It is well established that ethanol-inducible cytochrome P450 2E1 (CYP2E1) is induced by alcohol drinking in humans and animal models. In the past, we have cloned the genes for human and rat CYP2E1 and demonstrated multiple regulatory mechanisms: induction via transcription after birth, mRNA stabilization in ketotic states, activation of mRNA translation and protein stabilization by CYP2E1 substrates, suppression via transcription by YH439, mRNA degradation and protein degradation by carbon tetrachloride. Increased CYP2E1 after chronic alcohol consumption or certain pathological states leads to elevated production of acetaldehyde, reactive oxygen species, free radical metabolites and lipid peroxides while reducing cellular anti-oxidants such as glutathione. Therefore, cells or tissues with increased CYP2E1 become more susceptible to damage or cell death. In fact, other investigators have already demonstrated that hepatoma HepG2 cells or PC12 cells transfected with the CYP2E1 cDNA are far more sensitive to cell death than the non-transfected counterparts after exposure to various CYP2E1 substrates such as ethanol, acetaminophen (AAP), carbon tetrachloride, and arachidonic acid. Despite numerous studies on cell death by CYP2E1 substrates, the signaling mechanisms by which these CYP2E1 substrates exert their toxicological effects are unknown. Therefore, we continued to investigate the signaling mechanisms during apoptosis caused by various CYP2E1 substrates. We have been particularly interested in the potential role of mitogen activated protein (MAP) kinases involved in the early signal transduction pathway during apoptosis and the enzymes involved in the cell survival pathway. These enzymes include: c-Jun N-terminal protein kinase (JNK), p38 MAP kinase, extracellular-signal regulated protein kinase (ERK), phophatidylinositol 3-kinase, Akt protein serine/threonine kinase and their upstream kinases or downstream target proteins including various caspases and bax/bcl-2 proteins involved in cell apoptosis/survival processes. Our initial hypotheses were: CYP2E1 substrates and their metabolites would activate the JNK and p38 MAP kinase while suppressing the enzymes involved in the cell survival pathway. In addition, inhibitors of CYP2E1 and other enzymes elevated during apoptosis effectively prevent cell death caused by CYP2E1 substrates. Our results showed that treatment of target cells (C6 glioma and PC12 cells) with AAP or other CYP2E1 substrates caused time- and concentration-dependent apoptosis of these cells as evidenced by DNA fragmentation and fluorescent staining of the apoptotic cells. In these cells, c-jun N-terminal protein kinase activity (JNK) was selectively and transiently activated after treatment with AAP or 4-hydoxynonenal (HNE), a cytotoxic lipid aldehyde. Unexpectedly, these toxic compounds did not activate p38 MAP kinase or affect the ERK activity. The selective and transient activation of the JNK in target cells was critical for their apoptosis since blockade of the JNK pathway by transfecting the cDNA of a dominant negative mutant of JNK or SEK-1 significantly blocked the apoptosis. In addition, our results showed that 10 uM YH439, a transcriptional inhibitor of the CYP2E1 gene, not only reduced the CYP2E1 and JNK activities but also suppressed the apoptosis caused by CYP2E1 substrates, indicating the critical role of CYP2E1-dependent metabolism during apoptosis. The non-involvement of p38 MAP kinase in the apoptosis was further confirmed by the use of its selective inhibitor, SB203580. Our results, therefore, are in contrast with other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor alpha and interleukin 1-beta, all of which can activate P38 MAP kinase along with the JNK. Our results from in vitro cultured cells were replicated in in vivo models where AAP and carbon tetrachloride selectively and transiently activated the JNK and its upstream kinases, SEK-1 and MEK. Because of the selective activation of JNK by CYP2E1 substrates, we are investigating the potential activation of a phospho-protein phosphatase, which specifically dephosphorylates phopho-P38 MAP kinase, after treatment with CYP2E1 substrates.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Intramural Research (Z01)
Project #
1Z01AA000036-14
Application #
6431356
Study Section
(LMBB)
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Alcohol Abuse and Alcoholism
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Moon, Kwan-Hoon; Upreti, Vijay V; Yu, Li-Rong et al. (2008) Mechanism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-mediated mitochondrial dysfunction in rat liver. Proteomics 8:3906-18
Song, Byoung-Joon; Moon, Kwan-Hoon; Olsson, Nils U et al. (2008) Prevention of alcoholic fatty liver and mitochondrial dysfunction in the rat by long-chain polyunsaturated fatty acids. J Hepatol 49:262-73
Moon, Kwan-Hoon; Abdelmegeed, Mohamed A; Song, Byoung-Joon (2007) Inactivation of cytosolic aldehyde dehydrogenase via S-nitrosylation in ethanol-exposed rat liver. FEBS Lett 581:3967-72
Moon, Kwan-Hoon; Hood, Brian L; Kim, Bong-Jo et al. (2006) Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats. Hepatology 44:1218-30
Kim, Bong-Jo; Hood, Brian L; Aragon, Richard A et al. (2006) Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells. Proteomics 6:1250-60
Lee, Yun-Sik; Wan, Jie; Kim, Bong-Jo et al. (2006) Ubiquitin-dependent degradation of p53 protein despite phosphorylation at its N terminus by acetaminophen. J Pharmacol Exp Ther 317:202-8
Wan, Jie; Ernstgard, Lena; Song, Byoung J et al. (2006) Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats. J Pharm Pharmacol 58:51-61
Moon, Kwan-Hoon; Kim, Bong-Jo; Song, Byoung J (2005) Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation. FEBS Lett 579:6115-20
Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap et al. (2004) Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism. J Biol Chem 279:39968-74
Ryu, Jisung; Lee, Moon-Soon; Na, Jin Gyun et al. (2004) Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants. Environ Toxicol Chem 23:1004-11

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