Glutathione adduct of methylmercury activates the Keap1-Nrf2 pathway in SH-SY5Y cells.

Methylmercury (MeHg) reacts readily with GSH, leading to the formation of a MeHg-SG adduct that is excreted into extracellular space through multidrug-resistance-associated protein (MRP), which is regulated by the transcription factor Nrf2. We previously reported that MeHg covalently modifies Keap1 and activates Nrf2 in human neuroblastoma SH-SY5Y cells. In the study presented here, we examined whether the MeHg-SG adduct could also modulate the Keap1-Nrf2 pathway because the formation of the Hg-S bond is believed to be reversible in the presence of a nucleophile. SH-SY5Y cells exposed to the synthetic ethyl monoester of the MeHg-SG adduct (which is hydrolyzed by cellular esterase(s) to give the MeHg-SG adduct) exhibited a concentration-dependent cellular toxicity that was enhanced by pretreatment with a specific MRP inhibitor. As expected, the MeHg-SG adduct was able to modify cellular proteins in the SH-SY5Y cells and purified Keap1. We also found that this prodrug, as well as MeHg, causes the cellular Keap1 in the cells to be modified, resulting in Nrf2 activation and, thereby, the upregulation of the downstream genes. These results suggest that the MeHg-SG adduct is not electrophilic but that it modifies protein thiols (including Keap1) through S-transmercuration and that rapid Nrf2-dependent excretion of the MeHg-SG adduct is essential in decreasing the cytotoxicity of MeHg.