Biochemical and Biophysical Research Communications
Cytoprotective role of Nrf2/Keap1 system in methylmercury toxicity
Section snippets
Materials and methods
Materials. MeHg was purchased from Nacalai tesque (Kyoto, Japan); anti-glutamate cysteine ligase modifier subunit (GCLM), GCL catalytic subunit (GCLC), MRP2, 5′-nucleotidase (5′-NT) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSTA1 and GSTM1 from Oxford Biomedical Research (Oxford, MI); anti-MRP1 from Alexis Biochemicals (San Diego, CA); anti-Actin from Sigma (St. Louis, MO). All other reagents and chemicals used were of the highest grade available.
Binding of Keap1 protein with MeHg.
MeHg binds to a recombinant Keap1 protein
Since activation of Nrf2 is attributed to Keap1 modification through its reactive thiols, covalent interaction of recombinant Keap1 protein with MeHg was examined. As shown in Fig. 1, MeHg was bound to recombinant Keap1 protein following separation of the samples with MeHg by SDS–PAGE in the absence of 2-mercaptoethanol.
MeHg activates the transcription factor, Nrf2 in SH-SY5Y cells
The LC50 value of MeHg after exposure of human neuroblastoma SH-SY5Y cells was estimated by MTT assay was 1.29 ± 0.5 μM. In the subsequent experiments, we used subcytotoxic
Discussion
The present study indicates that MeHg, a ubiquitous environmental pollutant, is able to activate Nrf2, thereby upregulating downstream proteins such as GCL and GSTs. The results also that activation of Nrf2 confers, on SH-SY5Y cells and primary mouse hepatocytes, potent resistance to MeHg. Since the Nrf2 regulatory protein, Keap1 contains reactive thiols whose alteration is responsible for Nrf2 activation, a likely mechanism for MeHg action was its interaction with Keap1 thiols. In previous
Acknowledgments
We thank Prof. Akira Naganuma, Graduate School of Pharmaceutical Sciences, Tohoku University for kind donation of SH-SY5Y cells, and Prof. Ken Itoh, Center for advanced Medical Research, Hirosaki University School of Medicine for kind providing of ARE-luciferase and mNrf2/pcDNA3 and helpful advice in the experimental procedure.
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These authors contributed equally to this study.