The hepatotoxic metabolite of acetaminophen directly activates the Keap1-Nrf2 cell defense system.
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.


CONCLUSIONS
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.
Copple
Ian M
IM
Department of Pharmacology and Therapeutics, School of Biomedical Sciences, The University of Liverpool, Liverpool, UK.
Goldring
Christopher E
CE
Jenkins
Rosalind E
RE
Chia
Alvin J L
AJ
Randle
Laura E
LE
Hayes
John D
JD
Kitteringham
Neil R
NR
Park
B Kevin
BK
D000082:Acetaminophen / Q000378:metabolism* / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000494:pharmacology*; D000072019:Kelch-Like ECH-Associated Protein 1; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D016227:Benzoquinones; D003598:Cytoskeletal Proteins; D007097:Imines; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D000082:Acetaminophen; C028473:N-acetyl-4-benzoquinoneimine; D005978:Glutathione