Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1.
Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.
Fields
W R
WR
Biochemistry Department, Bowman Gray School of Medicine, Wake Forest University Comprehensive Cancer Center, Medical Center Boulevard, Winston-Salem, NC 27157, USA.
Morrow
C S
CS
Doehmer
J
J
Townsend
A J
AJ
D016604:Aflatoxin B1 / Q000633:toxicity*; D000478:Alkylation; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D051381:Rats; D014162:Transfection
D016604:Aflatoxin B1; D019362:Cytochrome P-450 CYP2B1; D005982:Glutathione Transferase