4.1 BPA may induce pyroptosis by NLRP3/Caspase-1/GSDMD signaling pathway
Pyroptosis is a newly identified form of inflammatory cell death triggered by caspase-1, which is characterized by caspase-1-dependence, the accumulation of GSDMD fragments, the formation of membrane pores and the leakage of intracellular contents, such as IL-18, IL-1β, LDH, etc. (Pi et al., 2021). Studies have shown that pyroptosis is involved in various diseases, particularly neurodegenerative diseases (Voet et al., 2019; Zhao et al., 2019). It is reported that polystyrene microplastics, which continuously pollute the environment, can induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/caspase-1 signaling pathway triggered by oxidative stress (Hou et al., 2021). Moreover, the activation of NLRP3 plays an important role in caspase-1-dependent pyroptosis. For example, one study has shown that the activation of NLRP3 is critical for the cell pyroptosis induced by propofol (Sun et al., 2019). Caspase-1 is responsible to transform pro-IL-1β to IL-1β (the active form), which is thus named IL-1β converting enzyme (Zhao et al., 2012). Activated caspase cleaves the connection between the N-terminal and C-terminal domains of GSDMD protein and releases the active N-terminal domain, which destroys the cell membrane and causes pyroptosis (Ding et al., 2016). In this study, BPA treatments can not only significantly change the mRNA levels of NLRP3, GSDMD, ASC, IL-1β, IL-18 and caspase-1 and the protein levels of Bcl-2, Bax, Bak1, caspase-3, NLRP3, caspase-1, GSDMD and IL-1β, but also increase the secretion of IL-1β, IL-18, caspase-1 and LDH in neuroblastoma cells, especially in IMR-32 cells from male. All results suggest that BPA induces pyroptosis of nervous cells, and male IMR-32 cells are more susceptible to BPA than female SK-N-SH cells. Moreover, even 1 nM BPA treatments can lead to the similar effects and consequently promote the pyroptosis of IMR-32 cells. These results suggest that BPA treatments can potently induce pyroptosis of IMR-32 and SK-N-SH cells. In the same time, our study also shows that the BPA-induced pyroptosis not only triggers nervous cell death but also mediates the releases of IL-1β, IL-18, caspase-1 and LDH, which will lead to an excessive proinflammatory process. Based on the above factors, LDH release is considered to be one of the main characteristics of pyroptosis (Fink et al., 2008). As an inhibitor of pyroptosis, YVAD is reported to restrain the caspase-1 activation, reduce the secretion of IL-1β and IL-18 and suppress the occurrence of pyroptosis in cells (Yue et al., 2019; Zamani et al., 2020). In this study, the caspase-1 mRNA and protein levels were significantly inhibited after YVAD treatment as expected, which further confirms that YVAD inhibits the pyroptosis of IMR-32 and SK-N-SH cells by inhibiting the activation of caspase-1. Simultaneously, the differences of mRNA expressions (i.e., NLRP3, GSDMD, ASC, IL-1β and IL-18), protein expressions (i.e., NLRP3, GSDMD and IL-1β) and GSDMD fluorescence expression between the BPA-treated group and the BPA + YVAD treated group suggest that BPA can induce pyroptosis in IMR-32 and SK-N-SH cells via NLRP3/Caspase-1/GSDMD signaling pathway.
Our previous studies has shown that BPA can promote ROS production, leading to oxidative stress and apoptosis in IMR-32 and SK-N-SH cells (Wang et al., 2021). ROS is involved in pyroptosis and has a significant action in regulating pyroptosis, which has been confirmed in many studies (Chen et al., 2016; Zhang et al., 2021). Excessive ROS may activate the NLRP3 inflammasome signaling pathway (Sun et al., 2019). In addition, ROS can mediate the NLRP3/Caspase-1/GSDMD signaling pathway involved in the process of cytotoxicity (Yang et al., 2020). Therefore, we speculated that ROS might also be involved in the BPA-induced pyroptosis of IMR-32 and SK-N-SH cells via the NLRP3/Caspase-1/GSDMD pathway. In our study, if cells were co-treated with BPA and EGCG, the differential expressions of mRNA (i.e., NLRP3, GSDMD, ASC, IL-1β, IL-18 and caspase-1) and proteins (i.e., NLRP3, caspase-1, GSDMD and IL-1β) in IMR-32 and SK-N-SH cells between the BPA-treated groups and the BPA + EGCG-treated groups were reduced or eliminated. At the same time, the secretion of IL-1β, IL-18, caspase-1 and LDH were also reduced. In addition, the level of MMP was increased. As inflammasomes induce the maturation and the release of IL-1β and IL-18, we preliminarily speculate that the BPA-induced ROS activate the NLRP3 inflammasome, and then resulting in pyroptosis. As a ROS scavenger, EGCG can inhibit caspase-1-dependent pyroptosis in IMR-32 and SK-N-SH cells.
4.2 Pyroptosis of neuroblastoma cells by BPA contributes to apoptosis
Pyroptosis is a caspase-1-dependent form of programmed inflammatory cell death. Apoptosis is a caspase-3-dependent and non-inflammatory programmed form (Rogers et al., 2017), regulated by anti-apoptotic and pro-apoptotic families (i.g., caspase family and Bcl-2 family) (Siddiqui et al., 2015). Caspase-3 is often activated by the death receptor mediated apoptotic pathway through the mitochondrial apoptotic pathway (Li and Yuan, 2008), which is controlled by the Bcl-2 family proteins. The activation of two Bcl-2 family members (i.e., Bak and Bax) can form pores on the mitochondrial outer-membrane, resulting in the release of mitochondrial inter-membrane components, and finally activate the occurrence of caspase cascade (Czabotar et al., 2014; Degterev and Yuan, 2008; Delbridge et al., 2016). However, pyroptosis can also be induced by pro-apoptotic caspase-3 (Rogers et al., 2017; Wang et al., 2017). Therefore, understanding the relationship of pyroptosis with apoptosis is helpful to clarify the mechanism of BPA apoptosis.
