Role of Mitochondrial Cytochrome P450 2E1 in Healthy and Diseased Liver

Cytochrome P450 2E1 (CYP2E1) is pivotal in hepatotoxicity induced by alcohol abuse and different xenobiotics. In this setting, CYP2E1 generates reactive metabolites inducing oxidative stress, mitochondrial dysfunction and cell death. In addition, this enzyme appears to play a role in the progression of obesity-related fatty liver to nonalcoholic steatohepatitis. Indeed, increased CYP2E1 activity in nonalcoholic fatty liver disease (NAFLD) is deemed to induce reactive oxygen species overproduction, which in turn triggers oxidative stress, necroinflammation and fibrosis. In 1997, Avadhani’s group reported for the first time the presence of CYP2E1 in rat liver mitochondria, and subsequent investigations by other groups confirmed that mitochondrial CYP2E1 (mtCYP2E1) could be found in different experimental models. In this review, we first recall the main features of CYP2E1 including its role in the biotransformation of endogenous and exogenous molecules, the regulation of its expression and activity and its involvement in different liver diseases. Then, we present the current knowledge on the physiological role of mtCYP2E1, its contribution to xenobiotic biotransformation as well as the mechanism and regulation of CYP2E1 targeting to mitochondria. Finally, we discuss experimental investigations suggesting that mtCYP2E1 could have a role in alcohol-associated liver disease, xenobiotic-induced hepatotoxicity and NAFLD.

Cytochrome P450 2E1 Predicts Liver Retrieval from Donation After Circulatory Death Using Air-Ventilated Normothermic Machine Perfusion

The optimal oxygen concentration is unclear for normothermic machine perfusion (NMP) of livers from donation after circulatory death donors (DCD). Our purposes were to investigate the effect of air-ventilated NMP on liver retrieval from DCD rats, and to analyze the underlying mechanism. Normothermic liver perfusion was performed using the NMP system with either air ventilation or oxygen ventilation for 2h in the rat liver following warm ischemia and cold ischemia preservation. Proteomics and metabolomics were used to reveal the significant molecular networks. The bioinformation analysis was validated by administering peroxisome proliferator activator receptor-γ (PPARγ) antagonist and agonist via ex vivo perfusion circuit in the air-ventilated NMP. Results showed that air-ventilated NMP conferred a better functional retrieval and a less inflammatory response in the rat DCD liver; integrated proteomics and metabolomics analysis indicated that intrahepatic docosapentaenoic acid (DPA) downregulation and upregulation of cytochrome P450 2E1 (CYP2E1) expression and activity were associated with DCD liver retrieval with air-ventilated NMP; PPARγ antagonist worsened liver function under air-oxygenated NMP whereas PPARγ agonist played the opposite role. In conclusion, air-ventilated NMP confers a better liver retrieval from DCD rats through the DAP-PPARγ-CYP2E1 axis; CYP2E1 activity provides a biomarker of liver retrieval from DCD.

Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis–transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

Drug Repurposing in Alternative Medicine: Sochehwan, a Polyherbal Traditional Korean Digestant, Protects against Alcoholic Steatohepatitis by Regulating Cytochrome P450 2E1 Expression

Sochehwan (SCH) is an herbal prescription from traditional oriental medicine and is currently used to treat digestive ailments. In a previous study, SCH was found to have the potential to attenuate metabolic syndrome (MetS) by activating AMPK and downstream signaling. From the view of drug repurposing, the efficacy of SCH on alcoholic liver injury is implied in classic medical texts but is yet to be proven. C57BL/6J mice were pre-treated with SCH orally for 5 days and challenged by providing a pair-fed Lieber DeCarli diet containing alcohol for 20 days. Hepatic enzyme and triglyceride levels and endoplasmic reticulum (ER) stress-related markers were analyzed. Moreover, mitogen-activated protein kinases (MAPKs) and cytochrome P450 2E1 (CYP2E1) levels were determined. CYP2E1-transfected HepG2 cells were used to test the cytoprotective efficacy of SCH against the adverse effects of alcohol in vitro. In mice, SCH administration notably reduced hepatic enzyme activity and neural lipid levels. Furthermore, ER-stress markers and MAPK phosphorylation were reduced due to ROS suppression, which was attributed to decreased CYP2E1 expression in liver tissue. In addition, SCH successfully protected CYP2E1-transfected HepG2 cells against ethanol. Our findings suggest SCH attenuated alcohol-induced liver injury by inhibiting CYP2E1 expression and indicate drug repurposing should be considered as a valuable option for drug development in traditional herbal medicines.

