Received for publication June 27, 2007.
Revised August 9, 2007.
Accepted for publication August 27, 2007.

Activation of Nrf2 by Toxic Bile Acids Provokes Adaptive Defense Responses to Enhance Cell Survival at the Emergence of Oxidative Stress

Abstract

Oxidative stress, causing necrotic and apoptotic cell death, is associated with bile acid toxicity. Using liver (HepG2, Hepa1c1c7, and primary human hepatocytes) and intestinal (C2bbe1, a Caco-2 subclone) cells, we demonstrated that toxic bile acids, such as lithocholic acid (LCA) and chenodeoxycholic acid, induced the NF-E2-related factor 2 (Nrf2) target genes, especially glutamate-cysteine ligase subunits (GCLM and GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, and thioredoxin reductase 1. Nrf2 activation and induction of Nrf2 target genes were also evident in-vivo in the liver of CD-1 mice treated 7-8 h or 4 d with LCA. Silencing of Nrf2 via small-interfering RNA suppressed basal and bile acid-induced mRNA levels of the above genes. Consistent with this, overexpression of Nrf2 enhanced, but of dominant-negative Nrf2 attenuated, Nrf2 target gene induction by bile acids. The activation of Nrf2-ARE (antioxidant responsive element) transcription machinery by bile acids was confirmed by increased nuclear accumulation of Nrf2, enhanced ARE-reporter activity, and increased Nrf2 binding to ARE. Importantly, Nrf2 silencing increased cell susceptibility to LCA toxicity, as evidenced by reduced cell viability, and increased necrosis and apoptosis. Concomitant with GCLC/GCLM induction, cellular glutathione (GSH) was significantly increased in bile acid-treated cells. Cotreatment with N-acetyl-L-cysteine, a GSH precursor, ameliorated LCA toxicity, whereas cotreatment with buthionine sulfoximine, a GSH synthesis blocker, exacerbated it. In summary, this study provides molecular evidence linking bile acid toxicity to oxidative stress. Nrf2 is centrally involved in counteracting such oxidative stress by enhancing adaptive antioxidative response, particularly GSH biosynthesis, and hence cell survival.

Key words: Phase II enzymes, Quinone oxidoreductase, Regulation - transcriptional, Regulation - xenobiotic, Apoptosis, Glutathione, Mitochondrial toxins, Oxidative stress/antioxidants, Toxicant-induced gene express, Oxidative stress

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