Research in this lab and others has provided evidence for an important role for expression of glutathione S-transferases (GSTs) (Phase II detoxification) in normal cellular defenses against reactive electrophilic metabolites of carcinogens, and as part of the biological response induced by chemopreventive agents. We have employed a transgenic cell modeling approach to examine the specific contributions of GSTs expressed individually and in combination with relevant genes, including members of the cytochrome P450 (Phase I activation) and the multidrug resistance protein (Phase III efflux) multigene families. We have found that protection against cytotoxicity and/or genotoxicity (DNA adducts or mutagenesis) can be quite different, depending in some cases primarily on GST characteristics and expression level, or in other cases on the nature of the carcinogen, whether it requires activation, and the P450 activation enzyme co-expressed with the GST. We propose to continue these investigations with our existing single-and dual-transfected V79 cell lines that stably express human GSTP1, GSTM1, or GSTA1, alone and also in combination with human P450-1A1 or-1B1, with addition of -1A2. We will focus on a limited set of polycyclic aromatic hydrocarbon (PAH) substrates for these P450s and GSTs, Benzo[a]Pyrene (and its two 7,8-dihydrodiol enantiomers); the more potent DiBenzo[a,I]Pyrene (and its intermediate 11,12-diol metabolites); and 5-methylchrysene. We will also examine metabolism and toxicities of estradiol, reportedly activated by these CYP isozymes and detoxified by hGSTP1, in these cell lines.
In Aim #1 we will examine the unique metabolic interactions between each P450 and GST combination, with both cytotoxicity/apoptosis, DNA adducts, and mutagenicity as endpoints.
Aim # 2 will model cell-cell interactions, asking whether binary mixtures of these cell lines show simple additivity, or synergy or antagonism in the presence of PAHs (e.g. via exchange of stable intermediates), and if GST expression is more effective in concert with one or the other P450 in the two different cell lines co-cultured.
Aim #3 will determine the mechanisms that underlie the large differences observed in GST protection against the cytotoxic vs. genotoxic effects of certain carcinogens.
In Aim #4 we will determine the mechanism for the intriguing observation that expression of GSTs with high efficiency for 4-nitroquinoline oxide or 4-hydroxynonenal conjugation confer paradoxical sensitivity instead of protection. These studies will enhance our knowledge of the factors that govern chemoprotective functions of GSTs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES010175-05A1
Application #
6870081
Study Section
Chemo/Dietary Prevention Study Section (CDP)
Program Officer
Thompson, Claudia L
Project Start
2000-02-01
Project End
2009-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
5
Fiscal Year
2005
Total Cost
$306,731
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