Deltamethrin induces apoptosis in cerebrum neurons of quail via promoting endoplasmic reticulum stress and mitochondrial dysfunction

Deltamethrin (DLM) is a widely used and highly effective insecticide. DLM exposure is harmful to animal and human. Quail, as a bird model, has been widely used in the field of toxicology. However, there is little information available in the literature about quail cerebrum damage caused by DLM. Here, we investigated the effect of DLM on quail cerebrum neurons. Four groups of healthy quails were assigned (10 quails in each group), respectively given 0, 15, 30, and 45 mg/kg DLM by gavage for 12 weeks. Through the measurements of quail cerebrum, it was found that DLM exposure induced obvious histological changes, oxidative stress, and neurons apoptosis. To further explore the possible molecular mechanisms, we performed real‐time quantitative PCR to detect the expression of endoplasmic reticulum (ER) stress‐related mRNA such as glucose regulated protein 78 kD, activating transcription factor 6, inositol requiring enzyme, and protein kinase RNA (PKR)‐like ER kinase. In addition, we detected ATP content in quail cerebrum to evaluate the functional status of mitochondria. The study showed that DLM exposure significantly increased the expression of ER stress‐related mRNA and decreased ATP content in quail cerebrum tissues. These results suggest that chronic exposure to DLM induces apoptosis of quail cerebrum neurons via promoting ER stress and mitochondrial dysfunction. Furthermore, our results provide a novel explanation for DLM‐induced apoptosis of avian cerebrum neurons.

induce neurotoxicity in many species including human, and DLM exposure is related to neurodegenerative diseases. 6,7 Endoplasmic reticulum (ER) is one of the membrane organelles in eukaryotic cells, integrating a variety of functions such as regulation of calcium (Ca 2+ ), synthesis and maturation of proteins, biosynthesis of lipids, and maintenance of cell homeostasis. 8 ER stress, as a protective stress response in eukaryotic cells, can be activated by the imbalanced homeostasis. 9 Once ER stress arises, excessive improperly folded proteins accumulate in the ER lumen. 10 The unfolded protein response (UPR) then occurs, which is sensed and activated by three transmembrane proteins, activating transcription factor 6 (ATF6), inositol requiring enzyme (IRE), and protein kinase RNA (PKR)-like ER kinase (PERK). 11 URP plays a crucial role in the restoration of ER homeostasis. However, if ER stress is sustained or serious, UPR will trigger apoptosis. 12 In addition, ER stress increases the accumulation of reactive oxygen species (ROS) in cells. 13 ROS can induce oxidative stress, which can lead to apoptosis. 14 Mitochondria, as essential organelles in cells, play a vital role in energy production and calcium homeostasis regulation. More importantly, mitochondria are the regulatory center of apoptosis, controlling apoptosis via releasing Cytochrome C (Cyt C), activating cysteine proteases, changing calcium flux, decreasing ATP pool, and producing excess ROS. 15 The mitochondrial pathway, one of the classic apoptotic pathways, is easily activated by numerous stimuli, thereby inducing apoptosis. 16,17 Mitochondria and ER are important target organelles for studying apoptosis and they are closely related to cell survival. 18 As a crucial part of cerebrum, neuron is the most basic structure and function unit of nervous system. Neuronal damage is a crucial mechanism of neurological diseases. 19 According to report, DLM can increase the risk of Parkinson's disease, learning disabilities, and Alzheimer's disease. 20 DLM interacts with cell Na + channels causing an increase of intracellular Ca 2+ and activation of the ER stress pathway. 21 Simultaneously, high concentration of Ca 2+ might trigger mitochondrial permeability transition pore opening. 22 Cyt C, apoptosis-inducing factor (AIF) and other apoptotic factors enter the cytoplasm through the open mitochondrial permeability transition pore. Cyt C and AIF activate the caspase pathway causing apoptosis. 23 However, there may be a connection between mitochondrial apoptotic pathway and ER stress apoptotic pathway, which makes them jointly involved in apoptosis process. DLM-induced neuronal apoptosis in rats, mice, and fish has been reported. 21,24 However, there are a few studies on DLM-induced quail neuron apoptosis, 25 and the specific mechanism of DLM-induced neuronal apoptosis remains unclear.
In this study, we aim to investigate the effect of long-term DLM exposure on neurons of quail cerebrum, and explore the role of ER stress and mitochondrial dysfunction in the progress of quail neuronal apoptosis. We hope to provide a new insight for further elucidating the specific mechanism of avian neuronal apoptosis caused by DLM.

