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

Abstract

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-⌬-(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. (HEPATOLOGY 2008;48:1292-1301

Silencing Glycogen Synthase Kinase-3β Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1

“…GCL-c mRNA levels have also been shown to be up-regulated by APAP treatment as a defense to counter GSH depletion caused by NAPQI (32). The transcription of GCL-c is regulated by Nrf-2, a transcription factor that translocates to the nucleus following activation by oxidative stress or covalent binding of NAPQI (32,33). In certain contexts GSK-3␤ has been shown to phosphorylate Nrf-2, which causes Nrf-2 to move out of the nucleus, thus preventing Nrf-2 transcriptional activity (i.e.…”

Aldo-keto reductase-7A protects liver cells and tissues from acetaminophen-induced oxidative stress and hepatotoxicity

“…33 Nrf2 has been shown to be strongly induced and activated by APAP or NAPQI. [35][36][37] Furthermore, in Nrf2-deficient mice, increased sensitivity to APAP resulted in greater severity in hepatic damage and increased lethality. 32,[38][39][40] Conversely, mice deficient for Keap1 were more resistant to toxic doses of acetaminophen, 41 whereas activation of Nrf2 by oleanolic acid led to protection against APAP-induced hepatotoxicity.…”

Targeting the Ataxia Telangiectasia Mutated-null phenotype in chronic lymphocytic leukemia with pro-oxidants

“…38 Most importantly, using a pro-oxidant based strategy, we show that ATM-null CLL cells are significantly more sensitive than ATM-wt tumors and non-tumor cells to agents previously shown to stimulate NRF2-mediated adaptation to stress. 39,40 Thus both NAPQI and parthenolide represent novel clinically applicable approaches for the treatment of ATM-null CLL. Of note, acetaminophen, the NAPQI precursor, has previously been used in a phase I trial for the treatment of metastatic melanoma 41 as an approach to enhance the specificity of anti-cancer agents which deplete glutathione.…”

“…39,40 Thus both NAPQI and parthenolide represent novel clinically applicable approaches for the treatment of ATM-null CLL. Of note, acetaminophen, the NAPQI precursor, has previously been used in a phase I trial for the treatment of metastatic melanoma 41 as an approach to enhance the specificity of anti-cancer agents which deplete glutathione.…”