The
transcription factor Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. We have demonstrated previously that
acetaminophen activates Nrf2 in mouse liver following administration of non-hepatotoxic and hepatotoxic doses in vivo, implying that Nrf2 may have an important role in the protection against
drug-induced liver injury. Nrf2 activation has been proposed to occur through the modification of
cysteine residues within Keap1, the cytosolic repressor of Nrf2. We hypothesized that
acetaminophen activates Nrf2 via the formation of its reactive metabolite
N-acetyl-p-benzoquinoneimine (
NAPQI), which may disrupt the repression of Nrf2 through the modification of
cysteine residues within Keap1. Here, we show that
NAPQI can directly activate the Nrf2 pathway in mouse liver cells, inducing an adaptive defense response that is antagonized by RNA interference targeted against Nrf2. Furthermore, mass spectrometric analysis shows that
NAPQI selectively modifies
cysteine residues in Keap1, both in
recombinant protein in vitro and in cells ectopically expressing Keap1. Using this cell-based model, we demonstrate that activation of Nrf2 by
NAPQI and a panel of probe molecules [
dexamethasone 21-mesylate, 15-deoxy-Delta-((12,14))-prostaglandin J(2),
2,4-dinitrochlorobenzene, and
iodoacetamide] correlates with the selective modification of
cysteine residues located within the intervening region of Keap1. However, substantial depletion of
glutathione (to less than 15% of basal levels) by
buthionine sulfoximine, which does not directly modify Keap1, is also sufficient to activate Nrf2.
CONCLUSION: Nrf2 can be activated via the direct modification of
cysteine residues located within the intervening region of Keap1, but also via the substantial depletion of
glutathione without the requirement for direct modification of Keap1. It is possible that both of these mechanisms contribute to the activation of Nrf2 by
acetaminophen.