Aldehyde Dehydrogenase 2 Deficiency Aggravates Lung Fibrosis through Mitochondrial Dysfunction and Aging in Fibroblasts.

Idiopathic pulmonary fibrosis, a fatal interstitial lung disease, is characterized by fibroblast activation and aberrant extracellular matrix accumulation. Effective therapeutic development is limited because of incomplete understanding of the mechanisms by which fibroblasts become aberrantly activated. Here, we show aldehyde dehydrogenase 2 (ALDH2) in fibroblasts as a potential therapeutic target for pulmonary fibrosis. A decrease in ALDH2 expression was observed in patients with idiopathic pulmonary fibrosis and bleomycin-treated mice. ALDH2 deficiency spontaneously induces collagen accumulation in the lungs of aged mice. Furthermore, young ALDH2 knockout mice exhibited exacerbated bleomycin-induced pulmonary fibrosis and increased mortality compared with that in control mice. Mechanistic studies revealed that transforming growth factor (TGF)-ß1 induction and ALDH2 depletion constituted a positive feedback loop that exacerbates fibroblast activation. TGF-ß1 down-regulated ALDH2 through a TGF-ß receptor 1/Smad3-dependent mechanism. The subsequent deficiency in ALDH2 resulted in fibroblast dysfunction that manifested as impaired mitochondrial autophagy and senescence, leading to fibroblast activation and extracellular matrix production. ALDH2 overexpression markedly suppressed fibroblast activation, and this effect was abrogated by PTEN-induced putative kinase 1 (PINK1) knockdown, indicating that the profibrotic effects of ALDH2 are PINK1- dependent. Furthermore, ALDH2 activated by N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1) reversed the established pulmonary fibrosis in both young and aged mice. In conclusion, ALDH2 expression inhibited the pathogenesis of pulmonary fibrosis. Strategies to up-regulate or activate ALDH2 expression could be potential therapies for pulmonary fibrosis.

To determine the frequency of aldehyde dehydrogenase type 2 (aldh2) deficiency in aplastic anaemia: A single center experience from pakistan.

BACKGROUND: Aplastic anaemia is a rare bone marrow failure syndrome and is defined by pancytopenia associated with a hypo-cellular bone marrow with no increase in reticulin and the absence of any abnormal infiltrate. The objective of the study was to determine the frequency of Aldehyde Dehydrogenase type 2 (ALDH2) deficiency in patients with Aplastic Anaemia and investigate its correlation with patient and disease characteristics. It was a descriptive cross-sectional study conducted at Armed Forces Bone Marrow Transplant Centre Rawalpindi from 01-08-2022-01-02-2023, over 6 months. METHODS: A total of 56 patients who were diagnosed with aplastic anaemia during this period, fulfilling inclusion criteria were enrolled. Patients were genotyped as GG (homozygous) and GA (heterozygous). GG had normal ALDH2, while GA were patients with ALDH2 deficiency. Data was collected on the patient's demographics, type and severity of anaemia, type of hematopoietic stem cell transplant (HSCT) and frequency of ALDH2 deficiency. Results were analyzed for ALDH2 deficiency and its correlation with patient and disease characteristics was investigated. RESULTS: A total of 56 patients were included in the study. The median age of the patients was 28 years (20-39). According to the type of aplastic anaemia, 2 (3.6%) had Fanconi anaemia and 54 (96.4%) had acquired aplastic anaemia. In our study, 18 (32.1%) patients had undergone HSCT while the remaining 38 (67.9%) could not undergo HSCT. The frequency of the presence of ALDH2 deficiency was 2 (3.6%). There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant. CONCLUSIONS: We concluded from our study the frequency of ALDH2 was rare in patients with aplastic anaemia. There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant.

Intake of Lactobacillus rhamnosus GG (LGG) fermented milk before drinking alcohol reduces acetaldehyde levels and duration of flushing in drinkers with wild-type and heterozygous mutant ALDH2: a randomized, blinded crossover controlled trial.

Alcohol consumption leads to acetaldehyde accumulation, especially in people with mutant aldehyde dehydrogenase 2 gene (ALDH2). Novel strategies to promote acetaldehyde detoxification are required to prevent alcohol-related toxicity. Probiotic bacteria such as Lactobacillus rhamnosus GG (LGG) were shown to have in vitro capacity to detoxify acetaldehyde. This randomized, blinded, placebo-controlled cross-over trial investigated the effect of LGG fermented milk in people with ALDH2 polymorphisms after moderate alcohol intake. Ten healthy wild-type and ten heterozygous mutant ALDH2 Thai men were block randomized into two groups. Each group consumed a different sequence of 150 mL fermented milk containing 108 CFU mL-1 LGG and lactic-acidified milk (placebo), followed by five glasses of beer (0.4 g ethanol per kg body weight), with a one-week wash-out. Consuming LGG fermented milk before alcohol reduced areas under the response curves of blood and salivary acetaldehyde in wild-type and heterozygous mutant ALDH2 individuals (p < 0.05 and p < 0.01, respectively). Interestingly, participants with mutant ALDH2 responded better than wild-type participants for salivary acetaldehyde (90% vs. 70%, p < 0.001). Their durations of flushing were reduced when consuming LGG milk. Regardless of ALDH2 status, 105 CFU mL-1 LGG was retained in saliva at least 3.5 h after milk consumption. In conclusion, intake of LGG fermented milk before drinking alcohol reduces blood and salivary acetaldehyde levels and duration of flushing in drinkers with wild-type and heterozygous mutant ALDH2. The addition of exogenous capacity to detoxify acetaldehyde using the probiotic product could be a potential strategy to promote the alleviation of exposure to reactive and carcinogenic acetaldehyde associated with alcohol drinking in individuals with defective ALDH2 enzyme.

Interplay between APC and ALDH1B1 in a newly developed mouse model of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. Mutations in the adenomatous polyposis coli (APC) gene are pivotal in colorectal tumorigenesis. Recently, we demonstrated that aldehyde dehydrogenase 1B1 (ALDH1B1) knockdown dramatically reduced colon tumor growth in a mouse xenograft model. The purpose of the present preliminary study is to examine the effect of loss of ALDH1B1 in CRC development in an inducible colon-specific Apc mouse model. METHODS: ApcW/FCdx2ERT2-Cre mice develop uni-allelic inactivation of Apc specifically in colon epithelial cells following tamoxifen treatment. Aldh1b1-/- KO mice were crossed with ApcW/FCdx2ERT2-Cre mice. Six-month-old male ApcW/FCdx2ERT2-Cre/Aldh1b1-/-, and ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice were treated with tamoxifen (50 mg/kg, i.p.) for three consecutive days. ApcW/F/Aldh1b1-/- and ApcW/F/Aldh1b1+/+ mice were treated with corn oil (i.e., tamoxifen vehicle control) for three consecutive days. Eighteen days later, mice were sacrificed and their colons examined microscopically, macroscopically and histologically for the presence of adenoma. RESULTS: All ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ and ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice treated with tamoxifen developed colorectal adenoma. The ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice showed a significant decrease in the total volume of all ileal and colonic adenomas, and decreased incidence of large colonic adenoma compared to ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice. Immunohistochemical analysis of p53 and ß-catenin showed a trend toward decreased expression score in colonic adenomas of ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice. CONCLUSION: The present preliminary study suggests that deletion of ALDH1B1 may protect against the full development of colorectal cancer. Further mechanistic studies are required to elucidate how ALDH1B1 contributes for colorectal cancer.

Neuronal Calcium Imaging, Excitability, and Plasticity Changes in the Aldh2-/- Mouse Model of Sporadic Alzheimer's Disease.

BACKGROUND: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer's disease brain, including increases in amyloid-ß, hyperphosphorylated tau, inflammation, calcium dysregulation, and oxidative stress. These are often noted prior to the development of behavioral, cognitive, and non-cognitive deficits. However, the extent to which these pathological changes function together or independently is unclear. OBJECTIVE: Little is known about the temporal relationship between calcium dysregulation and oxidative stress, as some reports suggest that dysregulated calcium promotes increased formation of reactive oxygen species, while others support the opposite. Prior work has quantified several key outcome measures associated with oxidative stress in aldehyde dehydrogenase 2 knockout (Aldh2-/-) mice, a non-transgenic model of sporadic Alzheimer's disease. METHODS: Here, we tested the hypothesis that early oxidative stress can promote calcium dysregulation across aging by measuring calcium-dependent processes using electrophysiological and imaging methods and focusing on the afterhyperpolarization (AHP), synaptic activation, somatic calcium, and long-term potentiation in the Aldh2-/- mouse. RESULTS: Our results show a significant age-related decrease in the AHP along with an increase in the slow AHP amplitude in Aldh2-/- animals. Measures of synaptic excitability were unaltered, although significant reductions in long-term potentiation maintenance were noted in the Aldh2-/- animals compared to wild-type. CONCLUSION: With so few changes in calcium and calcium-dependent processes in an animal model that shows significant increases in HNE adducts, Aß, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.

Investigating the health disparities in the association between lifestyle behaviors and the risk of head and neck cancer.

Many studies have reported a positive association between lower socioeconomic status (SES) and higher head and neck cancer (HNC) risk. Fewer studies have examined the impact of SES on the association between alcohol or cigarette use and HNC risk. The current case-control study (1104 HNC cases and 1363 controls) investigated the influence of education, a SES indicator, on the association between HNC and the use of alcohol, cigarettes, or betel quids in Taiwan, a country with universal health care. Our results showed a larger increase in HNC risk associated with alcohol among those with lower educational level (odds ratio [OR] = 2.07; 95% confidence interval [CI], 1.53-2.80) than those with higher educational level (OR = 1.38; 95% CI, 1.04-1.85) (heterogeneity-P = .03). Educational level had an influence on the association between alcohol use and HNC risk among those with genetic susceptibility (ALDH2-deficient) to the carcinogenic effect of alcohol. The association between cigarette or betel quid use and HNC risk was similar between the high and low educational groups. National policies and social interventions have led to the decline in the prevalence of cigarette and betel quid users in Taiwan. In contrast, due to the lack of adequate alcohol control policies, alcohol consumption in Taiwan has continued to rise. A higher impact of alcohol on HNC risk among lower SES individuals even with universal health care could be the result of insufficient alcohol control policies in Taiwan.

Aldehyde dehydrogenase 2 deficiency significantly exacerbates tert-butyl alcohol-induced toxicity in mice.

Owing to the use of ethyl tert-butyl ether (ETBE) as a fuel additive, the possible adverse effects of ETBE exposure have become a public concern. Our previous study showed that ETBE-induced toxicity in aldehyde dehydrogenase 2 (Aldh2) gene knockout (KO) mice was caused by its primary metabolite acetaldehyde, which was toxic. However, it is unclear whether tert-butyl alcohol (TBA), another main metabolite of ETBE, plays a role in ETBE-induced toxicity. To investigate this relationship, we analyzed the changes of TBA concentrations in tissues after ETBE exposure, and then evaluated the toxicity after direct TBA treatment in both KO and wild-type (WT) mice. An exposure to 500 ppm ETBE via inhalation resulted in the formation of its three metabolites, TBA, 2-methyl-1,2-propanediol and ethanol, whose concentrations in the liver, brain, fat and testis of male KO mice were significantly higher than the corresponding concentrations observed in male WT mice. Direct treatment to TBA (20 mg/mL of drinking water) caused significant changes in relative organ weights and histopathology, and increased levels of genetic damages in both types of mice. These toxic effects were also seen in KO mice exposed to a lower concentration of TBA (5 mg/mL), which was associated with increased oxidative stress in serum (reduced glutathione and reduced glutathione/oxidized glutathione ratio decreased). Our findings indicate that ALDH2 is involved in the metabolism of ETBE and TBA, and ALDH2 deficiency could greatly increase the sensitivity to TBA-induced toxicity.

Systemic Adeno-Associated Virus-Mediated Gene Therapy Prevents the Multiorgan Disorders Associated with Aldehyde Dehydrogenase 2 Deficiency and Chronic Ethanol Ingestion.

Aldehyde dehydrogenase type 2 (ALDH2), a key enzyme in ethanol metabolism, processes toxic acetaldehyde to nontoxic acetate. ALDH2 deficiency affects 8% of the world population and 35-45% of East Asians. The ALDH2*2 allele common genetic variant has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the oxidizing ability of the enzyme resulting in systemic accumulation of acetaldehyde with ethanol ingestion. With chronic ethanol ingestion, mutations in ALDH2 are associated with a variety of hematological, neurological, and dermatological abnormalities, and an increased risk for esophageal cancer and osteoporosis. Based on our prior studies demonstrating that a one-time administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) prevents the acute effects of ethanol administration (the "Asian flush syndrome"), we hypothesized that AAVrh.10hALDH2 would also prevent the chronic disorders associated with ALDH2 deficiency and chronic ethanol ingestion. To assess this hypothesis, AAVrh.10hALDH2 (1011 genome copies) was administered intravenously to two models of ALDH2 deficiency, Aldh2 knockout homozygous (Aldh2-/-) and knockin homozygous (Aldh2E487K+/+) mice (n = 10 per group). Four weeks after vector administration, mice were given drinking water with 10-15% ethanol for 12 weeks. Strikingly, compared with nonethanol drinking littermates, AAVrh.10hALDH2 administration prevented chronic ethanol-induced serum acetaldehyde accumulation and elevated liver malondialdehyde levels, loss of body weight, reduced hemoglobin levels, reduced performance in locomotor activity tests, accumulation of esophageal DNA damage and DNA adducts, and development of osteopenia. AAVrh.10hALDH2 should be considered as a preventative therapy for the increased risk of chronic disorders associated with ALDH2 deficiency and chronic alcohol exposure.

Acetaldehyde dehydrogenase 2 deficiency exacerbates cardiac fibrosis by promoting mobilization and homing of bone marrow fibroblast progenitor cells.

Cardiac fibrosis is a common feature of various cardiovascular diseases. Previous studies showed that acetaldehyde dehydrogenase 2 (ALDH2) deficiency exacerbated pressure overload-induced heart failure. However, the role and mechanisms of cardiac fibrosis in this process remain largely unknown. This study aimed to investigate the effect of ALDH2 deficiency on cardiac fibrosis in transverse aortic constriction (TAC) induced pressure overload model in mice. Echocardiography and histological analysis revealed cardiac dysfunction and enhanced cardiac fibrosis in TAC-operated animals; ALDH2 deficiency further aggravated these changes. ALDH2 chimeric mice were generated by bone marrow (BM) transplantation of WT mice into the lethally irradiated ALDH2KO mice. The proportion of circulating fibroblast progenitor cells (FPCs) and ROS level in BM after TAC were significantly higher in ALDH2KO mice than in ALDH2 chimeric mice. Furthermore, FPCs were isolated and cultured for in vitro mechanistic studies. The results showed that the stem cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4) axis played a major role in the recruitment of FPCs. In conclusion, our research reveals that increased bone marrow FPCs mobilization and myocardial homing contribute to the enhanced cardiac fibrosis and dysfunction induced by TAC in ALDH2 KO mice via exacerbating accumulation of ROS in BM and myocardial SDF-1 expression.

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation.

BACKGROUND: Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. METHODS: We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation. RESULTS: By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARß2) upon EtOH treatment of WT and Acss2-/- ESCs, but not Aldh2-/- ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation. CONCLUSIONS: ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.

ALDH2 deficiency promotes alcohol-associated liver cancer by activating oncogenic pathways via oxidized DNA-enriched extracellular vesicles.

