Neuronal cells have highly developed ER which makes
Neuronal oligomycin synthesis have highly developed ER, which makes them susceptible to loss of ER function following exposure to various agents, which leads to apoptosis (Lindholm et al., 2006, Katayama et al., 2004). Multiple pathways may be involved in ER stress-initiated apoptosis. Among them, the PKR-like endoplasmic reticulum kinase (PERK) pathway has been well-studied (Armstrong et al., 2010). This pathway leads to phosphorylation of eukaryotic initiation factor 2a (eIF2a) and enhances translation of mRNAs such as ATF4. eIF2a-ATF4 signaling has a pro-apoptotic function that involves the activation of ER chaperone glucose regulated protein 78 (GRP78), also known as binding protein (BiP), phosphorylated eIF2a, ATF4, and C/EBP homologous protein (CHOP) (Armstrong et al., 2010, Jiang and Wek, 2005, Qing et al., 2012).
Recently, an in vitro study has shown that exposure to TDCIPP can induce apoptosis in PC12 cells (Ta et al., 2014). However, the role of ER stress and related signaling pathways in TDCIPP-induced neurotoxicity is poorly understood. In the present study, a human neuroblastoma cell line (SH-SY5Y) was used as an in vitro model to determine the cytotoxic mechanisms of TDCIPP. We investigated cell viability and cell apoptosis, ROS generation and change in [Ca2+]i, and related protein expression patterns in ROS–mediated ER stress signaling pathways.
Materials and methods
Discussion The toxicity of TDCIPP has received much attention because of its presence in human samples. Several recent in vivo and in vitro studies have reported that exposure to TDCIPP results in several neurotoxic effects, including apoptosis (Ta et al., 2014, Behl et al., 2015). However, little information is available about the cellular signaling mechanisms that mediate the neurotoxic effects of TDCIPP. In the present study, SH-SY5Y neuroblastoma cells were selected as a simple, rapid, and cost-effective system to characterize these signaling pathways and to evaluate the toxicity of TDCIPP in the nervous system. Our results suggested that TDCIPP could indeed cause apoptosis. The initiation of apoptosis that we observed involved the generation of ROS and elevated [Ca2+]i. Furthermore, we found that TDCIPP-induced toxicity is associated with ER stress pathways, and the generation of ROS could occur upstream of TDCIPP-induced ER stress. We found that the IC50 of TDCIPP in SH-SY5Y cells was about 100μM. The measured IC50 is comparable to that reported from a study in the same cells exposed to the organophosphorus insecticide, chlorpyrifos (Lee et al., 2012a), suggesting that the toxicity of TDCIPP is equivalent to that of chlorpyrifos in SH-SY5Y cells. Further, our western blot results showed the induction of apoptosis markers (e.g. caspase 3). On the other hand, a loss of ΔΨm was observed, indicating that TDCIPP induced the apoptotic process via a mitochondria-mediated caspase-dependent pathway. These results are consistent with those of a recent study showing that chlorpyrifos (100μM), a typical organophosphorus pesticide, can induce SH-SY5Y cell apoptosis via a mitochondria-dependent pathway (Ki et al., 2013). To elucidate the cell signaling involved in the apoptotic pathway, we examined Bcl-2 and Bax genes and proteins. The members of the Bcl-2 family of proteins are important regulators that inhibit apoptotic cell death, whereas Bax genes are pro-apoptotic genes associated with the mitochondrial membrane (Garrido et al., 2006). The Bax/Bcl-2 ratio is critical for the regulation of apoptosis (Boussabbeh et al., 2015). In the present study, RT-qPCR showed that TDCIPP treatment significantly reduced the expression of Bcl-2 and increased Bax gene expression in SH-SY5Y cells, leading to an increased Bax/Bcl-2 ratio. The increase in Bax could be responsible for the concomitant execution phase of apoptosis, including disruption of the mitochondrial membrane and activation of caspase-3. Caspase-3 is a key executor of apoptosis, and is a prominent caspase that is activated downstream in apoptosis pathways (Kuribayashi et al., 2006). Ta et al. also found that TDCIPP (5–50μM) reduced PC12 cell viability and increased apoptosis (Ta et al., 2014). Taken together, these findings suggest that apoptosis could be one of the mechanisms of TDCIPP neurotoxicity.