Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.

摘要

Prostate cancer has become a major health problem for men in the Western world, yet the etiology of this disease is still largely unknown. Epidemiological evidence shows inverse associations with fruit and vegetable intake and strong positive associations with dietary fat intake and red meat consumption. Although fat is believed to be the major component in red meat responsible for its observed association with prostate cancer, other compounds present in meats may also contribute to prostate carcinogenesis.; Heterocyclic amines are pyrolysis products that have been isolated from cooked fish and meat and are highly mutagenic in the Ames assay and carcinogenic in rodents. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant of the mutagenic heterocyclic amines found in cooked meats, forms DNA adducts and induces mutations and carcinomas in the rat prostate.; The hypothesis that glutathione S-transferase P1 (GSTP1) could protect human prostate-derived cells against PhIP-related cytotoxicity and DNA damage was addressed in this thesis. The most common genetic alteration seen in human prostate carcinomas is silencing of GSTP1 through CpG island hypermethylation. GSTP1 can act as a carcinogen detoxication enzyme and was previously shown to inhibit the binding of an activated PhIP metabolite to DNA in vitro. These studies provide strong evidence that the human prostate may be a target for PhIP-related toxicities and that compromised GSTP1 expression enhances cytotoxicity and DNA damage.; In summary, human prostate-derived cells and human prostate tissues can activate N-OH-PhIP to a DNA damaging species. In LNCaP cells, N-OH-PhIP cytotoxicity could be modulated by inhibitors of acetylation, sulfation, glucuronidation, and β-glucuronidase as well as the complexing molecule chlorophyllin. Cytotoxicity could also be partially inhibited by restored GSTP1 expression, as determined by 30–40% increases in the concentration toxic to 50% of the cells. DNA adduct analysis by LC/ESI-MS showed that GSTP1 expressed in LNCaP cells could inhibit the formation of PhIP-DNA adducts by approximately 50%, and these levels were comparable to adducts seen in human prostate samples exposed to N-OH-PhIP in culture. Using LNCaP cytosols, activation of N-OH-PhIP to a DNA binding species was found to occur through an enzymatic reaction that requires physiological levels of ATP. (Abstract shortened by UMI.)