Apoptosis is a process of cells organizing self-destruction (Chen et al., 2007). Numerous studies have demonstrated that DLM can cause neuronal apoptosis (Huang et al., 2019; Liu et al., 2018; Strungaru et al., 2019). In this study, we explored the relationship among quail cerebrum neuronal apoptosis, mitochondrial, and ER. The mitochondrial apoptosis pathway is regulated by the members of the Bcl-2 family including pro-apoptotic (Bax) and anti-apoptotic (Bcl-2, Bcl-xL) proteins, while ER apoptosis pathway is activated by long-term or serious ER stress (Li et al., 2017; Logue et al., 2013). Here, we found that DLM decreased Bcl-2 protein expression, suggesting that DLM promotes neuronal apoptosis of quail cerebrum related with mitochondrial. Indeed, this was confirmed by TUNEL staining of cerebrum tissue from quail. Thus, DLM exposure induces neuronal apoptosis in quail cerebrum.
ER stress pathway is a new type of apoptosis pathway (Yang et al., 2020). Exposed to DLM, neurons may undergo ER stress (Hossain et al., 2020). Severe or long-term ER stress impairs ER function, and then apoptosis will be induced by activating ER stress-mediated apoptosis signaling pathway (Xu et al., 2020). At this time, the three activated transmembrane proteins ATF-6, IRE, and PERK will promote the production of C/EBP-homologous protein (CHOP), and then trigger apoptosis. Normally, the three membrane proteins are combined with chaperone GRP78/Bip and keep inactive. However, when ER stress outbreak, ATF-6, IRE, and PERK will be separated from GRP78/Bip and be activated. IRE1 and ATF-6 are transfered into the nucleus and bind to the ER stress response element to initiate the transcription and expression of CHOP. The transcription factor ATF-4 which is the downstream of PERK can bind to the AARE domain of the CHOP and promote CHOP expression, then down-regulate the expression of anti-apoptotic protein Bcl-2 (Rauti et al., 2020; Wang et al., 2021b). In our experiment, the findings indicated that DLM exposure obviously induced ER stress and further triggered neuronal apoptosis in quail cerebrum. Therefore, ER stress gets involved in the progress of DLM-induced apoptosis of quail cerebrum and plays an important role.
Mitochondria is the main source of ROS (Patel et al., 2020). The destruction of mitochondria will lead to the accumulation of a large number of ROS, which causes more serious mitochondrial dysfunction (Awoyemi et al., 2020; Han et al., 2021). MDA, SOD, and GSH are important indicators for detecting oxidative stress (Abdel-Daim et al., 2020). The release of ROS can cause oxidative stress in cells and it can trigger apoptosis through a variety of signaling pathways. When mitochondria undergo apoptosis, the expression of Bcl-2 will be inhibited (Zhang et al., 2020b). Simultaneously, the permeability of the mitochondrial membrane changes, then AIF and Cyt C are released from the mitochondria into the cytoplasm. AIF can be transported into the nucleus and cause DNA fragmentation (Li et al., 2020b). AIF and Cyt C are both pro-apoptotic factors (Han et al., 2018). These factors activate the caspase signaling pathway and undergo a series of enzyme-linked reactions to apoptosis. Our results showed that DLM induced mitochondrial damage of quail cerebrum neurons, causing oxidative stress of neurons. Meanwhile, the expressions of mitochondrial apoptosis-related factors AIF and Cyt C were increased. The TEM and the ATP assay results further confirmed DLM-induced mitochondria dysfunction in quail cerebrum. Hence, mitochondria dysfunction is involved in the process of neuronal apoptosis in quail cerebrum induced by chronic DLM exposure.
The use of pesticides is closely related to human health (Oudejans et al., 2020). DLM is a highly effective insecticide, but with a defect of polluting the environment. As a representative animal of poultry, quail has excellent advantages of high sensitivity, easy to feed, and small size. Besides, the toxic effects of quails are closer to the toxic effects of toxic substances in the natural environment, because of their idiographic genetic conditions. Therefore, quail has high great research value in pesticide safety evaluation (Bean et al., 2019). Studies on the effects of DLM neurotoxic have been reported in mammals, fish, and insects, but no reports have been reported on birds. According to report, in mouse cells, DLM can continuously increase the content of Ca2+ in cells, and the imbalance of Ca2+ homeostasis will promote ER stress (Choi et al., 2007). The response of ER stress will produce a large amount of CHOP, which inhibits the expression of Bcl-2 located on the mitochondrial membrane, thereby promoting apoptosis of the mitochondrial pathway (Wu et al., 2007). With the gradual loss of ER function, ER will further release Ca2+ into the cell cytoplasm (McFarland et al., 2020). High concentrations of Ca2+ can directly act on mitochondria, causing mitochondrial dysfunction and triggering mitochondrial-related apoptosis (Quintana et al., 2020). However, the effect of DLM on intracellular Ca2+ in cells of quail has not been reported to date. The specific mechanism of ER induced by DLM in quails is still unclear. In our study, the results suggest that DLM caused neuronal apoptosis of quail cerebrum. However, the mechanism is still unclear whether neuronal apoptosis of quail is caused by ER stress and mitochondrial dysfunction working together or separately (Fig. 7). Hence, we speculate that DLM induces neurotoxicity in quail cerebrum neurons, which is similar to DLM-induced cytotoxicity by increasing intracellular Ca2+ concentration in other animals.