BACKGROUND & AIMS: Excessive alcohol consumption is one of the major causes of hepatocellular carcinoma (HCC). Approximately 30-40% of the Asian population are deficient for aldehyde dehydrogenase 2 (ALDH2), a key enzyme that detoxifies the ethanol metabolite acetaldehyde. However, how ALDH2 deficiency affects alcohol-related HCC remains unclear. METHODS: ALDH2 polymorphisms were studied in 646 patients with viral hepatitis B (HBV) infection, who did or did not drink alcohol. A new model of HCC induced by chronic carbon tetrachloride (CCl4) and alcohol administration was developed and studied in 3 lines of Aldh2-deficient mice: including Aldh2 global knockout (KO) mice, Aldh2*1/*2 knock-in mutant mice, and liver-specific Aldh2 KO mice. RESULTS: We demonstrated that ALDH2 deficiency was not associated with liver disease progression but was associated with an increased risk of HCC development in cirrhotic patients with HBV who consumed excessive alcohol. The mechanisms underlying HCC development associated with cirrhosis and alcohol consumption were studied in Aldh2-deficient mice. We found that all 3 lines of Aldh2-deficient mice were more susceptible to CCl4 plus alcohol-associated liver fibrosis and HCC development. Furthermore, our results from in vivo and in vitro mechanistic studies revealed that after CCl4 plus ethanol exposure, Aldh2-deficient hepatocytes produced a large amount of harmful oxidized mitochondrial DNA via extracellular vesicles, which were then transferred into neighboring HCC cells and together with acetaldehyde activated multiple oncogenic pathways (JNK, STAT3, BCL-2, and TAZ), thereby promoting HCC. CONCLUSIONS: ALDH2 deficiency is associated with an increased risk of alcohol-related HCC development from fibrosis in patients and in mice. Mechanistic studies reveal a novel mechanism that Aldh2-deficient hepatocytes promote alcohol-associated HCC by transferring harmful oxidized mitochondrial DNA-enriched extracellular vesicles into HCC and subsequently activating multiple oncogenic pathways in HCC. LAY SUMMARY: Alcoholics with an ALDH2 polymorphism have an increased risk of digestive tract cancer development, however, the link between ALDH2 deficiency and hepatocellular carcinoma (HCC) development has not been well established. In this study, we show that ALDH2 deficiency exacerbates alcohol-associated HCC development both in patients and mouse models. Mechanistic studies revealed that after chronic alcohol exposure, Aldh2-deficient hepatocytes produce a large amount of harmful oxidized mitochondrial DNA via extracellular vesicles, which can be delivered into neighboring HCC cells and subsequently activate multiple oncogenic pathways, promoting HCC.

ALDH2 deficiency inhibits Ox-LDL induced foam cell formation via suppressing CD36 expression.

Foam cell formation plays an important role in the initiation and progression of atherosclerosis. Aldehyde dehydrogenase 2 (ALDH2), a key enzyme for aldehyde metabolism, is associated with coronary artery disease and affects atherosclerotic plaque vulnerability. However, the role of ALDH2 in foam cell formation remains unclear. Using peritoneal macrophages from ALDH2-deficient and control mice, we found that ALDH2 deficiency suppressed foam cell formation induced by oxidized low-density lipoproteins (ox-LDL) but not acetylated low-density lipoproteins (ac-LDL) ex vivo. After incubation with ox-LDL, ALDH2-deficient macrophages expressed lower levels of CD36 but the expression of other lipid metabolism-related proteins including SRA, LOX-1, ABCA-1, ABCG-1 and ACAT-1 was not changed in ALDH2-/- macrophages. Using CD36 inhibitor, we confirmed that CD36 contributes to the effect of ALDH2 on foam cell formation. PPARγ was downregulated in ox-LDL treated ALDH2-/- macrophages. 4-HNE was increased by ALDH2 deficiency and high concentration of 4-HNE suppressed the expression of PPARγ. These data suggest that ALDH2 plays an important role in foam cell formation via 4-HNE/PPARγ/CD36 pathway.

Ethanol induces skin hyperpigmentation in mice with aldehyde dehydrogenase 2 deficiency.

Alcohol induces various cutaneous changes, such as palmar erythema and jaundice. However, alcohol-induced skin hyperpigmentation due to melanin deposition has not been reported. Aldehyde dehydrogenase 2 (ALDH2), one of 19 human ALDH isozymes, metabolizes endogenous and exogenous aldehydes to their respective carboxylic acids. Reduced ALDH2 greatly affects acetaldehyde metabolism, leading to its accumulation in the body after the consumption of alcohol and the consequent development of a wide range of phenotypes. In the present study, we report a novel phenotype manifesting in a mouse model with the altered expression of ALDH2. Aldh2 knockout (Aldh2+/- and Aldh2-/-) and wild-type (Aldh2+/+) mice were fed a standard solid rodent chow and a bottle of ethanol solution at concentrations of 0%, 3%, 10%, or 20% (v/v) for more than 10 weeks. The intensity of their skin pigmentation was evaluated by macroscopic observation. Ethanol-exposed Aldh2+/- and Aldh2-/- mice exhibited dose-dependent skin pigmentation in areas of hairless skin, including the soles of the paws and tail; no such changes were observed in wild-type mice. The intensity of skin pigmentation correlated with the number of Aldh2 alleles that were altered in the mice (i.e., 0, 1 and 2 for Aldh2+/+, Aldh2+/-, Aldh2-/-, respectively). Interestingly, the skin pigmentation changes reversed upon the discontinuation of ethanol. The histological examination of the pigmented skin demonstrated the presence of melanin-like deposits, mainly in the epidermis. In conclusion, we report a novel finding that the intake of ethanol induces skin hyperpigmentation in an ALDH2 activity-dependent manner.

