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" Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity "
Rahman, Mohammad Arifur
Kumar, Santosh
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Latin Dissertation
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English
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| Record Number
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1051716
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TL50833
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| Main Entry
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Rahman, Mohammad Arifur
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| Title & Author
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Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity\ Rahman, Mohammad ArifurKumar, Santosh
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| College
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The University of Tennessee Health Science Center
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| Date
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2019
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| Degree
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Ph.D.
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2019
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| Note
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104 p.
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| Abstract
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Cytochrome P450 2E1 (CYP2E1)-mediated hepatic and extra-hepatic toxicity is of significant clinical importance. Diallyl sulfide (DAS) has been shown to prevent xenobiotics such as alcohol- (ALC/ETH), acetaminophen- (APAP) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. The objective of the first project was to find potent DAS analogs which can replace DAS as a research tool or as potential adjuvant therapy in CYP2E1-mediated pathologies. At first, we selected seven commercially available compounds that are similar to DAS (DAS analogs) based on computational docking study. We performed ligand-CYP2E1 docking study to determine the binding mode and binding energy. The analysis suggested a relative potential for these DAS analogs as CYP2E1 inhibitor. We then performed comprehensive inhibition kinetics of DAS analogs and determined the relative IC50, Ki, and types of inhibition compared to that of DAS. The results showed that compared to DAS, diallyl ether (DE) and allyl methyl sulfide (AMS) have lower Ki values (3.1 and 4.4 µM, respectively vs. 6.3 µM for DAS) and IC50 values (6.3 and 11.4 µM, respectively vs 17.3 µM for DAS). However, thiophene (TP) showed similar inhibitory capacities to that of DAS, and four other DAS analogs showed lower potency than DAS. From this project, we found relatively more potent inhibitors of CYP2E1 which can potentially replace DAS. Next, we investigated these analogs of DAS for their improved toxicity profiles and their effectiveness in reducing ALC- and APAP-induced toxicity, and HIV-1 replication. In this project, we evaluated the toxicity and efficacy of these analogs using hepatocytes, monocytes, and astrocytes where CYP2E1 plays an important role in xenobiotic-mediated toxicity. Our results showed that thiophene, allyl methyl sulfide, diallyl ether, and 2-prop-2-enoxyacetamide are significantly less cytotoxic than DAS in these cells. Moreover, these analogs reduced ALC- and APAP-induced toxicity in hepatocytes and HIV-1 replication in monocytes more effectively than DAS. Overall, our findings are significant in terms of using these DAS analogs as a tool in vitro and in vivo, especially to examine chronic xenobiotic-induced toxicity and disease pathogenesis that occurs through the CYP2E1 pathway. We also investigated the role of plasma exosomal CYP2E1 in mediating ALC- and APAP-induced toxicity in hepatic and monocytic cells. Cellular CYP2E1 is well-known to mediate ALC- and APAP-induced toxicity in hepatic and extra-hepatic cells. Although exosomes have been gaining importance in understanding the mechanism of intra- and inter-cellular communication, the functional role of drug metabolizing CYP enzymes in human plasma exosomes are yet to be explored. We recently reported that human plasma-derived exosomes contain substantial level of functional CYP2E1. In this project, we investigated the potential role of plasma exosomal CYP2E1 in mediating ALC and APAP induced toxicity. We treated hepatic and extra-hepatic (monocytic) cells with exosomes ± ALC/APAP. We observed that the plasma exosomes containing CYP2E1 cargo further exacerbated ALC- and APAP-induced toxicity in both hepatic and monocytic cells. Further, both exosomes- and ALC/APAP-induced toxicity was reduced/abolished by CYP2E1 inhibitor, DE. However, only ALC-, but not exosome-induced toxicity was reduced/abolished by CYP2E1 siRNA. These findings suggest that ALC/APAP-induced toxicity in the presence of exosomes are mediated, at least in part, by CYP2E1 enzyme. To validate these in vitro findings, we characterized plasma exosomal contents in a binge-drinking animal model and their effect on ALC/APAP-induced toxicity in monocytic cells. Our results showed that ALC exposure caused a significant induction of the plasma exosomal CYP2E1 level in a binge drinking murine model. These exosomes containing increased levels of CYP2E1 caused significant toxicity in monocytic cells compared to exosomes derived from control mice. Overall, our results showed an important role of exosomal CYP2E1 in exacerbating ALC- and APAP-induced toxicity. The study is significant in terms of understanding the role of exosomal CYP2E1 in cell-cell interactions, and their effects on drug-induced toxicity. In summary, we have identified several superior inhibitors of CYP2E1 which can replace DAS as research tool and we have demonstrated for the first time that exosomal CYP2E1 may contribute to ALC- and APAP-induced cytotoxicity.
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| Descriptor
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Pharmaceutical sciences
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Pharmacology
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Toxicology
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Kumar, Santosh
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The University of Tennessee Health Science Center
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