Aldh2 Knockout Mice Research Articles

Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) Protects against Binge Alcohol-Mediated Gut and Brain Injury.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) metabolizes acetaldehyde to acetate. People with ALDH2 deficiency and Aldh2-knockout (KO) mice are more susceptible to alcohol-induced tissue damage. However, the underlying mechanisms behind ALDH2-related gut-associated brain damage remain unclear. Age-matched young female Aldh2-KO and C57BL/6J wild-type (WT) mice were gavaged with binge alcohol (4 g/kg/dose, three doses) or dextrose (control) at 12 h intervals. Tissues and sera were collected 1 h after the last ethanol dose and evaluated by histological and biochemical analyses of the gut and hippocampus and their extracts. For the mechanistic study, mouse neuroblast Neuro2A cells were exposed to ethanol with or without an Aldh2 inhibitor (Daidzin). Binge alcohol decreased intestinal tight/adherens junction proteins but increased oxidative stress-mediated post-translational modifications (PTMs) and enterocyte apoptosis, leading to elevated gut leakiness and endotoxemia in Aldh2-KO mice compared to corresponding WT mice. Alcohol-exposed Aldh2-KO mice also showed higher levels of hippocampal brain injury, oxidative stress-related PTMs, and neuronal apoptosis than the WT mice. Additionally, alcohol exposure reduced Neuro2A cell viability with elevated oxidative stress-related PTMs and apoptosis, all of which were exacerbated by Aldh2 inhibition. Our results show for the first time that ALDH2 plays a protective role in binge alcohol-induced brain injury partly through the gut-brain axis, suggesting that ALDH2 is a potential target for attenuating alcohol-induced tissue injury.

Host ALDH2 deficiency aggravates nonalcoholic steatohepatitis through gut-liver axis

Nonalcoholic steatohepatitis (NASH) is the major cause of liver dysfunction. Animal and population studies have shown that mitochondrial aldehyde dehydrogenase (ALDH2) is implicated in fatty liver disease. However, the role of ALDH2 in NASH and the underlying mechanisms remains unclear. To address this issue, ALDH2 knockout (ALDH2−/−) mice and wild-type littermate mice were fed a methionine-and choline-deficient (MCD) diet to induce a NASH model. Fecal, serum, and liver samples were collected and analyzed to investigate the impact of the gut microbiota and bile acids on this process. We found that MCD-fed ALDH2−/− mice exhibited increased serum pro-inflammation cytokines, hepatic inflammation and fat accumulation than their wild-type littermates. MCD-fed ALDH2−/− mice exhibited worsened MCD-induced intestinal inflammation and barrier damage, and gut microbiota disorder. Furthermore, mice receiving microbiota from MCD-fed ALDH2−/− mice had increased severity of NASH compared to those receiving microbiota from MCD-fed wild-type mice. Notably, the intestinal Lactobacillus was significantly reduced in MCD-fed ALDH2−/− mice, and gavage with Lactobacillus cocktail significantly improved MCD-induced NASH. Finally, we found that ALDH2-/- mice had reduced levels of bile salt hydrolase and specific bile acids, especially lithocholic acid (LCA), accompanied by downregulated expression of the intestinal FXR-FGF15 pathway. Supplementation of LCA in ALDH2−/− mice upregulated intestinal FXR-FGF15 pathway and alleviated NASH. In summary, ALDH2 plays a critical role in the development of NASH through modulation of gut microbiota and bile acid. The findings suggest that supplementing with Lactobacillus or LCA could be a promising therapeutic approach for treating NASH exacerbated by ALDH2 deficiency.

ALDH2 deficiency increases susceptibility to binge alcohol-induced gut leakiness, endotoxemia, and acute liver injury in mice through the gut-liver axis

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme responsible for metabolizing toxic acetaldehyde to acetate and acts as a protective or defensive protein against various disease states associated with alcohol use disorder (AUD), including alcohol-related liver disease (ARLD). We hypothesized that Aldh2-knockout (KO) mice are more susceptible to binge alcohol-mediated liver injury than wild-type (WT) mice through increased oxidative stress, gut leakiness and endotoxemia. Therefore, this study aimed to investigate the protective role of ALDH2 in binge alcohol-induced gut permeability, endotoxemia, and acute inflammatory liver injury by exposing Aldh2-KO or WT mice to a single oral dose of binge alcohol 3.5, 4.0, or 5.0 g/kg. Our findings showed for the first time that ALDH2 deficiency in Aldh2-KO mice increases their sensitivity to binge alcohol-induced oxidative and nitrative stress, enterocyte apoptosis, and nitration of gut tight junction (TJ) and adherent junction (AJ) proteins, leading to their degradation. These resulted in gut leakiness and endotoxemia in Aldh2-KO mice after exposure to a single dose of ethanol even at 3.5 g/kg, while no changes were observed in the corresponding WT mice. The elevated serum endotoxin (lipopolysaccharide, LPS) and bacterial translocation contributed to systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury through the gut-liver axis. Treatment with Daidzin, an ALDH2 inhibitor, exacerbated ethanol-induced cell permeability and reduced TJ/AJ proteins in T84 human colon cells. These changes were reversed by Alda-1, an ALDH2 activator. Furthermore, CRISPR/Cas9-mediated knockout of ALDH2 in T84 cells increased alcohol-mediated cell damage and paracellular permeability. All these findings demonstrate the critical role of ALDH2 in alcohol-induced epithelial barrier dysfunction and suggest that ALDH2 deficiency or gene mutation in humans is a risk factor for alcohol-mediated gut and liver injury, and that ALDH2 could be an important therapeutic target against alcohol-associated tissue or organ damage.

Aldehyde Dehydrogenase 2 (ALDH2) Elicits Protection against Pulmonary Hypertension via Inhibition of ERK1/2-Mediated Autophagy.

Pulmonary hypertension (PH) is caused by chronic hypoxia that induces the migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs), eventually resulting in right heart failure. PH has been related to aberrant autophagy; however, the hidden mechanisms are still unclear. Approximately 40% East Asians, equivalent to 8% of the universal population, carry a mutation in Aldehyde dehydrogenase 2 (ALDH2), which leads to the aggregation of noxious reactive aldehydes and increases the propensity of several diseases. Therefore, we explored the potential aspect of ALDH2 in autophagy associated with PH. In vitro mechanistic studies were conducted in human PASMCs (HPASMCs) after lentiviral ALDH2 knockdown and treatment with platelet-derived growth factor-BB (PDGF-BB). PH was induced in wild-type (WT) and ALDH2-knockout (ALDH2−/−) mice using vascular endothelial growth factor receptor inhibitor SU5416 under hypoxic conditions (HySU). Right ventricular function was assessed using echocardiography and invasive hemodynamic monitoring. Histological and immunohistochemical analyses were performed to evaluate pulmonary vascular remodeling. EdU, transwell, and wound healing assays were used to evaluate HPASMC migration and proliferation, and electron microscopy and immunohistochemical and immunoblot assays were performed to assess autophagy. The findings demonstrated that ALDH2 deficiency exacerbated right ventricular pressure, hypertrophy, fibrosis, and right heart failure resulting from HySU-induced PH. ALDH2−/− mice exhibited increased pulmonary artery muscularization and 4-hydroxynonenal (4-HNE) levels in lung tissues. ALDH2 knockdown increased PDGF-BB-induced PASMC migration and proliferation and 4-HNE accumulation in vitro. Additionally, ALDH2 deficiency increased the number of autophagosomes and autophagic lysosomes together with autophagic flux and ERK1/2-Beclin-1 activity in lung tissues and PASMCs, indicating enhanced autophagy. In conclusion, the study shows that ALDH2 has a protective role against the migration and proliferation of PASMCs and PH, possibly by regulating autophagy through the ERK1/2-Beclin-1 pathway.

