Molecular studies on the toxifying effects by genetically engineered cytochromes P450.
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Biomedical subjects
V Soballa
Publications and source records attributed to V Soballa.
Stable expression of human cytochrome P450 1B1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene.
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.
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Genetically engineered mammalian cells and applications.
In general, cells genetically engineered for stable and defined expression of xenobiotic-metabolizing enzymes are useful tools whenever a metabolism-related problem in toxicology and pharmacology is to be solved. It is the genetic and phenotypic nature of a given cell that determines its applicability. Mammalian cells have useful characteristics not given in bacterial, yeast or insect cells, which also may express xenobiotic-metabolizing enzymes. It is the problem to be solved and the question to be answered which determine the optimal choice for the best-suited expression system. There may even be subtle differences between mammalian cells of different species and organ origin, which might play a role in choosing a mammalian expression system. Thus, the level and specificity of the xenobiotic-metabolizing enzyme, the experimental testing conditions, and the biological endpoints present in a chosen cell are the most important criteria to be observed in the application of the mammalian expression systems.
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Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells.
V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP have been shown to serve as analytical tools for studying metabolism related problems in toxicology and pharmacology. Here, the application of rat and human CYP1A1 and CYP1A2 is demonstrated for comparative studies on the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Live cells were cultivated for 2 days in the presence of these chemicals. Thereafter, the supernatant medium was checked for metabolites by gas chromatography and mass spectrometry. Marked cytochromes P450 and species dependent differences in the metabolite profiles were observed. Most important was the finding, that human cytochrome P450 1A1 almost exclusively oxidized benzo[a]pyrene in the 7,8,9,10-position, yielding the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene whereas the rat cytochrome P450 1A1 oxidized benzo[]pyrene in the 4,5-position and 7,8,9,10-position. The importance of this finding is underlined by results from cytotoxicity studies. Benzo[a]pyrene was twice as cytotoxic in the human cytochrome P450 1A1 than in the rat cytochrome P450 1A1 expressing V79 cells. Species and cytochrome P450 specific metabolite profiles were also observed for phenanthrene and benz[a]anthracene.
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