Expression of cytochrome P450 2E1 in normal human bronchial epithelial cells and activation by ethanol in culture

Abstract

Serum-free primary cultures of human bronchial epithelial cells and freshly isolated samples of human bronchial epithelium were used to investigate basal expression of the cytochrome P450 enzyme CYP2E1 and its activation or induction by ethanol in bronchial epithelial cells. The cultures consisted of > or =95% cells of epithelial characteristics as determined by transmission electron microscopy and immunohistochemical staining. Monolayers were obtained from explants over a period of several months via transfer of tissue into new dishes ('generations'1-5). Using RT-PCR analysis, basal expression of mRNAs coding for CYP2B7, CYP2F1 and CYP2E1 were detected in cultures from several donors. The basal expression of CYP2E1 protein and mRNA showed differences between the donors. The mRNA was detected even in cultures from higher generations and increased in some cultures over time. The CYP2E1 protein content was low and in most cultures of generations 2-5 could not be detected by immunoblot analysis of native protein extracts. Nevertheless, in some cases immunoreactive CYP2E1 protein was present in monolayers obtained from the fourth and fifth transfer (18-week 'generation'). CYP2E1 activity was measured via 6-hydroxylation of chlorzoxazone either by a destructive assay using cell lysate or by a non-invasive assay using the medium of cell cultures. In short-term cultured isolated bronchial epithelium, ethanol treatment increased CYP2E1 activity by up to 5-fold within 4 days but with inter-individual differences. In cells up to 4 weeks in culture, CYP2E1 activity remained inducible by a single dose of ethanol. Differentiated primary human cells in culture may be useful tools as model systems for the evaluation of CYP2E1-driven processes in man.

Xenobiotic-Induced Transcriptional Regulation of Xenobiotic Metabolizing Enzymes of the Cytochrome P450 Superfamily in Human Extrahepatic Tissues

“…Moreover, ethanol treatment increased CYP2E1 activity in short-term cultures of isolated bronchial epithelium [152]. CYP2E1 expression is controlled by methylation of dinucleotide CpG residues in the 5' end of the CYP2E1 gene in the lung [167].…”

“…Conflicting results have been published for some CYP enzymes concerning their expression in human lung tissue at the mRNA, protein and activity levels [22,146,150]. In addition, there are also CYP enzymes which have been detected at the mRNA level, but not at the protein level (such as CYP2F1 in bronchial epithelium [152]) or which have been detected at the protein, but not at the activity level (e.g. CYP3A4) [153].…”

“…CYP2E1 is expressed at the protein level in human lung or bronchial epithelial cells [150,152] and CYP2E1 activities (oxidation of butadiene, demethylation of N-nitrosodimethylamine, 6-hydroxylation of chlorzoxazone and hydroxylation of pnitrophenol) have been determined in human lung microsomes [165,166] and bronchial epithelial cells [152]. Moreover, ethanol treatment increased CYP2E1 activity in short-term cultures of isolated bronchial epithelium [152].…”

CAR and PXR expression and inducibility of CYP2B and CYP3A activities in rat and rabbit lungs

“…By far the greatest amount of information has been gathered concerning the induction of CYP 1A1; some reports have also shown that CYP 1B1, 2E1 and 2J may be inducible in the lung (Ding and Kaminsky, 2003;Hukkanen et al, 2002a;Runge et al, 2001;Xie et al, 2000).…”

A Toxicologist's Guide to the Preclinical Assessment of Hepatic Microsomal Enzyme Induction

“…In fact, posttranslational activity measurement is critically important for some isozymes. For example, the regulation of CYP2E1 expression is complex and appears to involve different mechanisms including gene transcription and posttranscriptional mRNA stabilization (Runge et al 2001). In addition to individual CYP isozyme activities, a complete assessment for induction should include cytochrome P450 content and NADPH-cytochrome P450 oxidoreductase (CytoR).…”