Effects of "Essential AD2" Supplement on Blood Acetaldehyde Levels in Individuals Who Have Aldehyde Dehydrogenase (ALDH2) Deficiency.

BACKGROUND: It is estimated that 1 billion people in the world have a point mutation in the gene encoding the aldehyde dehydrogenase 2 (ALDH2) enzyme, the primary enzyme responsible for the metabolism of acetaldehyde. The presence of this mutation is called ALDH2 deficiency. Because of limited ability to metabolize acetaldehyde, individuals with ALDH2 deficiency experience elevated levels of blood acetaldehyde after exposure to various common sources such as recreational alcohol. Because of higher levels of acetaldehyde, individuals with ALDH2 deficiency are at higher risk for numerous diseases, including liver cirrhosis, esophageal and gastric cancer, osteoporosis, and Alzheimer disease. STUDY QUESTION: The present trial was designed to study the effectiveness, safety, and tolerability of a nutritional supplement (Essential AD2). MEASURES AND OUTCOMES: The primary outcome was change in acetaldehyde levels in the blood after exposure to alcohol in individuals with ALDH2 deficiency before and after the use of study nutritional supplement. STUDY DESIGN: This was a 28-day open-label trial, comparing initial acetaldehyde levels after alcohol ingestion to levels after 28 days of a nutritional supplement (Essential AD2). The study consisted of 12 subjects genotyped to be heterozygous for the ALDH2 gene mutation. RESULTS AND CONCLUSIONS: ALDH2 deficient subjects showed a significant decrease in average blood acetaldehyde level 20 minutes after alcohol consumption (from 0.91 mg/dL to 0.71 mg/dL, P value = 0.02) after receiving 28 days of the nutritional supplement. Acetaldehyde levels taken at 10 minutes and 40 minutes also showed a decrease, although they were not statistically significant. In addition, safety tests looking at liver function tests showed a decrease in aspartate transaminase and alanine transaminase liver proteins from 27.3 to 15.2 and 20.9 to 13.2, respectively, over the 28 days. The treatment was well tolerated and no significant side effects were noted.

Effect of Alcohol Sensitivity in Healthy Young Adults on Breath Pharmacokinetics of Acetaldehyde After Mouth Washing with Alcohol.

BACKGROUND: Acetaldehyde is causally related to head and neck cancer. Individuals with aldehyde dehydrogenase 2 deficiency experience alcohol sensitivity and are referred to as "flushers" because of their skin-flushing response to high blood acetaldehyde levels after alcohol consumption. Acetaldehyde is produced in the oral cavity after local alcohol exposure without alcohol ingestion. However, the relationship between the oral acetaldehyde level after local alcohol exposure and alcohol sensitivity is unclear. Herein, sampling the exhaled breath, we evaluated the effect of alcohol sensitivity on the pharmacokinetics of ethanol (EtOH) and acetaldehyde in breath after mouth washing with alcohol. METHODS: Twenty-eight healthy young adults were divided into flusher and nonflusher groups based on an EtOH patch test. The subjects washed their mouths for 30 seconds with 40 ml of 5% v/v alcohol, and their breath samples were collected 12 times over 20 minutes after mouth washing and rinsing with water. EtOH and acetaldehyde concentrations in all breath samples were measured using sensor gas chromatography. RESULTS: Breath EtOH concentrations exponentially decreased in both groups after mouth washing with alcohol. Breath acetaldehyde concentrations showed an immediate increase, followed by an almost exponential decrease in both groups, but concentrations in the flusher group remained higher than those in the nonflusher group throughout the 20-minute measurement period. This was reflected in a peak concentration (Cmax ) of 808 ± 70 parts-per-billion (ppb) versus 1,715 ± 223 ppb, respectively (p = 0.001), and area under the curve values of 3,528 ± 1,399 ppb minutes versus 8,637 ± 1,293 ppb minutes, respectively (p = 0.002). CONCLUSIONS: This study revealed high concentrations of acetaldehyde in breath after local alcohol exposure in the oral cavity among flushers even without alcohol ingestion. This contributes to an increased risk among flushers of mutagenic DNA lesions in the mucosa of the upper digestive tract and cancer.

Protective effect of ALDH2 against cyclophosphamide-induced acute hepatotoxicity via attenuating oxidative stress and reactive aldehydes.

Cyclophosphamide (CY) is a widely used chemotherapeutic agent that is associated with severe side effects, such as hepatotoxicity and nephrotoxicity. However, the extent, mechanisms and potential prevention and treatment strategies of CY-induced acute hepatotoxicity and nephrotoxicity are largely unknown. In this study, we determined the existence and extent of CY-induced acute hepatotoxicity and nephrotoxicity, and demonstrated the effect of ALDH2 on CY-induced acute tissue toxicity and related mechanisms. Adult male C57BL/6J (wide-type, WT) and ALDH2-/- (KO) mice were divided into four groups: WT, WT + CY, KO + CY and WT + CY + Alda-1. Biochemical analysis showed that plasma ALT was increased by 35.8% in KO + CY group and decreased by 21.1% in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). However, there was no significant difference among WT, WT + CY and KO + CY groups regarding plasma renal marker enzymes, including blood urea nitrogen (BUN), creatinine and cystatin C (CysC). Levels of reactive oxygen species (ROS) and toxic aldehydes (acrolein, 4-hydroxynonenol and malondialdehyde) were increased significantly in KO + CY group and decreased significantly in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). These findings demonstrate that CY could induce acute hepatotoxicity without nephrotoxicity, and ALDH2 plays a protective role in CY-induced acute hepatotoxicity. The underlying mechanisms are associated with attenuating oxidative stress and detoxifying reactive aldehydes.

Targeting ALDH2 for Therapeutic Interventions in Chronic Pain-Related Myocardial Ischemic Susceptibility.

Clinical observations have demonstrated a link between chronic pain and increased ischemic heart disease mortality, but the mechanisms remain elusive. Reactive aldehydes have recently been confirmed as a new player in pain pathologies, while our previous study demonstrated that reactive aldehydes (4-HNE) induced carbonyl stress contributing to myocardial ischemic intolerance. The aim of this study was to explore whether chronic pain increases susceptibility to myocardial ischemia/reperfusion (MI/R) injury and to investigate the underlying mechanisms focusing on toxic aldehyde and carbonyl stress. Methods: Chronic pain was induced by chronic compression of the dorsal root ganglion (CCD). After 2 weeks CCD, aldehyde dehydrogenase (ALDH2) KO or wild-type (WT) littermate mice were then subjected to in vivo MI/R. Results: In CCD-WT mice, heightened nociception paralleled circulating aldehyde (4-HNE) accumulation and cardiac protein carbonylation. Mechanistically, CCD-induced 4-HNE overload provoked cardiac Sirtuin 1 (SIRT1) carbonylative inactivation and inhibited Liver kinase B1 (LKB1) - AMP-activated protein kinase (LKB1-AMPK) interaction, which resulted in exacerbated MI/R injury and higher mortality compared with non-CCD WT mice. ALDH2 deficiency further aggravated CCD-induced susceptibility to MI/R injury. Exogenous 4-HNE exposure in peripheral tissue mimicked chronic pain-induced aldehyde overload, elicited sustained allodynia and increased MI/R injury. However, cardiac-specific ALDH2 upregulation by AAV9-cTNT-mediated gene delivery significantly ameliorated chronic pain-induced SIRT1 carbonylative inactivation and decreased MI/R injury (minor infarct size, less apoptosis, and improved cardiac function). Conclusion: Collectively, chronic pain-enhanced carbonyl stress promotes myocardial ischemic intolerance by SIRT1 carbonylative inactivation and impairment of LKB1-AMPK interaction. ALDH2 activation and prevention of protein carbonylation may be a potential therapeutic target for myocardial ischemic vulnerability in chronic pain patients. Our results newly provided overlapping cellular mechanisms of chronic pain and myocardial dysfunction interplay.