| Animal and experimental protocol
Forty healthy male quails (average body weight of 80 ± 10 g, 21 days old) were purchased from Wanjia Farm in Harbin, China. The quails were managed in the animal room of Northeast Agricultural University with the standard laboratory conditions (temperature 22 ± 2 C, light 12 h/ dark 12 h cycle, relative humidity 55 ± 5%). Food and water were obtained freely throughout the entire experiment. 26,27 After a week of adaptive feeding, four groups of quails were randomly assigned (n = 10 per group): control group (normal saline administered by gavage), low dose (15 mg/kg DLM administered by gavage), medium dose (30 mg/kg DLM administered by gavage), and high dose (45 mg/kg DLM administered by gavage). Treatment duration was 12 weeks. The quails were euthanized and their cerebrum tissues were quickly dissected. 28 Some fresh cerebrum tissues were used for the analysis of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD). The remaining cerebrum tissues were stored in 4% paraformaldehyde solution and À80 C refrigerator. 29 Our animal experimental procedure was authorized by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China) and the IACUC number is 20200818.

| Histopathological analysis
Quail cerebrum tissues were soaked with 4% paraformaldehyde for 48 h, embedded in paraffin and sectioned in 0.5 μm thickness. Cerebrum tissue sections were fixed on glass slides, then dewaxed and stained with hematoxylin and eosin (H&E). Finally, the sections were fixed with neutral resin. In addition, the pathological changes were observed by a light microscope (BX-FM, Olympus Corp, Tokyo, Japan). 30 The score of the cerebrum injury was according to previously study. 31

| Transmission electron microscopy
Fresh cerebrum tissues of quails were cut into small pieces less than 1 mm 3 with a sharp blade and fixed in 2.5% glutaraldehyde for 24 h at 4 C. 32 These cerebrum tissues were fixed again with 1% osmic acid and rinsed with 0.1 mol/L phosphate-buffered saline (PBS) before and after fixation. The tissue blocks were dehydrated with different concentrations of ethanol at 4 C. After embedding and curing, the tissue blocks were cut into ultrathin sections. Then they were dyed with uranyl acetate and lead citrate. Finally, the ultrastructure of quail cerebrum was observed by a transmission electron microscope (TEM) Hitachi H-7650 (Tokyo, Japan).

| Measurement of oxidative stress bio-chemical markers
Quail cerebrum tissues were mixed with PBS buffer and homogenized on ice plate for 2 min with a portable disperser (S10, scientz, Ningbo, China). Then centrifugation was carried out at 3000 r/min for 15 min at 4 C. After centrifugation, the supernatant was used to measure the activity of SOD, and the contents of MDA and GSH in quail cerebrum with the corresponding commercial kits. 33,34

| Measurement of ATP concentration in cerebrum
The ATP content in cerebrum tissue of quail was determined by phosphomolybdic acid colorimetry. The cerebrum tissue was mixed with boiling distilled water to make homogenate. After boiling and centrifugation, the supernatant was taken for testing. According to the manufacturter's instructions, we added the corresponding reagents.
Finally, the absorbance was measured by spectrophotometer (PHILES, 156, Nanjing, China) with the wavelength of 636 nm. The protein concentration of cerebrum tissue was determined by BCA method. The results of ATP content were calculated by the known formula in the instructions of ATP kit.

| TUNEL assay
TUNEL assay was used to detect apoptosis. After the quail cerebrum was fixed by perfusion with 4% paraformaldehyde, paraffin sections were prepared routinely, and the section thickness was 4 μm. 35 We

| Quantitative real-time PCR
In order to extract total RNA from quail cerebrum tissue, Trizol reagent was used. 38,39 RNA concentration was determined by using ultramicro nucleic acid protein analyzer (Implen, N60, Germany), then the RNA was reverse transcribed into cDNA by a cDNA reverse transcription kit (Vazyme, Nanjing). As shown in Table 1, specific primers were synthe-  The data were presented as mean ± SEM. One-way ANOVA was performed to compare multiple groups following Dunnett's post-hoc test. P < 0.05 was considered statistically significant.

| DLM treatment exacerbated cerebrum injury in quail
As shown in Figure 1, the cerebrum tissue structure of the control group was normal, with normal neuronal morphology and diversity.
Compared with the control group, the neuronal morphology was changed and some neurons were shrunk in the DLM groups. In addition, some nuclei were disappeared and a large number of diseased neurons were appeared in the high dose DLM group.

| DLM treatment changed neurons ultrastructure in quail cerebrum
To determine the damage of neurons, we used TEM to observe the ultrastructure of quail cerebrum neurons. The normal morphology of nucleus, mitochondria, and ER could be observed in the control group. However, changes in nuclear morphology, mitochondrial swelling, and ER swelling were observed in DLMtreatment groups. In addition, the happening of nuclear lysis, mitochondrial swelling and ridge rupture, and ER swelling occurred significantly in the high dose group compared with the control group ( Figure 2).

| DLM treatment induced oxidative stress and reduced ATP level in quail cerebrum
To analyze the level of oxidative stress in the cerebrum tissues of quails, we detected the concentration of MDA and GSH, and the activity of SOD. As expected, DLM significantly increased the concentration of MDA ( Figure 3A). In addition, treatment with DLM significantly decreased GSH concentration and SOD activity compared with the control group ( Figure 3B and C). The ATP concentration of quail cerebrum was shown in Figure 3D. Compared with the control group, the ATP concentration of quail cerebrum in DLM-treatment groups decreased significantly and this effect was dose-related.

