Transcription factor Nrf2 protects HepG2 cells against CYP2E1 plus arachidonic acid-dependent toxicity.
Nrf2 is a transcription factor that regulates important antioxidant and phase II detoxification genes. Arachidonic acid (AA) causes CYP2E1-dependent toxicity in HepG2 cells. The ability of Nrf2 to protect against CYP2E1-dependent AA toxicity and its possible mechanism were evaluated. AA activates Nrf2 in CYP2E1-expressing HepG2 cells (E47 cells), increasing Nrf2 protein and mRNA levels, Nrf2 nuclear translocation, and Nrf2-ARE binding activity. These increases in Nrf2 are associated with elevated expression of Nrf2-regulated antioxidant genes. Overexpression of Nrf2 by transient transfection of plasmid Nrf2 confers resistance of E47 cells against AA toxicity. Blocking Nrf2 with small interfering RNA (siRNA)-Nrf2 potentiates the CYP2E1-dependent AA toxicity. This enhanced toxicity is accompanied by decreases of cellular GSH levels and increases in production of reactive oxygen species and lipid peroxidation. There is also a potentiation of mitochondrial damage in the presence of siRNA-Nrf2. The protective effects of Nrf2 against CYP2E1-dependent toxicity can be blocked by l-buthionine-(S,R)-sulfoximine, a specific inhibitor of glutamate-cysteine ligase, which is a rate-limiting enzyme in the synthesis of GSH and is regulated by Nrf2. Elevation of GSH by supplementing with glutathione ethyl ester can partially reverse the enhanced AA toxicity by siRNA-Nrf2. Moreover, in contrast to AA, l-buthionine-(S,R)-sulfoximine toxicity is not prevented by plasmid Nrf2 probably because protective GSH cannot be synthesized. Together, these results suggest that Nrf2, through up-regulation of glutamate-cysteine ligase and increase of GSH levels, protects against CYP2E1-dependent AA toxicity.
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Pengfei
P
Gong
Arthur I
AI
Cederbaum
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D016718:Arachidonic Acid / Q000633:toxicity*; D045744:Cell Line, Tumor; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation; D051379:Mice; D018345:Mice, Knockout; D051267:NF-E2-Related Factor 2 / Q000378:metabolism / Q000502:physiology*; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species; D014162:Transfection
D000975:Antioxidants; D051267:NF-E2-Related Factor 2; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D016718:Arachidonic Acid; D019392:Cytochrome P-450 CYP2E1; D005721:Glutamate-Cysteine Ligase