Abstract
(7S,8S)--Dihydroxy--7,8--dihydrobenzo[a]pyrene ((+)-BP-7,8-diol) is epoxidized to (7S,8R)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-syn-BPDE) by cytochrome P-450 isoenzymes and to (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((-)-anti-BPDE) by peroxyl free radicals. 32P postlabeling analysis of the diastereomeric BPDE-DNA adducts was used to investigate the pathways of (+)-BP-7,8-diol oxidation in mouse skin in vivo. The pattern of deoxynucleoside 3',5'-bisphosphate adducts in epidermal scrapings from female CD-1 mice indicated that cytochrome P-450 was the major oxidant. Similar results were obtained when the tumor-promoting phorbol ester tetradecanoylphorbolacetate (TPA) was coadministered with (+)-BP-7,8-diol. However, when animals were pretreated with TPA 24 h before coadministration of TPA and (+)-BP-7,8-diol, the pattern of BPDE-DNA adducts indicated that peroxyl radicals made a major contribution to (+)-BP-7,8-diol epoxidation. Peroxyl radical-dependent epoxidation was maximal when the time between the two TPA administrations was 24-72 h. No increase in (-)-anti-BPDE-DNA was observed when the non-tumor-promoting phorbol ester 4-O-methyl-TPA was substituted for TPA. The calcium ionophore A23187 stimulated peroxyl radical generation when substituted for the first, but not the second, TPA treatment. The antiinflammatory steroid fluocinolone acetonide inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the first but not the second TPA treatment. These findings demonstrate the existence of two independent pathways of metabolic activation of (+)-BP-7,8-diol in mouse epidermis, one dependent on cytochrome P-450 and the other dependent on peroxyl free radicals. The results also suggest that repetitive topical administration of tumor-promoting phorbol esters remodels epidermal metabolism leading to a significant increase in free radical generation.
Highlights
The major adduct cochromato- 7,8-diol could occur to the time between its application and graphs with (+)-syn-BPDE-N2-dG and theminor adduct co- the application of butylated hydroxy anisole (BHA), i.e. while the animals were alive
The relative abundance of DNA adducts detected by After the second TPA treatment themice were kept for 6 h postlabeling analysis suggests that cytochrome P-450 is the before BHA application and death
The present results provide strong evidence for the existence of two pathways of metabolic activation of BP-7,8-diol in mouse skin in vivo
Summary
BHA is both an antioxidant anda substrate for cytochrome P-450 so it prevents (+)-BP"I&diol oxidation by either pathwas determinedby the bicinchoninicacid protein assay using reagents way [31,32].When animalswere treated with BHA and killed from Pierce Chemical Co. immediately after coadministration of (+)-BP-7,8-diol and TPA, postlabeling analysis of the epidermal DNA revealed no adduct spots (Fig. 3c). The levels of Experiments were performed in which the time beboth adducts increase with time and dose of (+)-BP-7,8-diol tween the two TPA treatments was varied from 3 to 72h.
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