Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

National Institute On Alcohol Abuse And Alcoholism

Investigators

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Abstract

My lab has been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on promoting tissue injury by alcohol and other potentially toxic substances. Alcohol-induced oxidative and nitrative (nitro-oxidative) stress can inhibit the ALDH2 activity, resulting in accumulation of toxic acetaldehyde and lipid peroxides. In addition, CYP2E1-mediated nitro-oxidative stress can stimulate different types of post-translational modification (PTM) of proteins, contributing to mitochondrial dysfunction, endoplasmic reticulum (ER) stress and tissue/organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, acetylation, adduct formation, etc. All these PTMs generally occur shortly after exposure to alcohol and nonalcoholic substances or under pathological conditions. In the past, we showed a causal role of CYP2E1 in stimulating various PTMs and tissue injury by evaluating the time-dependent events between PTMs and actual cellular damage in wild-type (WT) compared to the age and sex-matched Cyp2e1-null mice in the presence or absence of a protective agent, including a specific CYP2E1 inhibitor chlormethiazole (CMZ). We also observed that these PTMs and functional alterations of covalently-modified proteins are observed in the liver and other tissues such as gut. In fact, we recently reported the critical role of CYP2E1 in binge alcohol-mediated gut barrier dysfunction (leaky gut), endotoxemia, and inflammatory liver injury in rats and WT mice by studying the pattern of various PTMs of the intestinal tight junction (TJ), adherens junction (AJ) proteins and their decreased levels. Binge alcohol exposure or fructose in drinking water significantly decreased the levels of gut TJ/AJ proteins while it increased the levels of intestinal CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, serum endotoxin and fecal albumin contents, suggesting elevated gut leakiness and endotoxemia. However, Cyp2e1-null mice were resistant to leaky gut and inflammatory liver injury by binge alcohol and the two nonalcoholic substances. These results demonstrate both direct or indirect, permissive role of CYP2E1 in promoting gut leakiness, endotoxemia and fibrotic liver disease. In FY2023, we have reported the importance of ALDH2 in protecting against binge alcohol-induced gut leakiness and acute liver injury. Oral administration of a single dose of 3.5 or 4 g/kg of alcohol caused gut and liver injury in Aldh2-KO but not in the corresponding WT mice. Our results showed that alcohol exposure at 3.5 or 4.0 g/kg increased nitration of gut TJ/AJ proteins, leading to their degradation with elevated gut barrier dysfunction, systemic endotoxemia, and liver injury in Aldh2-KO mice but not in the WT. Similar results of increased epithelial barrier dysfunction with decreased TJ/AJ proteins were also observed in ethanol-exposed T84 colon cells. Confocal image, immunoblot and biochemical analyses revealed that ethanol exposure decreased TJ/AJ proteins with elevated cell permeability of T84 cells. Treatment with a chemical Aldh2 activator Alda-1 prevented these changes while an Aldh2 inhibitor Daidzin treatment further aggravated the ethanol-mediated intestinal permeability of T84 cells. Additionally CRISPR/Cas9-mediated ALDH2 deletion in T84 cells increased alcohol-mediated cell death and paracellular permeability. These results suggest the important role of ALDH2 in protecting against alcohol-induced gut leakiness and liver injury, as frequently observed in people with a dominant negative mutation in Aldh2 gene. Thus, ALDH2 can be a potential target for preventing alcohol-associated tissue injury. Based on these results, we have also extended our mechanistic studies on the alcohol-induced brain damage in Aldh2-KO mice. A manuscript is being prepared on the new results. One lab member received an NIH ODS Scholar Award to study the beneficial effect of physiologically relevant doses of melatonin (MT) against thioacetamide (TAA)-mediated liver fibrosis. We observed liver fibrosis determined by picrosirius-red staining and immunoblot analyses of collagen, vimentin and alpha-smooth muscle actin after male rats were treated with TAA twice a week for up to 4 weeks. We observed increased apoptosis of hepatocyte apoptosis with elevated serum ALT and AST levels. However MT pretreatment significantly decreased the levels of TAA-mediated hepatic apoptosis and fibrosis in a time- and MT-dose dependent manner. In this rat fibrosis model, we did not observe changes in nitrated or phosphorylated proteins. However, the levels of acetylated proteins in TAA-exposed rat livers were markedly elevated and MT pretreatment significantly decreased their levels. Based on the selective increments of acetylated proteins, we determined the levels of NAD+-dependent seven Sirtuin (Sirt) isoforms and histone deacetylase (HDAC) activity. Only Sirt1 and its activity were decreased in TAA-exposed rats but the Sirt1 level and its activity were restored by MT pretreatment. In contrast, only small changes in HDAC and acetyltransferase activities were observed after TAA exposure. We observed that many critical proteins, such as p53, NF-kB, FOXO1, and SOD2, were acetylated and directly involved in TAA-mediated hepatotoxicity and fibrosis. Furthermore, TAA exposures exacerbated liver fibrosis in liver-specific Sirt1-KO mice, supporting our results with rats. These results demonstrated the critical role of Sirt1-dependent protein acetylation in liver fibrosis. In parallel, we also studied the mechanisms of TAA-mediated acute liver injury by focusing on the role of oxidative PTMs in promoting mitochondrial dysfunction and acute liver injury. Our results showed that a single injection of TAA increased the oxidative stress and PTMs which negatively affected the activities of a few mitochondrial proteins, leading to mitochondrial dysfunction and eventually death of liver cells in WT mice. However, acute liver injury was virtually not observed in the TAA-exposed Cyp2e1-null mice, demonstrating the direct role of CYP2E1 in TAA-metabolism and subsequent mitochondrial dysfunction and acute liver injury. We are ready to submit one manuscript on these results in the near future. In collaboration with a former lab member Dr. Young-Eun Cho, we also studied the molecular mechanism by which ellagic acid prevented dextran sulfate sodium (DSS)-induced tissue injury. Histology and biochemical analyses showed that DSS in drinking water for 7 days caused gut, liver, and brain injury through the gut-liver-brain axis. Immunoblot analyses showed the decreased levels of many gut TJ/AJ proteins that were nitrated and degraded by ubiquitin-dependent proteolysis in DSS-exposed mice. DSS treatment also caused liver and brain injury in mice through the NF-kB and MAPK pathways. However, treatment with a physiologically relevant dose of ellagic acid significantly prevented DSS-mediated gut, liver, and brain injury. One manuscript on these new results was submitted for publication. All these experimental rodent and cell studies not only represent examples of our mechanistic and translational research with a safe dietary supplement to prevent liver injury and fibrosis caused by alcohol, TAA, or DSS, but also suggest a conserved mechanism of gut and liver injury in different disease models through the gut-liver axis. Furthermore, we also published a few review articles on the important roles of gut dysbiosis in promoting gut leakiness, leading to systemic endotoxemia and various neurological diseases, including those caused by alcohol and traumatic brain injury through the gut-brain axis.