Tributyltin activates the Keap1-Nrf2 pathway via a macroautophagy-independent reduction in Keap1.

Tributyltin (TBT) is an environmental chemical, which was used as an antifouling agent for ships. Although its use has been banned, it is still persistently present in ocean sediments. Although TBT reportedly causes various toxicity in mammals, few studies on the mechanisms of biological response against TBT toxicity exist. The well-established Keap1-Nrf2 pathway is activated as a cytoprotective mechanism under stressful conditions. The relationship between TBT and the Keap1-Nrf2 pathway remains unclear. In the present study, we evaluated the effect of TBT on the Keap1-Nrf2 pathway. TBT reduced Keap1 protein expression in Neuro2a cells, a mouse neuroblastoma cell line, after 6 hr without altering mRNA expression levels. TBT also promoted the nuclear translocation of Nrf2, a transcription factor for antioxidant proteins, after 12 hr and augmented the expression of heme oxygenase 1, a downstream protein of Nrf2. Furthermore, TBT decreased Keap1 levels in mouse embryonic fibroblast (MEF) cells, with the knockout of Atg5, which is essential for macroautophagy, as well as in wild-type MEF cells. These results suggest that TBT activates the Keap1-Nrf2 pathway via the reduction in the Keap1 protein level in a macroautophagy-independent manner. The Keap1-Nrf2 pathway is activated by conformational changes in Keap1 induced by reactive oxygen species or electrophiles. Furthermore, any unutilized Keap1 protein is degraded by macroautophagy. Understanding the novel mechanism governing the macroautophagy-independent reduction in Keap1 by TBT may provide insights into the unresolved biological response mechanism against TBT toxicity and the activation mechanism of the Keap1-Nrf2 pathway.

Cytosine arabinoside induces phosphorylation of histone H2AX in hippocampal neurons via a noncanonical pathway.

Cytosine arabinoside (Ara-C), an anticancer drug, is known to inhibit DNA replication in mitotic cells. Ara-C is also considered to induce DNA damage, leading to neuronal cell death. To identify the mechanism by which Ara-C kills neurons, we assessed the levels of phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks (DSBs), in hippocampal neurons cultured for 48 h with Ara-C. There was a time-dependent increase in the percentage of cells accumulating γ-H2AX, but TUNEL staining did not indicate the formation of DSBs. The nuclear spread of γ-H2AX remained after Ara-C was withdrawn. These features of Ara-C-induced γ-H2AX formation were quite distinct from those observed in proliferating pheochromocytoma cells. Furthermore, Ara-C-induced γ-H2AX formation appeared to utilize cyclin-dependent kinase 7, but not ataxia telangiectasia mutated (ATM) or ATM and Rad3 related, which are well-known kinases in γ-H2AX formation. Taken together, our findings indicated that Ara-C stimulated γ-H2AX formation in neurons without DSB formation and utilization of canonical kinases, leading to neuronal cell death.