Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents.
Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2(-/-) MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 micromol/l sulforaphane was very substantially lower in Nrf2(-/-) MEFs than in wild-type cells, and the rebound leading to a approximately 1.9-fold increase in glutathione that occurred 12-24 h after Nrf2(+/+) MEFs were treated with sulforaphane was not observed in Nrf2(-/-) fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 micromol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, alpha,beta-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2(+/+) MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2(-/-) MEFs were typically approximately 50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 micromol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2(+/+) MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione.
Higgins
Larry G
LG
Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK.
Kelleher
Michael O
MO
Eggleston
Ian M
IM
Itoh
Ken
K
Yamamoto
Masayuki
M
Hayes
John D
JD
D000222:Adaptation, Physiological / Q000187:drug effects* / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D005609:Free Radicals / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D017879:Isothiocyanates / Q000633:toxicity; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051267:NF-E2-Related Factor 2 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010545:Peroxides / Q000633:toxicity*; D013454:Sulfoxides; D013861:Thiocyanates / Q000633:toxicity*; D015262:Xenobiotics / Q000633:toxicity
D005609:Free Radicals; D017879:Isothiocyanates; D051267:NF-E2-Related Factor 2; D010545:Peroxides; D013454:Sulfoxides; D013861:Thiocyanates; D015262:Xenobiotics; C016766:sulforaphane; D005978:Glutathione