Ethanol concentration induces production of 3,4-dihydroxyphenylacetic acid and homovanillic acid in mouse brain through activation of monoamine oxidase pathway

First, we aimed to investigate ex vivo the effects of ethanol (EtOH) on levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and their metabolites in the frontal cortex, hippocampus, and striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Animals were treated intraperitoneally with saline (control) or EtOH (1.0, 2.0, or 3.0 g/kg). Brain samples were collected 60 and 120 min after EtOH injection, and monoamines and their metabolites were measured by HPLC-ECD. We found in both WT and Aldh2-KO mice that 3.0 g/kg EtOH increased the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and decreased the level of 3-methoxytyramine (3-MT). A 2.0 g/kg dose of EtOH also increased HVA, but there was not a consistent effect within the brain regions of Aldh2-KO and WT mice. There were inconsistent findings of genotype differences in the levels of DA, 5-HT, and their metabolites in the brain regions tested. None of the EtOH doses altered NE, DA, 5-HT, or 5-hydroxyindoleacetic acid contents in any of the brain regions studied. Second, we tested whether EtOH-induced increases in DOPAC and HVA are mediated by increased monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) activity. To test this, we used the MAO blocker clorgyline (2.0 and 4.0 mg/kg) and the COMT blocker tolcapone (15 and 30 mg/kg) alone or in combination with EtOH (3.0 g/kg). Clorgyline alone increased 3-MT and decreased DOPAC and HVA levels, whereas tolcapone alone increased DOPAC and decreased 3-MT and HVA levels. Surprisingly, the combination of EtOH with clorgyline (4.0 mg/kg) or tolcapone (30 mg/kg) further decreased 3-MT and increased DOPAC and HVA levels, an effect that reversed the inhibitor-induced decreases in HVA. These results suggest that a high concentration of EtOH can accelerate DA metabolism, as evidenced by the increase in DOPAC and HVA, and this effect is likely a consequence of increased degradation of DA by MAO.

Aldehyde dehydrogenase 2 deficiency promotes skeletal muscle atrophy in aged mice.

Aldehyde dehydrogenase 2 (ALDH2) detoxifies acetaldehyde produced from ethanol. A missense single nucleotide polymorphism (SNP) rs671 in ALDH2 exhibits a dominant-negative form of the ALDH2 protein. Nearly 40% of people in East Asia carry an inactive ALDH2*2 mutation. Previous studies reported that ALDH2*2 is associated with increased risk of several diseases. In this study, we examined the effect of ALDH2 deficiency on age-related muscle atrophy and its underlying mechanisms. We found that ALDH2 deficiency promotes age-related loss of muscle fiber cross-sectional areas, especially in oxidative fibers. Furthermore, ALDH2 deficiency exacerbated age-related accumulation of 4-hydroxy-2-nonenal (4-HNE), a marker of oxidative stress in the gastrocnemius muscle. Similarly, mitochondrial reactive oxygen species (ROS) production increased in aged ALDH2-knockout mice, indicating that ALDH2 deficiency induced mitochondrial dysfunction. In summary, ALDH2 deficiency promotes age-related muscle loss, especially in oxidative fibers, which may be associated with an increased accumulation of oxidative stress via mitochondrial dysfunction.

Aldh2 gene reduces oxidative stress in the bladder by regulating the NF-κB pathway in a mouse model of ketamine-induced cystitis.

Aldehyde dehydrogenase 2 (aldh2) serves an important role in the development of organ injury. Therefore, the present study investigated the effects of aldh2 on the oxidative stress response in a mouse model of ketamine-induced cystitis (KIC). A total of 60 8-week-old male Institute of Cancer Research wild-type (WT) mice and 45 aldh2 knock-out (KO) mice were randomized to receive low-dose ketamine (30 mg/kg), high-dose ketamine (60 mg/kg) or normal saline (controls). At 4, 8 and 12 weeks post-injection, bladder tissues were harvested and used to investigate the protective mechanisms of aldh2 on bladder function. The results demonstrated that aldh2 KO mice exhibited significant weight loss following chronic ketamine injection compared with that in WT mice. Furthermore, ketamine treatment increased the urination rate (P<0.05), pathological score (P<0.05), levels of the oxidative stress product malondialdehyde (P<0.05) in addition to reducing the expression of the anti-oxidative stress enzyme superoxide dismutase (P<0.05) and glutathione-SH (P<0.05). Oxidative stress in aldh2 KO mice was also found to significantly enhance the expression of proteins associated with the NF-κB signaling pathway, which promoted the expression of inducible nitric oxide synthase (P<0.05) and cyclooxygenase-2 (P<0.05) further. Finally, aldh2 KO mice demonstrated higher severity of fibrosis in the submucosal and muscular layers of the bladder. In conclusion, the present study suggests that aldh2 serves a protective role in preventing inflammation and fibrosis in KIC.

Alpha-lipoic acid protects against pressure overload-induced heart failure via ALDH2-dependent Nrf1-FUNDC1 signaling

Alpha-lipoic acid (α-LA), a well-known antioxidant, was proved to active ALDH2 in nitrate tolerance and diabetic animal model. However, the therapeutic advantage of α-LA for heart failure and related signaling pathway have not been explored. This study was designed to examine the role of α-LA–ALDH2 in heart failure injury and mitochondrial damage. ALDH2 knockout (ALDH2−/−) mice and primary neonatal rat cardiomyocytes (NRCMs) were subjected to assessment of myocardial function and mitochondrial autophagy. Our data demonstrated α-LA significantly reduced the degree of TAC-induced LV hypertrophy and dysfunction in wild-type mice, not in ALDH2−/− mice. In molecular level, α-LA significantly restored ALDH2 activity and expression as well as increased the expression of a novel mitophagy receptor protein FUNDC1 in wild-type TAC mice. Besides, we confirmed that ALDH2 which was activated by α-LA governed the activation of Nrf1–FUNDC1 cascade. Our data suggest that α-LA played a positive role in protecting the heart against adverse effects of chronic pressure overload.

Disruption of the aldehyde dehydrogenase 2 gene increases the bone anabolic response to intermittent PTH treatment in an ovariectomized mouse model.

Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that oxidizes the acetaldehyde produced by alcohol metabolism. This variant not only affects the response to alcohol but is also associated with several diseases, such as esophageal cancer, myocardial infarction, and particularly osteoporosis. In our previous study, we reported that compared to wild-type (WT) mice, Aldh2 knockout (KO) mice naturally have a strong bone formation ability, and high expression of parathyroid hormone receptor (PTHR1) in osteocytes. The effect of the Aldh2 gene on bone metabolism in response to intermittent PTH treatment is unknown. The purpose of this study was to clarify the effect of the Aldh2 gene on the bone anabolic response to intermittent PTH treatment in ovariectomized mice. Female KO and WT mice were ovariectomized at 8weeks of age. At 14weeks of age, the KO and WT mice were divided into vehicle-treated (Veh) and PTH-treated (PTH) groups (i.e., the WT-Veh, WT-PTH, KO-Veh and KO-PTH groups). PTH (1-34) and vehicle were subcutaneously administered to each group at a dose of 40μg/kg body weight (BW) five times per week for 4weeks. Micro-CT showed that the bone volume (BV), trabecular number (Tb.N), connectivity density (Conn.D), and cortical thickness (Ct.Th) values in the KO-PTH mice were significantly higher than those in the KO-Veh mice. Histomorphometric analysis showed that the BV, Tb.N, and mineral apposition rate (MAR) values in the KO-PTH group were significantly higher than those in the KO-Veh group. The mRNA expression level of PTHR1 in the KO-PTH group was significantly increased and that of p21 in the KO-PTH group was significantly decreased compared with the levels in the KO-Veh group. The expression of PTHR in osteocytes from the KO-PTH group was also significantly increased compared with that in osteocytes from the KO-Veh group. Furthermore, cell cultures revealed that the ALP+CFU-f/total CFU-f percentage was significantly higher in the KO-PTH group than in the KO-Veh group. We concluded that in ovariectomized Aldh2 KO mice, the bone anabolic response to intermittent PTH treatment was significantly enhanced compared to that in WT mice, which may be mediated by the high expression level of PTHR1.

Aldh2 Attenuates Stem Cell Factor/Kit-Dependent Signaling and Activation in Mast Cells.

Mitochondrial aldehyde dehydrogenase (ALDH2) metabolizes endogenous and exogenous aldehydes and protects cells against oxidative injury. Inactivating genetic polymorphisms in humans are common and associate with alcohol flush reactions. However, whether mast cell Aldh2 activity impacts normal mast cell responses is unknown. Using bone marrow-derived mast cells from Aldh2 knockout mice, we found evidence for a role of mast cell Aldh2 in Kit-mediated responses. Aldh2-deficient mast cells showed enhanced Kit tyrosine kinase phosphorylation and activity after stimulation with its ligand (stem cell factor) and augmentation of downstream signaling pathways, including Stat4, MAPKs, and Akt. The activity of the phosphatase Shp-1, which attenuates Kit activity, was reduced in Aldh2−/− mast cells, along with an increase in reactive oxygen species, known to regulate Shp-1. Reduced Shp-1 activity concomitant with sustained Kit signaling resulted in greater proliferation following Kit engagement, and increased mediator and cytokine release when Aldh2−/− mast cells were co-stimulated via Kit and FcεRI. However, FcεRI-mediated signaling and responses were unaffected. Therefore, our findings reveal a functional role for mast cell intrinsic Aldh2 in the control of Kit activation and Kit-mediated responses, which may lead to a better understanding of mast cell reactivity in conditions related to ALDH2 polymorphisms.

Targeting liver aldehyde dehydrogenase-2 prevents heavy but not moderate alcohol drinking

Aldehyde dehydrogenase 2 (ALDH2), a key enzyme for detoxification the ethanol metabolite acetaldehyde, is recognized as a promising therapeutic target to treat alcohol use disorders (AUDs). Disulfiram, a potent ALDH2 inhibitor, is an approved drug for the treatment of AUD but has clinical limitations due to its side effects. This study aims to elucidate the relative contribution of different organs in acetaldehyde clearance through ALDH2 by using global- (Aldh2-/-) and tissue-specific Aldh2-deficient mice, and to examine whether liver-specific ALDH2 inhibition can prevent alcohol-seeking behavior. Aldh2-/- mice showed markedly higher acetaldehyde concentrations than wild-type (WT) mice after acute ethanol gavage. Acetaldehyde levels in hepatocyte-specific Aldh2 knockout (Aldh2Hep-/-) mice were significantly higher than those in WT mice post gavage, but did not reach the levels observed in Aldh2-/- mice. Energy expenditure and motility were dramatically dampened in Aldh2-/- mice, but moderately decreased in Aldh2Hep-/- mice compared to controls. In the 2-bottle paradigm and the drinking-in-the-dark model, Aldh2-/- mice drank negligible volumes from ethanol-containing bottles, whereas Aldh2Hep-/- mice showed reduced alcohol preference at high but not low alcohol concentrations. Glial cell- or neuron-specific Aldh2 deficiency did not affect voluntary alcohol consumption. Finally, specific liver Aldh2 knockdown via injection of shAldh2 markedly decreased alcohol preference. In conclusion, although the liver is the major organ responsible for acetaldehyde metabolism, a cumulative effect of ALDH2 from other organs likely also contributes to systemic acetaldehyde clearance. Liver-targeted ALDH2 inhibition can decrease heavy drinking without affecting moderate drinking, providing molecular basis for hepatic ALDH2 targeting/editing for the treatment of AUD.

Moderate to high ethanol and acetaldehyde administration decreases c-fos protein expression in the hippocampus of Aldh2-knockout and C57BL/6N mice

Background: Ethanol (EtOH) and acetaldehyde (AcH) have long been associated with many adverse effects in the central nervous system. c-fos has been used as an indirect index of neural activity.&#x0D; Method: Here, we investigated the effects of systemic administration of EtOH and AcH on the expression of c-fos protein in the hippocampus of Aldh2-knockout (Aldh2-KO) mice. The animals received an intraperitoneal injection of saline (control), EtOH (1.0, 2.0 and 4.0 g/kg) or AcH (50, 100 and 200 mg/kg), and the expression of c-fos protein was measured by Western blotting.&#x0D; Result: We found that EtOH at doses of 2.0 and 4.0 g/kg decreased c-fos in wild-type (WT) mice, whereas EtOH at all three doses tested (1.0-4.0 g/kg) decreased c-fos in Aldh 2-KO mice. Likewise, AcH at doses of 100 and 200 mg/kg had a significant effect in lowering c-fos protein in both WT and Aldh2-KO mice.&#x0D; Conclusion: Our observations suggest that moderate to high EtOH and AcH can decrease the expression of c-fos protein in the mouse hippocampus. This effect may support, at least in part, the link between c-fos and spatial memory deficits following EtOH and AcH exposure as we have observed in our previous study.&#x0D; Bangladesh Journal of Medical Science Vol.19(1) 2020 p.83-89

Gene Therapy Correction of Aldehyde Dehydrogenase 2 Deficiency

Aldehyde dehydrogenase 2 (ALDH2) deficiency causes “Asian flush syndrome,” presenting as alcohol-induced facial flushing, tachycardia, nausea, and headaches. One of the most common hereditary enzyme deficiencies, it affects 35%–40% of East Asians and 8% of the world population. ALDH2 is the key enzyme in ethanol metabolism; with ethanol challenge, the common ALDH2*2 (E487K) mutation results in accumulation of toxic acetaldehyde. ALDH2*2 heterozygotes have increased risk for upper digestive tract cancers, compounded by smoking and drinking alcohol. We hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the human ALDH2 coding sequence (AAVrh.10hALDH2) would correct the deficiency state. AAVrh.10hALDH2 was administered intravenously to Aldh2 knockout (Aldh2−/−) and Aldh2 E487K knockin homozygous (Aldh2E487K+/+) mice. Following acute ethanol ingestion, untreated ALDH2-deficient mice had elevated acetaldehyde levels and performed poorly in behavioral tests. In contrast, treated Aldh2−/− and Aldh2E487K+/+ mice had lower serum acetaldehyde levels and improved behavior. Thus, in vivo AAV-mediated ALDH2 therapy may reverse the deficiency state in ALDH2*2 individuals, eliminating the Asian flush syndrome and reducing the risk for associated disorders.

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

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. 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. 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. 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. 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.

Abstract 4360: ALDH2 and the TGF-β/SMAD3 adaptor, Sptbn1 suppress hepatic steatosis and obesity

