Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

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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 inactivated the ALDH2 activity, resulting in elevated amounts of 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 organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, adduct formation, etc that usually occur after exposure to alcohol and nonalcoholic substances or under pathological conditions. In the past, we showed an important role of CYP2E1 in stimulating various PTMs in rodent tissues and demonstrated their causal roles in tissue injury by evaluating the time-dependent events of PTMs and actual tissue injury in the presence or absence of an antioxidant N-acetylcysteine (NAC) or a specific CYP2E1 inhibitor chlormethiazole (CMZ). However, PTMs and functional alterations of covalently-modified proteins are not restricted to the liver. Our recent results indicate that similar alterations can take place in extra-hepatic tissues such as gut and brain. Therefore, we have also characterized the nitrated and/or p-JNK-target proteins in gut and brain of wild-type (WT) mice, WT rats and HIV-1 transgenic (Tg) rats where elevated levels of nitroxidative stress and p-JNK are observed after exposure to binge alcohol, fructose, or a high fat diet (HFD) containing high cholesterol. We previously reported the critical role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and inflammatory liver injury in WT mice and rats but not in Cyp2e1-null mice. We have continuously studied the mechanism of barrier dysfunction by investigating the role of different PTMs of the junctional complex proteins including tight junction (TJ), adherent junction (AJ), and desmosome in alcohol-induced gut leakiness and endotoxemia. Binge alcohol exposure significantly increased the levels of intestinal CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, serum endotoxin and fecal albumin contents, suggesting elevated apoptosis of enterocytes, gut leakiness and endotoxemia. Differential mass-spectral analyses of the TJ-enriched fractions of gut epithelial cells showed that several TJ, AJ and desmosome proteins were decreased in alcohol-exposed rats compared to controls. Immunoblots revealed that the levels of TJ proteins (claudin-1, occludin and ZO-1), AJ proteins (-catenin and E-cadherin) and desmosome plakoglobin were markedly decreased in binge-alcohol exposed rats, WT mice, and autopsied human ileums but not in Cyp2e1-null mice. We are studying the causal role of nitration or phosphorylation of junctional complex proteins in elevated gut leakiness, leading to endotoxemia and liver injury. We recently reported an important role of CYP2E1 in nonalcoholic steatohepatitis (NASH) and aging-related liver inflammation and fibrosis. We continued studying the role of CYP2E1 in liver fibrosis promoted by HFD containing high cholesterol for 24 weeks. Liver histology showed that only WT fed HFD (WT-HFD) developed NASH and fibrosis with elevated levels of fibrosis marker proteins but not in HFD-fed Cyp2e1-null mice. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in HFD-fed WT. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in HFD-fed WT that also showed the highest levels of serum endotoxin, TLR-4 levels, and inflammatory markers. These results demonstrate that CYP2E1 plays a permissive role for other proteins/genes to stimulate HFD-mediated liver fibrosis. From 2016, we have started a new project to characterize extracellular vesicles (EVs) in circulating blood secreted from the damaged liver in mice and rats after exposure to binge alcohol (ethanol) or acetaminophen (APAP). The amounts of total and liver-specific proteins in circulating EVs from mice exposed to APAP were significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs revealed that the amounts of liver-specific proteins were increased in APAP-exposed mice compared to those of controls. Consistently, binge ethanol exposure markedly elevated liver-specific proteins in circulating EVs from WT mice and patients with alcoholic hepatitis, compared to their respective controls. The number of EVs and the amounts of EV CYP2E1 and other P450 isoforms were markedly elevated in both alcoholics and alcohol-exposed rats and WT mice, but not in the corresponding Cyp2e1-null mice. The amounts of EV CYP2E1 depended on increased oxidative and ER stress, since their levels were decreased by co-treatment with NAC or CMZ, but increased by an ER stress inducer thapsigargin. Moreover, cell death rates were elevated when recipient primary hepatocytes were exposed to exogenous EVs from alcohol-exposed rodents and alcoholics, showing that exogenous EVs from alcohol-exposed mammals are functional in cell communication and can promote cell death by activating the apoptosis signaling pathway. These results demonstrate an important role of CYP2E1 in elevating EV CYP2E1 through increased oxidative and ER stress. Elevated EV-CYP2E1 can be used as a potential biomarker for drug- and alcohol-mediated liver injury. Due to our experience in CYP2E1-related nitroxidative stress, PTMs, and apoptosis signaling pathway in the liver, we have extended our study on the mechanisms of aging-dependent neuronal apoptosis and neurodegeneration in HIV-1 Tg rats. Histological and immunohistochemical analyses showed decreased density of neuronal cells with elevated astrogliosis in 5-month old HIV-1 Tg rats compared to the age- and gender-matched WT. Increased levels of nitroxidative stress marker proteins, such as CYP2E1, iNOS, the stress-activated JNK and p38K, nitrated proteins, hyper-phosphorylated tau, and amyloid plaques, were consistently observed in HIV-1 Tg rats. Moreover, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyper-phosphorylation and tauopathy, accompanied with elevated levels of many apoptosis-related proteins, including Bax and cleaved (activated) caspase-3. These results collectively indicate that nitroxidative stress accompanied by activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyper-phosphorylation are responsible for increased neuronal cell death and neurodegeneration in 5-month old HIV-Tg rats. Based on our basic mechanistic studies, we conducted translational research by evaluating the beneficial effects of dietary supplements, including walnut and indole-3-carbinol (I3C), against alcoholic fatty liver disease (AFLD) and NASH in animal models. Daily supplementation with physiologically relevant levels of walnut (20% energy-derived) or I3C (40 mg/kg/day orally) significantly prevented HFD (45% energy-derived) or AFLD by activating sirtuin-1 and AMP-kinase with restoration of antioxidant glutathione, while decreasing CYP2E1, lipid peroxidation, and protein nitration. Walnut or I3C also decreased the elevated levels of activated p-JNK, p-p38K and hepatocyte apoptosis. These hepatic improvements by walnut or I3C also coincided with reduction of HFD- or alcohol-induced inflammation of adipose tissues and macrophage infiltration. Finally, we also plan to study the beneficial effects of n-3 fatty acids on liver steatosis and obesity in rodent models.