We have previously demonstrated multiple regulatory mechanisms for ethanol inducible cytochrome P450 2E1 (CYP2E1): induction via transcription, mRNA stabilization, activation of mRNA translation and protein stabilization, suppression via transcription, mRNA degradation and protein degradation. We recently reported a transcriptional suppression of CYP2E1 gene by an exogenous compound, YH439. During this fiscal year, we studied the biological role of CYP2E1 in acetaldehyde- protein adduct formation in rats chronically treated with alcohol. The 37 kDa acetaldehyde adduct protein was detected in alcohol treated rats while it is absent in pair-fed control animals. During the ethanol treatment, the level of CYP2E1 activity was significantly elevated while other enzymes such as alcohol or aldehyde dehydrogenase involved in alcohol or acetaldehyde metabolism were unchanged. Co-treatment of YH439 with ethanol markedly reduced the level of the 37 kDa adduct, suggesting that this adduct is most likely produced by a CYP2E1- dependent mechanism. In addition, we tested the protective effect of YH439 on the apoptosis of C6 glioma cells caused by acetaminophen (AAP, Tylenol) and other CYP2E1 substrates. The major advantage of C6 glioma cells over other established cell lines is that we dont need to transfect CYP2E1 cDNA and select the stable transformants prior to use, since CYP2E1 is known to be expressed in C6 glioma cells despite very low level of expression. Treatment of AAP or other CYP2E1 substrates including ethanol caused time and dose-dependent apoptosis of C6 cells as evidenced by classical DNA fragmentation. In C6 cells, c-jun N- terminal protein kinase activity (JNK) was selectively and transiently activated after AAP treatment. The activity of p-38 protein kinase or mitogen activated protein kinase remained unchanged. The selective activation of the JNK pathway by AAP is similar to the mechanisms of cell death caused by 4-hydroxynonenal (HNE) or carbon tetrachloride, another substrates of CYP2E1. Furthemore, pretreatment of YH439 (10 uM) of C6 cells not only reduced the CYP2E1 level but also prevented the apoptosis observed at 18 and 36 hr post-AAP treatment. Consistent with the in vitro data, JNK was selectively activated in the mouse liver treated with AAP or carbon tetrachloride. To further elucidate the relationship between the c-jun kinase activation and apoptosis of cultured cells, we are studying the role of various caspases upon treatment of CYP2E1 substrates including ethanol and arachidonic acid. In addition, changes in the level of DNA-adducts in the CYP2E1- transfected cells and C6 glioma cells are being determined by HPLC. - neurosciences, health & behavior, molecular genetics, cirrhosis, hepatology, cell and molecular biology, alcohol, metabolism, apoptosis""""""""
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