No abstract

1 Omeprazole, a proton pump inhibitor therapeutically administered for the treatment of gastric ulcers, induces the expression of cytochromes P4501A1/2 (CYP1A1/2) through transcriptional activation mediated by the Ah (dioxin)-receptor. Primary cultures of hepatocytes isolated from rabbit, rat, mouse and human livers were compared for CYP1A1/2 mRNA inducibility by omeprazole (1 to 100 μM). 2 Primary cultures of human hepatocytes were the most sensitive to the inducing effects of omeprazole. Rabbit hepatocytes were the only other cells studied that showed induced CYP1A1/2 mRNA expression from a concentration lower than 100 μM (i.e., 10 μM). Rat hepatocytes were the least sensitive to omeprazole induction. The response of mouse hepatocytes to omeprazole treatment was variable, with CYP1A1/2 mRNA expression being induced in only two of the three cultures examined. 3 Differences in the time dependence of CYP1A1/2 mRNA expression were observed between species. In general, after treatment of hepatocytes with omeprazole the levels of CYP1A1 mRNA peaked prior to that of CYP1A2 mRNA. 4 Due to the interspecific variability of CYP1A mRNA inducibility by omeprazole, we conclude that human hepatocytes in culture are probably the only appropriate animal model for prediction of CYP1A induction in humans.

Our previous studies have demonstrated that a 2-[N-(acetoxyacetyl)amino]fluorene-transformed rat epithelial-like cell line, W8, contains a transcriptionally inactive alpha 2(I) gene with a hypermethylated promoter/first-exon region. We have cloned the rat promoter/first-exon region (-211 to +207) from W8 cells and their parent cell line, K16, which expresses alpha 2(I) collagen. There were no sequence differences between the clones from the two cell lines, indicating that a mutation was not responsible for transcriptional inhibition. The alpha 2(I) rat promoters were cloned upstream of the chloramphenicol acetyltransferase gene. Both constructs were equally active in both cell lines, suggesting that trans-activating factors for alpha 2(I) transcription are present in W8 cells. Finally, methylation of plasmids at all CpG sites with SssI methylase completely inhibited transcription using alpha 2(I) promoters, but methylation did not inhibit simian-virus-40 promoter-driven transcription. Certain methylation sites partially inhibit promoter activity. An HhaI methylation site inhibited transcriptional activity of the alpha 2(I) promoter 8-fold, whereas methylation at the HpaII site in the rat alpha 2(I) promoter did not decrease transcriptional activity. This provides further evidence that methylation at specific sites in the collagen alpha 2(I) promoter is responsible for the inactivation of transcription in W8 cells.

Interleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1β and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1β on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1–induced NF-κB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.