (Type B2) could be seen coursing over it by magnifying endoscopy with NBI. We diagnosed the lesion as a superficial hypopharyngeal cancer (Type 0-I+IIa) and performed endoscopic laryngo-pharyngeal surgery. The pathological diagnosis was poorly differentiated squamous cell carcinoma with ly1, v1, and prominent subepithelial invasion. Pharyngeal cancer is often found synchronously or metachronously in patients with esophageal cancer, presumably as a result of the shared risk factors between the two cancers, such as alcohol drinking, smoking and deficient ALDH2 activity. Endoscopic examination to detect any pharyngeal cancer must be performed in esophageal cancer patients with the aforementioned risk factors. Diagnosis of a pharyngeal elevated lesion is often difficult due to the presence of the tonsils and lymphoid follicles. Therefore, careful magnifying endoscopy with NBI should always be performed to confirm the presence/absence of blood vessels coursing over small and slightly elevated lesions.