A number of known chemopreventive agents are hypothesized to work in part via induction of glutathione-S-transferase (GST) expression. In order to understand the functions of GST in detoxification of cytotoxic and mutagenic electrophiles, the investigators have utilized transgenic cell lines to show that GST expression can provide efficacious protection against DNA alkylation and, in some cases, cytotoxicity caused by electrophilic carcinogens that are GST substrates, including 4-NQO, B[a]P, BPDE, AFB1, CDNB, and specific drugs. However, the results indicated that the factors governing protection by GST are complex and vary with different agents and endpoints. The studies outlined in this proposal will provide new information on the efficacy and specificity of human GSTP1 or GSTM1 protection against DNA adduct formation or cytotoxicity caused by exposure to PAHs activated in situ by co-expressed rat rCYP1A1 or human hCYP1A1. Heterologous expression of the GST isozymes in V79 cells previously stably transfected with rCYP1A1 or hCYP1A1 will be used as the experimental model system. Importantly, this information will be directly compared with the effects of GST isoenzyme expression on metabolite accumulation and with cellular end-points, such as cytotoxicity, in cells. They hypothesize that the efficacy of the GST system is dependent on multiple factors and not only the enzymatic efficiency with a particular substrate. Several of these factors will be examined in the next funding period, including the relationship between protection by transfected GST isozymes against the above end-points and 1) the level of GST protein expressed, 2) rates and site of activation vs. detoxification, and resultant metabolite profiles and/or levels, 3) cellular factors: glutathione (GSH) supply, and/or efflux of GSH-conjugates, and 4) genetic polymorphisms that affect the active site architecture of hGSTP1-1. These studies will provide a detailed understanding of key parameters affecting the efficacy of GST protection in the transfected cells, and should help to identify the mechanisms of differential protection observed against the various cellular injury end-points examined.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES010175-02
Application #
6350837
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Packenham, Joan P
Project Start
2000-02-01
Project End
2004-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
2
Fiscal Year
2001
Total Cost
$220,890
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Rudd, Lisa P; Kabler, Sandra L; Morrow, Charles S et al. (2011) Enhanced glutathione depletion, protein adduct formation, and cytotoxicity following exposure to 4-hydroxy-2-nonenal (HNE) in cells expressing human multidrug resistance protein-1 (MRP1) together with human glutathione S-transferase-M1 (GSTM1). Chem Biol Interact 194:113-9
Kabler, Sandra L; Seidel, Albrecht; Jacob, Juergen et al. (2009) Differential protection by human glutathione S-transferase P1 against cytotoxicity of benzo[a]pyrene, dibenzo[a,l]pyrene, or their dihydrodiol metabolites, in bi-transgenic cell lines that co-express rat versus human cytochrome P4501A1. Chem Biol Interact 179:240-6
Ahmad, Sarfaraz; Kabler, Sandra L; Rudd, Lisa et al. (2008) Cytotoxicity and mutagenicity of 5-methylchrysene and its 1,2-dihydrodiol in V79MZ cells modified to express human CYP1A1 or CYP1B1, in the presence or absence of human GSTP1 coexpression. Toxicol Lett 183:99-104
Kushman, Mary E; Kabler, Sandra L; Ahmad, Sarfaraz et al. (2007) Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1. Mutat Res 624:80-7
Kushman, Mary E; Kabler, Sandra L; Fleming, Melissa H et al. (2007) 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 hCYP Carcinogenesis 28:207-14
Kushman, Mary E; Kabler, Sandra L; Ahmad, Sarfaraz et al. (2007) Protective efficacy of hGSTM1-1 against B[a]P and (+)- or (-)-B[a]P-7,8-dihydrodiol cytotoxicity, mutagenicity, and macromolecular adducts in V79 cells coexpressing hCYP1A1. Toxicol Sci 99:51-7
Townsend, Alan J; Kabler, Sandra L; Doehmer, Johannes et al. (2002) Modeling the metabolic competency of glutathione S-transferases using genetically modified cell lines. Toxicology 181-182:265-9