Stable Expression of Human Cytochrome P450 1B1 in V79 Chinese Hamster Cells and Metabolically Catalyzed DNA Adduct Formation of Dibenzo[a,l]pyrene

Abstract

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

“…The several-fold greater potency of both compounds in the cytotoxicity assay with the hCYP1A1-expressing line than in the hCYP1B1-expressing line is consistent with results of an earlier study with these cell lines [19]. The greater cytotoxicity with hCYP1A1 activation may be due to polar metabolites of DBP other than DBPDE that were observed in a previous study with the V79MZhCYP1A1 cells, but not with the V79MZhCYP1B1 cells [18]. The (+/−) DB-11,12-dihydrodiol is substantially more potent in the cytotoxicity assay than the parent compound DBP with the V79hCYP1A1 cells, whereas both compounds have similar cytotoxicity in the V79MZh1B1 cells as indicated by the similar IC 50 values for each compound.…”

“…The activity of hCYP1B1 was 7.1 pmol/min/mg in the V79MZh1B1 cell line and 8.2 pmol/min/mg in the hGSTA1-expressing derivative line V79MZh1B1/hGSTA1-11. Although the specific activity of hCYP1A1 was higher than that of hCYP1B1 using the BOMCC substrate, the enzyme expression levels have been previously shown to be comparable in the V79MZh1A1 and V79MZh1B1 cell lines (18). The activity of hGSTA1-1 expressed in the double-transfected V79MZh1A1/hGSTA1-39 cells was 586 nmol/min/mg, and was 1101 ± 19 nmol/min/mg in V79MZh1B1/hGSTA1-11 cells (Table 1).…”

“…Both human CYP1A1 and CYP1B1 catalyze the activation of DBP to reactive metabolites that bind to cellular macromolecules [18,19]; therefore we developed transgenic model cell lines with either hCYP1A1 or hCYP1B1 expression, either alone or with co-expressed hGSTA1, for use in determining the efficacy of GST protection.…”

“…V79MZ cells transfected with human CYP1A1 or human CYP1B1 were developed by Dr. Johannes Doehmer and colleagues at the Technical Institute for Toxicology, Munich, Germany [18,28]. The human GSTA1 cDNA (generously provided by Dr. C-P. Tu, Penn State University) was ligated into the modified CMV promoter-driven ΔpCEP4 expression vector, and generation of the V79MZh1A1 or V79MZh1B1 cell lines stably expressing human glutathione-S-transferase A1 was by the method of calcium phosphate-mediated transfection followed by hygromycin selection, verification of similar growth rates, and GST analysis as described previously [26].…”

“…Human cytochrome P450-1A1 (hCYP1A1) and cytochrome P450-1B1 (hCYP1B1), and to a lesser extent other CYPs, are known to catalyze the stereospecific biotransformation of DBP to yield predominantly two of the four possible stereoisomers, (+)-syn-DBPDE (S,R,S,R) and (−)-anti-DBPDE (R,S,S,R) [15,16]. The (−)-anti-DBPDE stereoisomer was responsible for the majority of DBPDE-DNA adducts, and was produced as a greater proportion of the total metabolites by hCYP1B1 as compared to hCYP1A1 [17,18]. Interestingly, however, activation via hCYP1A1-mediated metabolism of DBP elicited stronger cytotoxic effects [19], possibly due to polar DNA adducts of metabolites produced only in cells expressing hCYP1A1.…”