title: Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.
abstract: The prostate has been identified as a target for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced carcinogenesis. Humans are exposed to PhIP through ingestion of well-done cooked meats, and there is evidence from epidemiological studies that implicates red meat consumption in prostate carcinogenesis. The alpha and pi class isoforms of glutathione S-transferases (GSTs) have been shown to inhibit adduction of activated PhIP metabolites to DNA in cell-free systems. In humans, silencing of GST pi(GSTP1) through CpG island hypermethylation is found in nearly all prostate carcinomas and is believed to be an early event in prostate carcinogenesis. We hypothesized that suppressed GSTP1 expression in prostate cells would increase their vulnerability to cytotoxicity and DNA adduct formation mediated by activated PhIP metabolites. To test this hypothesis, the human prostate adenocarcinoma cell line, LNCaP, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1 cells exposed to N-OH-PhIP, but not parent PhIP, for 24 h showed a dose-dependent decrease in cell viability. GSTP1-overexpressing cells had LC50s 30-40% higher than cells transfected with the vector alone. PhIP-DNA adducts isolated from LNCaP-derived cells and primary human prostate tissue cultures exposed to N-OH-PhIP were analyzed by liquid chromatography/electrospray ionization mass spectrometry. Primary cultures of human prostate tissue and LNCaP-GSTP1 cells had approximately 50% lower adduct levels than parental LNCaP and vector control cells. Bioactivation assays using LNCaP cytosols showed that enzymatic activation of N-OH-PhIP to a DNA binding species was dependent on ATP and could be inhibited by recombinant human GSTP1 in the presence of glutathione. This evidence confirms that N-OH-PhIP can be bioactivated to a DNA binding species in human prostate and human prostate-derived cells. These observations provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model system for studying the role of this enzyme in protection against N-OH-PhIP induced DNA damage in prostate carcinogenesis. Loss of GSTP1 expression in human prostate may, therefore, enhance its susceptibility to carcinogenic insult by compounds such as N-OH-PhIP. Conversely, induction of GSTs in early-stage prostate carcinogenesis may be a useful protective strategy.
authors.affiliation: Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, USA.
authors.forename: C P
authors.initials: CP
authors.lastname: Nelson
authors.forename: L C
authors.initials: LC
authors.lastname: Kidd
authors.forename: J
authors.initials: J
authors.lastname: Sauvageot
authors.forename: W B
authors.initials: WB
authors.lastname: Isaacs
authors.forename: A M
authors.initials: AM
authors.lastname: De Marzo
authors.forename: J D
authors.initials: JD
authors.lastname: Groopman
authors.forename: W G
authors.initials: WG
authors.lastname: Nelson
authors.forename: T W
authors.initials: TW
authors.lastname: Kensler
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics / Q000517:prevention & control; D000368:Aged; D001711:Biotransformation; D002273:Carcinogens / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics / Q000633:toxicity*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000096:biosynthesis*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011467:Prostate / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000517:prevention & control; D011725:Pyridines / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics / Q000633:toxicity*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D007093:Imidazoles; D007527:Isoenzymes; D011725:Pyridines; C060533:2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D004247:DNA; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase