Nuclear translocation of aryl hydrocarbon receptor in response to oxidative stress involves phosphorylation of mitogen activated protein kinases.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 3136 Enzymatic biotransformation of many chemicals to carcinogens is mediated by cytochrome P4501A1 (CYP1A1) that is regulated by the aryl hydrocarbon receptor (AhR). The AhR binds ligands, including 2‘3‘7‘8-tetrachlorodibenzodioxin (TCDD), benzo[ a ]pyrene (B[ a ]P) and β -napthoflavone (BNF), which induce its translocation into the nucleus. In the nucleus, AhR heterodimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and transcribes genes possessing the xenobiotic response element (XRE) 5′- GCGTG -3′. Recent evidence suggests that the AhR can translocate independently of traditional ligands in response to electrophiles, endogenous stimuli and some phytochemicals. The chemopreventive effect of benzyl isothiocyanate (BIT) and other phytochemicals may involve oxidative stress. This might enhance or diminish activity of carcinogen metabolizing and detoxification enzymes due to altered nuclear translocation and DNA binding affinity of AhR and NF-E2 related factor-2 (Nrf-2) for their respective response elements. We previously observed that AhR partially translocated to the nucleus in Hepa1 and HepG2 liver carcinoma cells treated with BIT. This occurred independently of ligands and suggests that oxidative stress might activate carcinogens. In this study, we determined whether phosphorylated ERK1/2 and p38 MAPK are required for AhR nuclear translocation in response to BIT. These kinases might provide a molecular switch between AhR and other proteins in vivo and may be essential for signal transduction in AhR-mediated transcription. Hepa1 cells were treated with 1.25, 2.5, 5 and 10 μM BIT or DMSO alone (≤0.2%v/v) and phosphorylated ERK1/2 and p38 MAPK detected using specific antibodies and western blot. In other experiments, cells were treated with 5 μM BIT or 10 μM BNF alone, or in combination with 10 μM U2016, a potent MEK inhibitor, which prevents phosphorylation of ERK1/2. Nuclear translocation of AhR was visualized using immunofluorescence and confocal microscopy, and confirmed by western blot of Hepa1 nuclear and cytoplasmic lysates. To assess AhR nuclear translocation following inhibition of p38 MAPK, cells were treated as described above except SB202190 was used to inhibit p38 MAPK. BIT induced phosphorylation of ERK1/2 and p38 MAPK, suggesting that these kinases function in the stress response to phytochemical treatments. Inhibition of ERK1/2 and p38 MAPK resulted in reduced nuclear translocation of AhR in both BIT and BNF treated cells. These data provide preliminary evidence that ERK1/2 and p38 MAPK might function in AhR-mediated signaling in response to phytochemicals. The results also provide insight into mechanisms that might be altered by phytochemicals thus affecting transcriptional regulation of carcinogen metabolizing and detoxification enzymes (Support: NIH MBRS/GM; RCMI RR033032).