Environmental pollutants are considered an important factor causing pyroptosis and apoptosis (Yu et al., 2021; Zhang et al., 2021). Moreover, cell pyroptosis has been proved to promote cell apoptosis (Shi et al., 2017). Pi et al. reported that molybdenum induced pyroptosis in duck renal tubular epithelial cells and YVAD reduced the molybdenum-induced apoptosis (Pi et al., 2021). We hypothesized that the relationship of apoptosis and pyroptosis in nervous cells induced by BPA might be similar to the previous reports. In our study, YVAD was used as a caspase-1 inhibitor to explore the relationship between the BPA-induced pyroptosis and apoptosis by detecting protein levels of Bcl-2, Bak-1, Bax and caspase-3, MMP and apoptosis ratio. Bak-1 and Bax can promote apoptosis (Shi et al., 2010). Bcl-2 can inhibit apoptosis, which is mainly located on the outer membrane of the mitochondria and regulates apoptosis through mitochondrial pathway (Birkinshaw and Czabotar, 2017). Caspase-3 is the executor caspase of apoptosis. MMP is a vital marker of intrinsic apoptosis (Park et al., 2020). Our results showed that protein levels of Bak-1, Bax and caspase-3 in the 10 µM or the 100 µM BPA-treated groups were significantly higher than those in the control group, but the protein level of Bcl-2 demonstrated the opposite trend in IMR-32 (or SK-N-SH) cells. Further studies showed that BPA treatments could decrease MMP and increase apoptosis ratio and LDH level. Moreover, YVAD could significantly improve the changes of apoptosis-related indicators induced by BPA. These results suggest that YVAD can decrease the BPA-induced apoptosis in neuroblastoma cells. Moreover, the activation of caspase-1 is involved in the BPA-induced apoptosis. Hence, we can conclude that apoptosis is related with pyroptosis in the BPA-induced neurotoxicity, and the inhibition of caspase-1 dependent pyroptosis may attenuate the BPA-induced apoptosis in neuroblastoma cells.
4.3 BPA induces damages in neuroblastoma cells in a nonmonotonic and gender-specific manner
In this study, we used IMR-32 and SK-N-SH cells as the model to investigate the gender-dependent neurotoxicity induced by various concentrations of BPA. Interestingly, a non-monotonic relationship between the BPA concentrations and cytotoxic effects was observed. In particular, low-dose BPA treatment showed more obvious cytotoxicity than medium or high dose. There was an inverted U-shaped curve between BPA exposure concentration and cytotoxicity, which may be attributed to the exogenous endocrine disrupting effects of BPA. Our results are in accordance with the previous studies that low-dose BPA or bisphenol S can cause significant impacts on rats, while the middle-dose BPA has no obvious effects (da Silva et al., 2019; Zhang et al., 2019). In general, concentrations used to assess toxic effects of pollutants often range from low to high doses. However, only high concentration exposure is used in the study of molecular mechanism if the dose-effect response is linear. Our study showed that it is important to set the appropriate concentration range of BPA according to the toxic effect to study its corresponding toxic mechanism. Otherwise, improper concentration selection will lead to meaningless mechanism or opposite results because its non-linear relationships between dose and effects. It is easier to draw meaningful conclusions by selecting concentrations of toxic effects.
As an environmental endocrine disruptor, BPA has been shown to activate several types of receptors, including ER (Perera et al., 2017; Pinto et al., 2019). Although our in vitro studies have shown that BPA treatment can lead to apoptosis and pyroptosis in different gender nervous cells, and further confirmed the previous in vivo data that BPA can gender-dependently disrupt dendritic development and neurotransmitter homeostasis in the rat hippocampus (Zhang et al., 2019), the exact mechanism of BPA gender-specific induction of apoptosis and pyroptosis of neuroblastoma cells remains unclear. In this study, at the transcriptional level, our results showed that the expression trends of IL-1β and caspase-1 levels were opposite in male and female neuroblastoma cells, while the expression trends at the translation level were consistent between both gender cells. And then we observed that the BPA-induced apoptosis and pyroptosis in IMR-32 and SK-N-SH cells were significantly attenuated after the ICI (an ER inhibitor) pretreatments. Therefore, BPA may induce apoptosis and pyroptosis of nervous cells through ER. The distribution and content of ER in the body may be related to the higher effects of BPA on males. Bisphenol AF, an analogue of BPA, is reported to promote the ER activation and induce apoptosis in some cell lines (Huang et al., 2021; Kojima et al., 2019). When the secretion of estrogen is reduced and the expression of ER is low, women are more prone to vascular damage (Trenti et al., 2018). BPA exposure resulted in a significant down-regulation of estrogen receptor β (ERβ) in male hippocampus, but not in female hippocampus (Xu et al., 2015). Therefore, the role of exogenous estrogens (including BPA) in ER activation and function (i.g., the regulation of ROS generation, apoptosis and pyroptosis) still needs to be further clarified by primary cells or animal experiments.