Mitochondrial aldehyde dehydrogenase-2 deficiency compromises therapeutic effect of ALDH bright cell on peripheral ischemia.

The autologous ALDH bright (ALDHbr) cell therapy for ischemic injury is clinically safe and effective, while the underlying mechanism remains elusive. Here, we demonstrated that the glycolysis dominant metabolism of ALDHbr cells is permissive to restore blood flow in an ischemic hind limb model compared with bone marrow mononuclear cells (BMNCs). PCR array analysis showed overtly elevated Aldh2 expression of ALDHbr cells following hypoxic challenge. Notably, ALDHbr cells therapy induced blood flow recovery in this model was reduced in case of ALDH2 deficiency. Moreover, significantly reduced glycolysis flux and increased reactive oxygen species (ROS) levels were detected in ALDHbr cell from Aldh2-/- mice. Compromised effect on blood flow recovery was also noticed post transplanting the human ALDHbr cell from ALDH2 deficient patients (GA or AA genotypes) in this ischemic hindlimb mice model. Taken together, our findings illustrate the indispensable role of ALDH2 in maintaining glycolysis dominant metabolism of ALDHbr cell and advocate that patient's Aldh2 genotype is a prerequisite for the efficacy of ALDHbr cell therapy for peripheral ischemia.

ALDH2-deficiency as genetic epidemiologic and biochemical model for the carcinogenicity of acetaldehyde.

Humans are cumulatively exposed to acetaldehyde from various sources including alcoholic beverages, tobacco smoke, foods and beverages. The genetic-epidemiologic and biochemical evidence in ALDH2-deficient humans provides strong evidence for the causal relationship between acetaldehyde-exposure due to alcohol consumption and cancer of the upper digestive tract. The risk assessment has so far relied on thresholds based on animal toxicology with lower one-sided confidence limit of the benchmark dose values (BMDL) typically ranging between 11 and 63 mg/kg bodyweight (bw)/day dependent on species and endpoint. The animal data is problematic for regulatory toxicology for various reasons (lack in study quality, problems in animal models and appropriateness of endpoints - especially cancer - for transfer to humans). In this study, data from genetic epidemiologic and biochemical studies are reviewed. The increase in the daily exposure dose to acetaldehyde in alcohol-consuming ALDH2-deficients vs. ALDH2-actives was about twofold. The acetaldehyde increase due to ALDH2 inactivity was calculated to be 6.7 µg/kg bw/day for heavy drinkers, which is associated with odds ratios of up to 7 for head and neck as well as oesophageal cancer. Previous animal toxicology based risk assessments may have underestimated the risk of acetaldehyde. Risk assessments of acetaldehyde need to be revised using this updated evidence.

Mitochondrial PKC-ε deficiency promotes I/R-mediated myocardial injury via GSK3ß-dependent mitochondrial permeability transition pore opening.

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)-induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase-2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross-clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2-/- ) and wild-type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin-related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N-acetylcysteine (NAC) or PKC-δ shRNA treatment on glycogen synthase kinase-3ß (GSK-3ß) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2-/- mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC-ε translocation was lower in ALDH2-/- mice than in WT mice, and PKC-δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre-treatment under I/R injury. In addition, PKC-ε inhibition caused activation of caspase9, caspase3 and Drp1Ser616 in response to I/R stress. Importantly, expression of phosphorylated GSK-3ß (inactive form) was lower in ALDH2-/- mice than in WT mice, and both were increased by NAC pre-treatment. I/R-induced mitochondrial translocation of GSK-3ß was inhibited by PKC-δ shRNA or NAC pre-treatment. In addition, mitochondrial membrane potential (∆Ψm ) was reduced in ALDH2-/- mice after I/R, which was partly reversed by the GSK-3ß inhibitor (SB216763) or PKC-δ shRNA. Collectively, our data provide the evidence that abnormal PKC-ε/PKC-δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK-3ß-dependent mPTP opening, which results in mitochondrial injury-triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2-/- mice following I/R stress.