Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

National Institute On Alcohol Abuse And Alcoholism

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Abstract

My lab members have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase-2 (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 inactivates the ALDH2 activity, resulting in the greater accumulation of toxic acetaldehyde and lipid peroxides, which can contribute to tissue injury. In addition, CYP2E1-mediated nitroxidative stress can stimulate different types of post-translational modifications (PTMs) of cellular proteins, leading to mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and apoptosis/necrosis of parenchymal cells, contributing to tissue/organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, acetylation, adduct formation, etc. All these PTMs usually take place shortly after exposure to potentially toxic agents, such as binge alcohol, CYP2E1 substrates, 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 by using wild-type (WT) versus the age- and sex-matched Cyp2e1- knockout (KO) mice. We also reported that these PTMs and functional alterations of covalently-modified proteins are observed in the liver and other extra-hepatic tissues, such as the gut and kidney. We recently reported the critical role of CYP2E1 in binge alcohol-mediated intestinal barrier dysfunction (leaky gut), endotoxemia, and inflammatory liver injury in rats and WT mice. In contrast, Cyp2e1-KO mice were resistant to binge alcohol-induced gut leakiness and hepatotoxicity. We continuously studied the mechanisms of gut epithelial barrier dysfunction by investigating the role of different PTMs (e.g., nitration) of the junctional complex proteins, such as tight junction (TJ), adherent junction (AJ), and desmosome, in alcohol-exposed rodents and T84 human colon cells. Binge alcohol exposure significantly decreased the amounts of intestinal TJ/AJ proteins, while it increased the levels of gut and liver CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, and serum endotoxin, suggesting elevated oxidative stress, gut leakiness, endotoxemia, and liver injury. Our results showed that gut TJ and AJ proteins were decreased through proteasomal degradation after they were nitrated and conjugated with ubiquitin in alcohol-exposed rodents and T84 colon cells. The mechanisms of leaky gut, endotoxemia and advanced liver disease (or fibrosis) by a nonalcoholic substance fructose were also studied. Our mechanistic studies revealed that treatment with 30% fructose in drinking water for 8 weeks caused gut leakiness, endotoxemia and liver fibrosis in rats and WT mice through increased apoptosis of enterocytes and nitration of the gut TJ/AJ proteins. Unexpectedly, the levels of CYP2E1 were increased in the fructose-exposed rats and WT mice possibly by its protein stabilization through an increased abundance of the ethanol-producing gut bacteria. However, Cyp2e1-null mice were resistant to fructose-induced leaky gut and liver disease. These results suggest another important role of CYP2E1 in fructose-induced gut leakiness, endotoxemia, and inflammatory liver disease. We recently reported a permissive role of CYP2E1 in aging-related liver and kidney fibrosis in 1617 month-old WT mice since the age- and sex-matched Cyp2e1-KO mice were normal without signs of liver/kidney fibrosis. The levels of nitrated proteins were significantly elevated in the aged WT mice, although the identities and functional roles of these nitrated proteins are unknown. To study the causal roles of the nitrated proteins in promoting liver fibrosis, we chose to analyze the nitrated proteins elevated in the middle-aged WT mice which did not show any signs of tissue fibrosis. Thus, we are in the middle of purifying the nitrated proteins by the method we developed to determine their identities and study their functional roles in liver fibrosis. In another study, we also observed liver fibrosis in thioacetamide (TAA)-exposed rats. Pretreatment with a physiologically relevant dose of melatonin (MT) significantly attenuated the levels of TAA-mediated liver fibrosis, although the preventive mechanisms of MT are unknown. Since we have experience in characterizing PTMs and are interested in finding causal factors in liver fibrosis, we have studied the patterns of different PTMs in the liver of TAA-exposed rats without or with MT pretreatment. Since changes in gut microbiota such as Ruminococcus were also reported to correlate with the degree of liver fibrosis possibly by regulating the levels of short-chain fatty acids, we are also in the middle of determining the composition and abundance of gut microbiota in TAA MT-exposed rats or aged-WT mice versus Cyp2e1-KO counterparts. Moreover, we studied the mechanisms of TAA-induced acute liver injury by focusing on the time-dependent PTMs, leading to mitochondrial dysfunction and hepatotoxicity. Our results showed that TAA promptly increased oxidative stress and protein nitration and/or phosphorylation, accompanied with suppression of the activities of a few mitochondrial proteins, including ALDH2, likely leading to mitochondrial dysfunction and hepatotoxicity observed at later time points in WT mice. However, these changes were virtually absent in the TAA-exposed Cyp2e1-KO mice, demonstrating the direct role of CYP2E1 in TAA-mediated mitochondrial dysfunction and acute liver injury. Two or three manuscripts on these results are being prepared. Based on the results of our mechanistic studies, we have conducted translational studies to evaluate the beneficial effects of natural phytochemicals on alcohol-mediated gut barrier dysfunction and inflammatory liver injury and investigated their molecular mechanisms of protection. In collaboration with a former lab member Dr. Young-Eun Cho, we studied the beneficial effect of physiologically relevant doses of ellagic acid (EA), a major component of pomegranate, on binge alcohol-mediated leaky gut and acute liver injury. Pretreatment with EA via oral administration for 2 weeks significantly prevented binge alcohol-mediated gut leakiness and alcoholic fatty liver, as determined by histology, measurements of serum markers of liver injury, immunoblots of gut TJ/AJ proteins, and other analyses. Our results clearly showed that binge ethanol exposure increased oxidative stress and apoptosis of enterocytes with decreased amounts of intestinal TJ/AJ proteins, leading to gut leakiness, endotoxemia, and acute liver injury in mice. Binge alcohol also markedly changed the composition and abundance of gut microbiota (i.e. gut dysbiosis), which can contribute to intestinal barrier dysfunction, as we recently reviewed. Pretreatment with a physiologically relevant dose of EA (60 mg/kg/day in mice), as an antioxidant, significantly prevented binge ethanol-mediated oxidative stress and cell death markers, gut barrier dysfunction, endotoxemia, and acute liver injury. Furthermore, EA pretreatment attenuated alcohol-mediated gut dysbiosis and this prevention may also contribute to improvement against gut leakiness and acute liver injury in mice. Our results represent another example of our translational study but also strongly indicate that alcohol-mediated gut leakiness and inflammatory fatty liver disease through the gut-liver axis can be alleviated with many safe, dietary antioxidants.