6533b7d3fe1ef96bd1260271

RESEARCH PRODUCT

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

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description

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYP1A1 mediated metabolism of polycyclic aromatic hydrocarbons (PAH). B[a]P and the purified (+)- and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer.