High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes.
Nrf2, which belongs to the basic leucine zipper (bZip) transcription factor family, has been implicated as a key molecule involved in antioxidant-responsive element (ARE)-mediated gene expression. In order to examine the role of Nrf2 in protection against xenobiotic toxicity, the sensitivity of nrf2 knockout mice to acetaminophen (N-acetyl-4-aminophenol (APAP)) was analyzed. The saturation of detoxification pathways after high levels of exposure to APAP is known to induce hepatotoxicity. Two factors important in its detoxification are UDP-glucuronosyltransferase (UDP-GT), an ARE-regulated phase-II drug-metabolizing enzyme, and glutathione (GSH), an antioxidant molecule whose synthesis depends on ARE-regulated gamma-glutamylcysteine synthetase (gammaGCS). Two- to 4-month-old male mice were orally administered a single dose of APAP at 0, 150, 300, or 600 mg/kg. Doses of 300 mg/kg APAP or greater caused death in the homozygous knockout mice only, and those that survived showed a greater severity in hepatic damage than the wild-type mice, as demonstrated by increased plasma alanine aminotransferase activity, decreased hepatic non-protein sulfhydryl (NPSH) content, and centrilobular hepatocellular necrosis. The high sensitivity of Nrf2-deficient mice was confirmed from observations made at 0, 2, 8, and 24 h after dosing with 300 mg/kg APAP; increased anti-APAP immunoreactivity was also noted in their livers at 2 h. Untreated homozygous knockout mice showed both a lower UDP-GT activity and NPSH content, which corresponded to decreased mRNA levels of UDP-GT (Ugt1a6) and the heavy chain of gammaGCS, respectively. These results show that Nrf2 plays a protective role against APAP hepatotoxicity by regulating both drug metabolizing enzymes and antioxidant genes through the ARE.
Enomoto
A
A
Toxicology Division II, Laboratory of Pathology, Institute of Environmental Toxicology, 4321 Uchimoriya, Mitsukaido, Ibaraki 303-0043, Japan. enomoto@iinet.ne.jp
Itoh
K
K
Nagayoshi
E
E
Haruta
J
J
Kimura
T
T
O'Connor
T
T
Harada
T
T
Yamamoto
M
M
D000082:Acetaminophen / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000633:toxicity*; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D056486:Chemical and Drug Induced Liver Injury / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006579:Heterozygote; D006720:Homozygote; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D012333:RNA, Messenger; D015534:Trans-Activators; D000082:Acetaminophen; D014453:Glucuronosyltransferase; D000410:Alanine Transaminase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione