TOXICOLOGICAL RELEVANCE OF HUMAN GST P-1 POLYMORPHISM

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DESCRIPTION: (Adapted from the Investigator's Abstract)The overall objective of this project is to define the significance of human glutathione S-transferase _ (hGSTP1-1) polymorphism in cellular detoxication of carcinogenic diol epoxides (DEs) of polycyclic aromatic hydrocarbons, which are widespread environmental pollutants and believed to be etiological factors in human chemical carcinogenesis. Recent preliminary studies indicate that the two polymorphic forms of hGSTP1-1, which differ in their primary structure by a single amino acid in position 104, significantly differ in their activity toward DEs. These observations led the investigators to hypothesize that the hGSTP1-1 polymorphism may be an important determinant of variable response of individuals to PAH-induced cancer. This hypothesis will be tested by a series of experiments proposed under four interrelated specific aims. In specific aim 1, they will compare the kinetic parameters and catalytic efficiencies of three naturally occurring polymorphic forms of hGSTP1-1 [hGSTP1-1(I104,A113), hGSTP1-1 (V104,A113) and hGSTP1-1(V104,V113)] and hGSTP1-1(I104,V113) form, prepared by site-directed mutagenesis, in the GSH conjugation of various bay-region and fjord-region DEs. In specific aim 2, we will investigate the effects of overexpression of hGSTP1-1 variants, through stable transfection in HepG2 cells, on cytotoxicity of anti-BPDE and anti-CDE [ultimate carcinogenic metabolite of benzo(a)pyrene and chrysene, respectively] and on formation of2 the predominant DNA adduct of anti-BPDE (trans-N dG-BPD adduct). Studies in specific aim 3 are designed to determine the correlation between expression of hGSTP1-1 variants in humans (by PCR using blood samples from normal healthy subjects as well as cancer patients) and GST activity toward anti-BPDE,2 trans-N dG-BPD adduct formation (in white blood cells of same individuals) and susceptibility to cancer (medical history) of the blood donors. Finally, specific aim 4 is to elucidate the molecular mechanism of differences in the kinetic properties of hGSTP1-1 variants in the GSH conjugation of DEs by X-ray crystallography and molecular modeling of their active sites. These studies should enhance our understanding of the protective mechanisms against carcinogenic DEs and also shed light on the physiological significance of hGSTP1-1 polymorphism in the detoxication of this class of ultimate carcinogens. In the long term, the information generated from this project may be valuable in formulating strategies for cancer prevention in humans and may help in identifying human population at risk for PAH-induced cancer. The proposed studies will assess a total of 408 cases plus and an equal number of controls. The population will consist of members of both genders and will include Caucasians, blacks and other minorities, as available and representative of the patient base at Mercy Hospital.