In-silico Inhibitory Potential of Triphala Constituents Against Cytochrome P450 2E1 for the Prevention of Thioacetamide-induced Hepatotoxicity

Background: Triphala, which is a combination of fruits of Terminalia chebula, Terminalia bellerica and Embilica officinalis generally recommended as herbal drug formulation in the Indian traditional medicine system. Study Design: To study the in-silico inhibitory potential of Triphala constituents against cytochrome P450 2E1 (CYP2E1) for the prevention of Thioacetamide-induced Hepatotoxicity Place and Duration of Study: The work has been performed at MUP's College of Pharmacy (B Pharm), Degaon, Risod, Washim, Maharashtra, India in between February 2021 to May 2021. Methodology: We have studied the inhibitory potential of Triphala on CYP2E1 by applying molecular docking tools. The major chemical constituents of Triphala i.e. gallic acid, chebulic acid, ellagic acid, epicatechin, syringic acid, and ascorbic acid were docked on CYP2E1. Results: Docking results revealed the very good inhibitory potential of Triphala in terms of binding affinity towards CYP2E1. All the chemical constituents have formed at least 2 and at most 6 hydrogen bonds with the crystal structure of CYP2E1. The binding energies (kcal/mol) of gallic acid, chebulic acid, ellagic acid, epicatechin, syringic acid, and ascorbic acid are -6.1, -7.1, -9.1, -8.3, -6.3, and -5.7, respectively. Ellagic acid has formed strong hydrogen bonds with Thr-303 and Thr-304 with bond length of 1.98 A0 and 2.26 A0 which confirms the excellent inhibition of CYP2E1. Conclusion: These findings can be used to control the CYP2E1-facilitated biotransformation and drug interactions in the development of new chemical entities. In future, these phytoconstituents can be used as lead molecules to overcome the cancer associated with oxidative stress resulting from the hyperactivity of CYP2E1.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

Combined Effects of Amino Acids in Garlic and Buna-Shimeji (Hypsizygus marmoreus) on Suppression of CCl4-Induced Hepatic Injury in Rats

The combination of the garlic-derived amino acid, S-allyl-l-cysteine sulfoxide (ACSO), and ornithine or arginine on CCl4-induced hepatic injury was examined. After investigating the effectiveness of the mixture of ACSO and ornithine or arginine in preventing hepatic injury in vivo, an extract rich in ACSO and ornithine was prepared by converting arginine in garlic to ornithine by arginase from Hypsizygus marmoreus (buna-shimeji), after screening the productivity of ornithine among 12 kinds of mushrooms. Co-administration of ACSO with ornithine or arginine suppressed the increase in aspartate transaminase, alanine transaminase, and thiobarbituric acid reactive substance, and the decrease in glutathione S-transferase and cytochrome p450 2E1 activities after CCl4 injection more effectively than a single administration of ACSO. All extracts prepared from garlic and buna-shimeji with low and high contents of ACSO and arginine or ornithine significantly suppressed CCl4-induced hepatic injury in rats. Considering that ACSO is tasteless, odourless, and enhances taste, and ornithine has a flat or sweet taste and masks bitterness, the extract rich in ACSO and ornithine from garlic and buna-shimeji could be considered a potential antioxidant food material that can be added to many kinds of food to prevent hepatic injury.