title: Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1.
abstract: Transgenic cell lines were constructed to study dynamic competition between activation versus detoxification of benzo[a]pyrene (B[a]P) and its metabolites. Transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone, or expressing hCYP1A1 in combination with human glutathione S-transferase P1 (hGSTP1), were used to determine how effectively GST protects against macromolecular damage or mutagenicity of B[a]P or its enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol [(+)B[a]P-7,8-diol] and (-)-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol]. Mutagenicity of B[a]P at the hprt locus was dose- and time-dependent in cells that expressed hCYP1A1. Mutagenicity was reduced in cells further modified to co-express hGSTP1. Dose-response and time-course studies indicated that mutagenicity was reduced up to 3-fold by hGSTP1 expression, compared with cells expressing hCYP1A1 alone. Mutagenicity induced by the B[a]P 7,8-dihydrodiols was also dose-dependent, and was reduced 2- to 5-fold by hGSTP1. Expression of hGSTP1 reduced B[a]P adducts in total cellular macromolecules by 3.8-fold, which correlated with the reduction in B[a]P mutagenicity and with reduction in the formation of the proximate metabolite B[a]P 7,8-dihydrodiols from B[a]P. However, measurement of total B[a]P metabolites bound to DNA isolated from cells incubated with [3H]-B[a]P revealed only 12, 33 and 24% reduction at 12, 24 and 48 h, respectively, by GSTP1 expression. Nevertheless, (32)P-post-labeling analysis demonstrated nearly total prevention of the known B[a]P-DNA adduct, N2-guanine-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), in cells co-expressing hGSTP1. This adduct, thought to be the most mutagenic of the stable B[a]P adducts, accounts for 15% or less of the total DNA adducts observed. These results indicate that the reduction in hCYP1A1-mediated B[a]P mutagenesis by hGSTP1 is probably largely due to prevention of the N2-guanine-BPDE adduct. However, the significant fraction (30-40%) of this mutagenesis and the majority of the total DNA binding that are not prevented together suggest formation by hCYP1A1 of a subset of mutagenic metabolites of B[a]P that are not effectively detoxified by hGSTP1.
authors.affiliation: Department of Biochemistry, Wake Forest University, School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Kushman
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Kabler
authors.forename: Melissa H
authors.initials: MH
authors.lastname: Fleming
authors.forename: Srivani
authors.initials: S
authors.lastname: Ravoori
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Gupta
authors.forename: Johannes
authors.initials: J
authors.lastname: Doehmer
authors.forename: Charles S
authors.initials: CS
authors.lastname: Morrow
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Townsend
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000478:Alkylation; D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts; D004249:DNA Damage; D051549:Glutathione S-Transferase pi / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000502:physiology; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*; D014162:Transfection
chemical_list: D018736:DNA Adducts; D009153:Mutagens; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D019363:Cytochrome P-450 CYP1A1; D007041:Hypoxanthine Phosphoribosyltransferase; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi