32P-Postlabeling Analysis of DNA Adduction in Mouse Skin following Topical Administration of (+)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene

Ashok P. Reddy, Donna Pruess-Schwartz, Chuan Ji, Peter Gorycki, Lawrence J. Marnett

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13 Scopus citations

Abstract

32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3′-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [γ-32P]ATP. 32P-Labeled deoxynucleoside 3′,5′-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol, (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-syn-BPDE] with DNA. The level of this adduct increased in a dose-and time-dependent fashion and was elevated in animals pretreated with β-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3′5′-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.

Original languageEnglish (US)
Pages (from-to)26-33
Number of pages8
JournalChemical Research in Toxicology
Volume5
Issue number1
DOIs
StatePublished - Jan 1 1992
Externally publishedYes

ASJC Scopus subject areas

  • Toxicology

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