Abstract Background: Previously we have demonstrated that TGF-β-deficient mutants derived from the loss of Smad3 and the Smad adaptor β2SP (Sptbn1) phenocopy a human cancer stem cell syndrome, are exquisitely sensitive to alcohol, with dysregulation DNA damage repair and have raised levels of aldehyde dehydrogenase (ALDH2). Moreover, β2SP and β2SP/Smad3 mutant mice develop multiple liver abnormalities that include steatosis, chronic hepatitis, inflammation, and cancer. ALDH2 is a key intermediate enzyme for aldehyde and alcohol metabolism. We, therefore, hypothesized that disruption of TGF-β signaling combined with ALDH2 deficiency would increase the susceptibility of liver cancer. Methods: Aldh2 knockout mice were intercrossed with Sptbn1 heterozygous knockout mice to explore the role of Aldh2 and Sptbn1 in liver steatosis and liver cancer. Conditional knockout of Sptbn1 mice was generated by the intercross of Albumin-Cre mice with mice carrying Loxp sites flanked exons of Sptbn1. Sptbn1 conditional mice were fed with high-fat diet or chow diet. Further analysis included blood lipid panel analysis and tissue profiles including western and RNA analysis, lipidomics and metabolomics analysis of liver tissues from the mutant mice and wild-type controls. Results: Interestingly, we found that Aldh2-/-Sptbn1+/- mice on a normal diet demonstrated early signs of truncal obesity. Within 6 months of age, we observed a greater than 23% increase in body weight, with predominantly abdominal fat accumulation in Aldh2-/-Sptbn1+/- mice compared to Aldh2-/- mice or wild type (WT) mice (Aldh2-/-Sptbn1+/- vs Aldh2-/- or WT, p&amp;lt;0.05). A marked increase in zone 3 hepatic micro and macro-steatosis with some inflammation was observed in liver tissues of mutant Aldh2-/-Sptbn1+/- mice as well as the conditional Sptbn1 knockout (Alb-Cre+Sptbn1F/+) mice in a high-fat diet. In addition to significant increased blood glucose levels in Aldh2-/-Sptbn1+/- mice compared with Aldh2-/- mice (165±23 VS 135±13, p&amp;lt;0.05), further analysis revealed that the fatty liver phenotype accompanied with abnormal expression of genes related to glucose uptake, metabolisms and homeostasis as demonstrated by decrease of glucose transporter and increased expression of Ceramide which is known to promote insulin resistance. Moreover, we also observed disruption of lipid metabolism as demonstrated by significantly increased serum and liver triglyceride and cholesterol ester levels in Aldh2-/-Sptbn1+/- mice. Conclusions: Our data suggest that the TGF-β pathway plays an important role in maintaining normal glucose metabolism homeostasis and patients with SPTBN1 and ALDH2 defects may have increased susceptibility to obesity as well as fatty liver (NASH), and cancer. Citation Format: Shuyun Rao, Sobia Zaidi, Wilma Jogunoori, Shoujun Gu, Jon White, Bibhuti Mishra, Chu-Xia Deng, Patricia Latham, Lopa Mishra. ALDH2 and the TGF-β/SMAD3 adaptor, Sptbn1 suppress hepatic steatosis and obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4360.

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.

Inhalation exposure to low levels of ethyl tertiary butyl ether: Its genetic effects were significantly modified by ALDH2 activity.

Previous experiments showed that high concentrations of ethyl tertiary butyl ether (ETBE) exposure (500-5,000 ppm) significantly resulted in DNA damages in aldehyde dehydrogenase 2 (Aldh2) knockout (KO) mice. This study was aimed to verify the genotoxic effects in three genetic types, Aldh2 KO, heterogeneous (HT), and wild type (WT), of mice exposed to lower concentrations of ETBE (50-500 ppm) by inhalation. Histopathology assessments in the livers, measurements of genotoxic biomarkers in blood and livers, and urinary 8-hydroxydeoxyguanosion (8-OH-dG) for the oxidative DNA damage of whole body were performed. Significant histopathological changes and DNA strand breaks both in hepatocytes and leukocytes were found in HT and KO male mice exposed to ≥200 ppm ETBE, but not in 50 ppm ETBE. 8-OH-dG levels either in liver or urine were higher in the HT and KO male mice exposed to ≥200 ppm ETBE. The pathological and genetic effects of ETBE were almost at the same extents for HT and KO mice. Thus, 50 ppm could be the no observed adverse effect level for ETBE in HT and KO male mice, which was far lower than the 500 ppm in WT mice. These results suggested that decrease and deficiency of ALDH2 activity would significantly increase the sensitivity to ETBE-induced genotoxicity as well as hepatotoxic effects after exposure even to low concentrations of ETBE. Environ. Mol. Mutagen. 60: 145-153, 2019. © 2018 Wiley Periodicals, Inc.

