Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

National Institute On Alcohol Abuse And Alcoholism

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Abstract

Members of my lab have 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 (nitroxidative) stress can inhibit ALDH2 activity, resulting in accumulation of toxic acetaldehyde and lipid peroxides. In addition, CYP2E1-mediated nitroxidative stress can stimulate different types of post-translational modification (PTM) of cellular 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 oxidative tissue injury by evaluating the time-dependent events of PTMs and actual cellular damage in the presence or absence of an antioxidant N-acetylcysteine (NAC) or a specific CYP2E1 inhibitor chlormethiazole (CMZ) and using wild-type (WT) versus age and sex-matched Cyp2e1-null mice. We also observed that these PTMs and functional alterations of covalently-modified proteins are observed in the liver and other tissues such as gut. Therefore, we also characterized the nitrated and/or p-JNK target proteins in the gut of WT mice compared to Cyp2e1-null mice, WT rats and HIV-1 transgenic (Tg) rats where elevation of nitrated proteins and p-JNK target proteins was observed after exposure to binge alcohol, fructose in drinking water, or a western-style fast food-high fat diet (FF-HFD) containing high cholesterol. We recently reported the critical involvement of CYP2E1 in binge alcohol-mediated intestinal 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), adherent junction (AJ), and desmosome and their roles in alcohol-induced gut injury and acute hepatotoxicity. Binge alcohol exposure 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 depending on the toxic agents, such as alcohol and nonalcoholic substances. In FY2022, we have also reported the importance of gut dysbiosis in causing gut leakiness in many disease states. In collaboration with a former lab member Dr. Young-Eun Cho we also studied the molecular mechanism by which plum extracts prevented dextran sulfate sodium (DSS)-induced intestinal colitis and liver injury. Our histology and biochemical analyses showed that pretreatment with plum extracts as a component of rodent chow significantly prevented DSS-mediated gut injury and hepatic inflammation. 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 and T84 colon cells. DSS treatment also caused acute liver injury in mice. However, DSS-mediated gut leakiness and liver injury were significantly prevented by plum extracts. Our results clearly showed that DSS-mediated gut leakiness and liver injury take place by similar mechanisms of binge ethanol-induced intestinal barrier dysfunction and liver injury. As described earlier, ALDH2 is a mitochondrial low Km enzyme involved in the metabolism of many reactive aldehydes, including acetaldehyde produced from oxidative alcohol metabolism. In fact, people with a ALDH2 gene mutation are known to be more susceptible to alcohol-induced tissue damage, including different types of cancer. Thus, we hypothesized that Aldh2-KO mice are more sensitive to binge alcohol-induced gut and liver injury than the WT mice. We therefore studied the mechanisms for greater susceptibility of Aldh2-KO mice to alcohol-induced gut and liver injury compared to WT mice. 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 WT mice. Similar results of increased epithelial barrier dysfunction were also observed in ethanol-exposed T84 colon cells. Confocal imaging, 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. 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 the Aldh2 gene. Based on our studies, ALDH2 can be a potential target for preventing AUD-associated tissue injury. 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 picro-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 levels of alanine and asparagine aminotransferase activities. However MT pretreatment significantly decreased the levels of TAA-mediated hepatic apoptosis and fibrosis in a time- and MT-dose dependent manner. In this 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 are trying to purify the acetylated proteins and determine their identities to further characterize their causal roles in TAA-mediated liver fibrosis. In parallel, we have 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 absent in the TAA-exposed Cyp2e1-null mice, demonstrating the direct role of CYP2E1 in TAA-mediated mitochondrial dysfunction and acute liver injury. We are preparing manuscripts on these results. These experimental rodent and cell studies not only represent examples of our translational research with a safe, dietary supplement to prevent liver injury and fibrosis caused by alcohol, DSS or TAA, but also suggest a conserved mechanism of gut and liver injury in different disease models through the gut and liver axis, as we recently reported.