| DLM treatment induced neuronal apoptosis in quail cerebrum
Apoptosis of cerebrum neurons in quail was determined by TUNEL staining. There were only a few TUNEL-positive cells in the control group. However, the number of apoptotic cells increased significantly in DLM-treatment groups and it was a dose-dependent relationship.
Furthermore, the apoptosis rate of neurons in high dose DLM group was the highest (Figure 4).

| DLM induced apoptosis via activating the ER pathway and exacerbating the mitochondrial damage pathway in quail cerebrum
The ER pathway and mitochondrial pathway are key cell life activities.
The new study provides evidence that the ER and mitochondrial pathway participate in the process of apoptosis. 46 With the further research, the study has shown that ER pathway and mitochondrial damage pathway are related to upstream related molecules. 47 We DLM groups were increased significantly compared to the control group and showed a dose-dependent relationship ( Figure 5D). In the mitochondrial apoptotic pathway, we detected the protein expressions of Bcl-2, Bax, and caspase-3. Furthermore, we measured the mRNA expression levels of AIF, Cyt C, Bcl-2, and caspase-3. In DLM groups, the protein expression levels of Bcl-2 was decreased significantly and the Bax protein was increased ( Figure 5A). The protein expression levels of caspase-3 ( Figure 5B) was increased significantly in a dose-dependent manner. As shown in Figure 4C, the mRNA expression levels of AIF, Cyt C, and caspase-3 were increased significantly and the mRNA expression levels of Bcl-2 decreased in DLM groups, and this effect was dose-related.

| PPI analysis
We constructed a PPI analysis network of cerebrum damage using the STRING 10 database (Figure 6) to confirm our conclusion. The

| DISCUSSION
Apoptosis is a process of cells organizing self-destruction. 48 Numerous studies have demonstrated that DLM can cause neuronal apoptosis. [49][50][51] 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 and Bcl-xL) proteins, while ER apoptosis pathway is activated by long-term or serious ER stress. 52,53 Here, we found that DLM decreased Bcl-2 protein expression and increased the expression of Bax protein, 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. 54 Exposed to DLM, neurons may undergo ER stress. 28 Severe or long-term ER stress impairs ER function, and then apoptosis will be induced by activating ER stress-mediated apoptosis signaling pathway. 55 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 sepa- The use of pesticides is closely related to human health. 70 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. 71 Studies on the effects of DLM neurotoxic have been reported in mammals, fish, and insects. [72][73][74] Furthermore, there were only a few studies about the exposure of pyrethroid induced the change of brain morphology. 75,76 According to report, in mouse cells, DLM can continuously increase the content of Ca 2+ in cells, and the imbalance of Ca 2+ homeostasis will promote ER stress. 77 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. 78 With the gradual loss of ER function, ER will further release Ca 2+ into the cell cytoplasm. 79 High concentrations of Ca 2+ can directly act on mitochondria, causing mitochondrial dysfunction and triggering mitochondrial-related apoptosis. 80 However, the effect of DLM on intracellular Ca 2+ 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 (Figure 7). The genome-wide gene expression profiling could be used to clarify the mechanism in the future. 81 Hence, we speculate that DLM induces neurotoxicity in quail cerebrum neurons, which is similar to DLMinduced cytotoxicity by increasing intracellular Ca 2+ concentration in other animals.

| CONCLUSION
In conclusion, DLM exposure induces apoptosis of cerebrum neurons in quails via promoting ER stress and mitochondrial dysfunction. Our study provides a novel explanation for DLM-induced apoptosis of avian cerebrum neurons.
F I G U R E 6 PPI analysis. Protein network of proteins regulated between ER stress-related genes, mitochondrial dynamics-related genes, and apoptosis-related gene expressed in the cerebrum of quail. PERK (Eif2ak3), ATF6 (Atf6), IRE1 (Ern1), GRP78 (Hspa5), AIF (Aifm1), Cyt C (Cycs), caspase-3 (Casp3), and Bcl-2 (Bcl2). Network nodes represent proteins splice isoforms or post-translational modifications are collapsed, that is, each node represents all the proteins produced by a single, protein-coding gene locus. Edges represent proteinprotein associations are meant to be specific and meaningful, that is, proteins jointly contribute to a shared function; this does not necessarily mean they are physically binding to each other, and different color of the edges represented different interactions which showed in the above F I G U R E 7 The mechanism on neuronal apoptosis induced by DLM in quail. DLM induces neuronal apoptosis via promoting ER stress and mitochondrial dysfunction

ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (31972754).

CONFLICT OF INTEREST
There are no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT
Research data are not shared.