The cap’n’collar transcription factor Nrf2 mediates both intrinsic resistance to environmental stressors and an adaptive response elicited by chemopreventive agents that determines susceptibility to electrophilic xenobiotics

Abstract

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 Nrf2 −/− 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), NaAsO 2, and various mutagens, including β-propiolactone, cisplatin, mechlorethamine and methyl methanesulfonate to ∼50% of that observed in equivalent wild-type cells. Exposure of Nrf2 +/+ fibroblasts, but not Nrf2 −/− fibroblasts, to a non-toxic dose (3 μmol/l) 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 μM Sul for 18 h prior to challenge with xenobiotics, conferred between 2.0- and 4.0-fold protection against isothiocyanates, reactive carbonyls, peroxides, quinones, NaAsO 2, and the anticancer nitrogen mustard chlorambucil, but pre-treatment with 3 μM 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 μM Sul, was dependent on glutathione because simultaneous pre-treatment with 5 μmol/l buthionine sulfoximine abolished the increased tolerance of these xenobiotics. However, inducible resistance towards menadione that occurred upon pre-treatment with 3 μM Sul was independent of glutathione and may be due to upregulation of Nqo1. Thus Nrf2 controls cellular resistance against electrophiles.