Alcohol inhibits T-cell glucose metabolism and hepatitis in ALDH2-deficient mice and humans: roles of acetaldehyde and glucocorticoids

ObjectiveAldehyde dehydrogenase 2 (ALDH2), a key enzyme to detoxify acetaldehyde in the liver, exists in both active and inactive forms in humans. Individuals with inactive ALDH2 accumulate acetaldehyde after alcohol...

Cortical bone loss due to skeletal unloading in aldehyde dehydrogenase 2 gene knockout mice is associated with decreased PTH receptor expression in osteocytes

Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that degrades and detoxifies the acetaldehyde produced by alcohol metabolism. In our previous study, we found that compared with wild-type mice (WT), climbing exercises did not increase trabecular bone mass in Aldh2 knockout mice (KO). The purpose of this study was to clarify the effect of the Aldh2 gene on cortical bone structure and on the dynamics of skeletal unloading. Eight-week-old male KO and WT were divided into ground control (GC) or tail suspension (TS) groups for one week (i.e., the KOGC, KOTS, WTGC and WTTS groups). We measured the bone mineral density (BMD) of the femur using dual-energy X-ray absorptiometry. We assessed the femoral morphometry using peripheral quantitative computed tomography (pQCT) and evaluated the femoral cortex histomorphometry, and cortical mRNA using quantitative RT-PCR and cortical bone immunohistostaining. No significant differences were found between the femoral BMD of WTGC and that of WTTS, but the BMD in KOTS was significantly lower than that of KOGC. The pQCT results revealed that the cortical BMD of the femoral diaphysis in KOTS was significantly lower than that of KOGC. Furthermore, the cortical bone area and cortical thickness were significantly lower in KOTS than in the other three groups. Cortical histomorphometric analysis revealed that the endosteal and periosteal bone formation parameters were significantly lower in KOTS than in KOGC. Bone formation signals such as parathyroid hormone receptor (PTHR) were significantly decreased in KOTS compared with the levels in KOGC. Cortical bone immunohistostaining revealed a significantly decreased expression of PTHR in the osteocytes of KOTS compared with the expression level in KOGC. Thus, we concluded that when the Aldh2 gene is disrupted, skeletal unloading suppresses bone formation to decrease cortical bone mass, which may be mediated by a decreased expression of PTH receptors in osteocytes.

Mitochondrial ALDH2 Deficiency Promotes Fibrosis and Reactive Oxygen Species Formation in the Myocardium of Aged Mice

Enhanced fibrosis and reactive oxygen species (ROS) generation are two major features of aged heart. Since a loss-of-function mutation of ALDH2 was found in nearly half of East Asian population, we explored the impact of ALDH2 deficiency on the process of aging in the heart. We found that the body weight increased profoundly in aged wild-type (WT) mice but not in ALDH2 knockout (KO) mice. However, the ratio of heart weight to body weight increased significantly only in KO mice. Echocardiographic assessment showed that left ventricular ejection fraction of aged WT mice was significantly decreased, which was further aggravated by ALDH2 deficiency. Further study revealed that the compromised cardiac function of aged KO mice might be resulted from PI3K/Akt pathway-mediated fibrosis formation, as shown by remarkable upregulation of collagen I, transforming growth factor-β, and α-smooth muscle actin expression. Aging-induced ROS generation was quantified by the expression of 4-hydroxy-2-nonenal-adducted proteins, which was notably increased in the myocardium of aged KO mice. Besides, the expression of cleaved caspase-3 and 8-oxoguanine DNA glycosylase also increased significantly in the myocardium of aged KO mice. Further study demonstrated that increased ROS generation stimulated a prominent upregulation of ROS scavenger SOD2 expression in the myocardium of aged WT mice but not in KO mice. Our findings suggest that mitochondrial ALDH2 deficiency accelerates aging in the heart by increasing fibrosis formation and oxidative stress.