Post-translational inhibition of cytochrome P-450 2E1 expression by chlomethiazole in Fao hepatoma cells.
Chlomethiazole (CMZ) is a sedative and anticonvulsant drug that has been shown to be an efficient transcriptional inhibitor of expression of rat hepatic ethanol-inducible cytochrome P-450 2E1 (CYP2E1). Recent results have shown that human CYP2E1 expression in vivo is almost completely inhibited in control subjects and in alcoholic patients treated with CMZ. In the present investigation, we evaluated the mode of action of CMZ on CYP2E1 expression in Fao rat hepatoma cells. Transcriptional activity of the CYP2E1 gene was monitored using reverse transcription-polymerase chain reaction-based quantification of CYP2E1 heterologous nuclear RNA (hnRNA) against a mimic DNA standard, mRNA was detected by Northern blotting, enzyme protein was detected by Western blotting, and CYP2E1-dependent catalytic activity was detected by assay of chlorzoxazone-6-hydroxylation. Six hours after CMZ treatment, the levels of both CYP2E1 protein and catalytic activity were concomitantly reduced at an IC50 value of about 5 microM. Ethanol treatment of the cells caused a 2-fold induction of CYP2E1 protein levels, which was inhibited by CMZ. Change of medium unexpectedly caused an increase in CYP2E1 gene transcription 4 h later, as monitored by quantitative determination of CYP2E1 hnRNA. However, CMZ failed to influence the expression of CYP2E1 hnRNA or mRNA both constitutively and after medium change, indicating no effect on gene transcription or mRNA synthesis/stability. Cycloheximide treatment of the cells did not abolish the inhibitory action of CMZ, further indicating an action at the post-translational level; in addition, CMZ inhibited CYP2E1 expression in V79 cells with stably expressed CYP2E1 under the control of the SV40 promoter. The data indicate that the CYP2E1 gene is transcriptionally activated in response to medium change and that CMZ, apart from a transcriptional inhibitor of CYP2E1 expression, acts in addition as an efficient high-affinity post-translational inhibitor of CYP2E1, probably due to an allosteric destabilization of the enzyme. This indicates a very rapid and effective CMZ-mediated inhibition of CYP2E1 in vivo.
Simi
A
A
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Ingelman-Sundberg
M
M
D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D015152:Blotting, Northern; D002719:Chlormethiazole / Q000494:pharmacology*; D002753:Chlorzoxazone / Q000494:pharmacology; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D000927:Anticonvulsants; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D002719:Chlormethiazole; D003513:Cycloheximide; D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone