Carcinogenesis Advance Access published online on August 2, 2006
Carcinogenesis, doi:10.1093/carcin/bgl125
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1 Department of Biochemistry, Wake Forest University, Winston-Salem NC, USA 27157; Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University, Winston-Salem NC, USA 27157
* To whom correspondence should be addressed. 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 to 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 hours, respectively, by GSTP1 expression. Nevertheless, 32P-postlabeling analysis demonstrated nearly total prevention of the known B[a]P-DNA adduct, N2-guanine-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 likely due largely 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.
Received July 19, 2005
Revised May 16, 2006
Accepted June 23, 2006
CARCINOGENESIS
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
Mary E. Kushman 1, Sandra L. Kabler 2, Melissa H. Fleming 2, Srivani Ravoori 3, Ramesh C. Gupta 4, Johannes Doehmer 5, Charles S. Morrow 1, and Alan J. Townsend 1 *
2 Department of Biochemistry, Wake Forest University, Winston-Salem NC, USA 27157
3 James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40292
4 James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40292; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292
5 GenPharmTox BioTech AG, Munich, Germany
Alan J. Townsend, E-mail: atown{at}wfubmc.edu
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