Transcription factor Nrf2 regulates genes encoding drug-metabolising enzymes and drug transporters, as well as enzymes involved in the glutathione, thioredoxin and peroxiredoxin antioxidant pathways. Using mouse embryonic fibroblast (MEF) cells from Nrf2(+/+) and Nrj2(-/-) mice, in conjunction with the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, we have shown that loss of Nrf2 diminishes the intrinsic resistance of mutant fibroblasts towards isothiocyanates (i.e. sulforaphane), epoxides (i.e. (2S,3S)-(-)-3-phenylglycidol, ethyl 3-phenylglycidate and styrene-7,8-epoxide), peroxides, hydroquinones and quinones (i.e. tert-butylhydroperoxide, tert-butylhydroquinone and 2,3-dimethoxynaphthoquinone), NaAsO2, and various mutagens, including beta-propiolactone, cisplatin, mechlorethamine and methyl methanesulfonate to similar to 50% of that observed in equivalent wild-type cells. Exposure of Nrf2(+/+) fibroblasts, but not Nrj2(-/-) fibroblasts, to a non-toxic dose (3 mu mol/1) of the chemopreventive agent sulforaphane (Sul) stimulated an adaptive response that, 18 h after first being subjected to the isothiocyanate, caused an induction of between 2- and 10-fold in the levels of mRNA for glutamate-cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, glutathione S-transferases and NAD(P)H:quinone oxidoreductase-1 (Nqo1); this was accompanied by an increase in total glutathione of between 1.5- and 1.9-fold. Pre-treatment of Nrf2(+/+) MEF cells with 3 mu M Sul for 18 h prior to challenge with xenobiotics, conferred between 2.0- and 4.0-fold protection against isothiocyanates, reactive carbonyls, peroxides, quinones. NaAsO2, and the anticancer nitrogen mustard chlorambucil, but pre-treatment with 3 mu M Sul produced no such increased tolerance in Nrf2(-/-) MEF cells. The inducible resistance towards acrolein, cumene hydroperoxide and chlorambucil, produced by pre-treating wild-type fibroblasts with 3 mu M Sul, was dependent on glutathione because simultaneous pre-treatment with 5 mu mol/1 buthionine sulfoximine abolished the increased tolerance of these xenobiotics. However, inducible resistance towards menadione that occurred upon pre-treatment with 3 mu M Sul was independent of glutathione and may be due to upregulation of Nqo1. Thus Nrf2 controls cellular resistance against electrophiles. (C) 2010 Published by Elsevier Ireland Ltd.
