Exploring the molecular mechanism of Parkinson's disease triggered by combined atrazine and simazine exposure

1 Background

Parkinson's disease (PD) ranks as the second most prevalent neurodegenerative disease characterized by a progressive decline in neurological function, trailing only Alzheimer's disease (Jiang et al., 2020). The main clinical manifestations of PD consist of asymmetrical bradykinesia, resting tremor, rigidity, and postural instability (Cabezas et al., 2014; Daviaud et al., 2014). The gradual loss of dopamine-producing neurons in the midbrain is the main contributor to motor dysfunction in PD (Grealish et al., 2010). PD is influenced by both genetic and environmental factors (Kim et al., 2022) and, in particular, pesticide exposure is a well-established environmental risk factor (Zhang et al., 2018).

Triazine herbicides are commonly used in the cultivation of crops like maize, sugarcane, and sorghum, and may accumulate in the soil, groundwater, and food products, posing potential health risks to agricultural practitioners (Buglak et al., 2019; Xu et al., 2019). Atrazine (ATR) and simazine (SIM) are typical triazine herbicides, which are used widely worldwide. At present, in vivo and in vitro experiments have shown that ATR or SIM exposure can damage dopaminergic neurons and cause PD-like symptoms (Hossain and Filipov, 2008; Walters et al., 2015; Li et al., 2017). Considering the chemical structural similarity of ATR and SIM, the combined exposure to these herbicides might be an important environmental factor contributing to the development of PD. However, there are few studies on the combination of ATR and SIM exposure, and the mechanisms that may cause PD are still unknown.

In this study, we obtained differentially expressed genes (DEGs) for PD by analyzing brain nigrostriatal microarray data from PD patients and controls. The differential targets of combined ATR and SIM exposure leading to PD were further screened by network pharmacology and validated by molecular docking, as well as molecular biological detection. Our findings suggest that the combined exposure of ATR and SIM could be associated with the onset of PD. This implies the necessity for additional investigation into the molecular mechanisms that underlie this connection.

2 Materials and methods

3 Results

3.1

3.1 DEG screening and network pharmacological analysis

In total, we identified 8416 mRNAs, of which 934 were differentially expressed (802 upregulated and 132 downregulated, Fig. 1a and b). Through the SwissTargetPrediction, CTD, STITCH, and GeneCards databases, we obtained 3063 targets for ATR, 31 targets for SIM, and 3076 targets of triazine herbicides after removing duplicate targets. Through Venn analysis, we finally obtained 151 intersection targets (122 upregulated and 29 downregulated, Fig. 1c). The intersectional targets were used to construct a PPI network using the STRING database, which included 151 nodes and 181 edges with an average node degree of 2.4 (Fig. 1d, Table S2).

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Fig. 1

DEG screening and network pharmacological analysis. (a) Volcano plot of DEGs between PD patients and control. (b) Heatmap of DEGs between PD patients and control. (c) The Venn map of triazine herbicide-related targets and PD-related targets. (d) The PPI network. The width of each edge is proportional to the combined score, whereas the color (from green to pink) and size of each node are proportional to interaction strength.

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3.2

3.2 GO and KEGG enrichment analysis

To investigate the potential mechanisms underlying triazine herbicide-induced PD, we conducted GO and KEGG pathway enrichment analyses. The overlapping targets were enriched in the BP terms “transmembrane receptor protein tyrosine kinase signaling pathway”, “response to hormone”, and “regulation of sequestering of calcium ion”; the CC terms “transcription regulator complex”, “asymmetric synapse”, and “neuron to neuron synapse”; and the MF terms “DNA-binding transcription activator activity”, “hormone binding”, and “phosphatase activity” (Fig. 2a–c). The KEGG pathway analysis revealed that the common targets were enriched in the terms “Calcium signaling pathway”, “Ras signaling pathway”, and “Estrogen signaling pathway” (Fig. 2d).

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Fig. 2

GO and KEGG enrichment analysis. (a) BP enrichment analysis, (b) CC enrichment analysis, (c) MF enrichment analysis, and (d) KEGG pathway enrichment analysis.

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3.3

3.3 Hub target identification

The top 10 targets were calculated by the closeness, degree, EPC, MCC, MNC, and radiality methods (Fig. 3a–f, Table S3). Through Venn analysis, cyclin-dependent kinase 2 (CDK2), cytochrome c (CYCS), Fos proto-oncogene (FOS), Src family tyrosine kinase (LCK), BDNF/NT-3 growth factors receptor (NTRK2), platelet-derived growth factor receptor beta (PDGFRB), progesterone receptor (PGR), and vascular endothelial growth factor A (VEGFA) were identified as the top eight hub targets (Fig. 3g), and PPI networks of CDK2, CYCS, FOS, LCK, NTRK2, PDGFRB, PGR, and VEGFA were constructed through the STRING database (Fig. 3h).

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Fig. 3

Hub target identification. Top 10 targets calculated through (a) the closeness method, (b) the degree method, (c) the EPC method, (d) the MCC method, (e) the MNC method, and (f) the radiality method. The width of each edge is proportional to the combined score, whereas the color (light green to purple). (g) A Venn map of the top eight core targets. (h) The PPI network of the eight targets.

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3.4

3.4 Molecular docking

To further investigate the ability of ATR and SIM to bind to the top eight core targets, we performed a molecular docking analysis. The binding energy between ATR and CDK2 was −7.38 kcal/mol (Fig. 4a), between ATR and CYCS it was −7.68 kcal/mol (Fig. 4b), between ATR and PGR it was −7.42 kcal/mol (Fig. 4c), between SIM and CDK2 it was −7.13 kcal/mol (Fig. 4d), between SIM and CYCS it was −7.56 kcal/mol (Fig. 4e), and between SIM and PGR it was −7.26 kcal/mol (Fig. 4f). The binding energies of the remaining targets to both ATR and SIM were greater than −7 (Table S4).

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Fig. 4

Molecular docking. (a) ATR-CDK2, (b) ATR-CYCS, (c) ATR-PGR, (d) SIM-CDK2, (e) SIM-CYCS, and (f) SIM-PGR.

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3.5

3.5 Cell viability assay and qPCR

To investigate the dopaminergic neurotoxicity effect of ATR and SIM alone or in combination, the SH-SY5Y cells were treated by various concentrations of ATR and SIM for 72 h, followed by a CCK-8 assay. We found that both ATR (Fig. 5 a-b, ANOVA: F (5,30) = 1.29, P < 0.01) and SIM (Fig. 5 c-d, ANOVA: F (5,30) = 0.36, P < 0.01) decreased the cell viability in a dose-dependent manner. The IC_50s for ATR and SIM in SH-SY5Y cells were 396.2 μM and 69.1 μM respectively. The qPCR analysis demonstrated that exposure to SIM (35 μM, 1/2 IC₅₀) alone or in combination with ATR (200 μM, 1/2 IC₅₀) for 72 h significantly increased CDK2 mRNA expression (Fig. 5 e, ANOVA: F (3,20) = 4.517, P < 0.05, SIM vs. control: P < 0.05, mixture vs. control: P < 0.01). ATR alone or combined with SIM for 72 h significantly elevated PGR mRNA levels (Fig. 5 f, ANOVA: F (3,20) = 6.293, P < 0.01, ATR vs. control: P < 0.05, mixture vs. control: P < 0.01). Conversely, SIM exposure alone or in combination with ATR for 72 h significantly reduced CYCS mRNA expression (Fig. 5 g, ANOVA: F (3,20) = 5.342, P < 0.01, SIM vs. control: P < 0.05, mixture vs. control: P < 0.01).

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Fig. 5

Cell viability assay and qPCR. (a) SH-SY5Y cellular model exposure to ATR, (b) Effects of ATR on the cytotoxicity of SH-SY5Y cells, (c) SH-SY5Y cellular model exposure to SIM, (d) Effects of SIM on the cytotoxicity of SH-SY5Y cells, (e) CDK2 mRNA expression level, (f) PGR mRNA expression level, and (g) CYCS mRNA expression level. Expression levels were standardized to GAPDH. Each column represents the mean ± S.E.M, *P < 0.05 vs. control, ^**P < 0.01 vs. control.

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4 Discussion

Although the majority of PD cases are believed to occur randomly, environmental factors may have a significant impact on the onset of the disease (Xia et al., 2017). According to epidemiological studies (Fernandez-Abascal et al., 2018), there may be a relationship between pesticide exposure and PD. Some pesticides, such as rotenone, have even been used to induce PD in animal models (Zhang et al., 2019). In the present study, we preliminarily explored the molecular biological mechanism of PD induced by combining ATR and SIM exposure through bioinformatics algorithms. We first screened aberrantly expressed mRNAs between PD patients and control by mining GSE20333 from GEO datasets, and acquired 934 DEGs (802 up-regulated and 132 down-regulated). Subsequently, a potential 151 ATR and SIM targets (122 up-regulated and 29 down-regulated) for the inducement of PD were predicted using network pharmacology. Additionally, GO and KEGG analyses were performed to investigate the biological characteristics of the potential targets, and eight hub targets were further screened for validation using molecular docking. Finally, we constructed ATR and SIM alone or in combination exposure cellular models to validate our screened results.

KEGG analysis showed the significant enrichment of DEGs in the calcium signaling and Ras signaling pathways. A previous study used spatial transcriptome profiling of the hippocampal single cell microzone and found that the calcium signaling pathway plays a key role in the PD mouse model (Jia et al., 2023). It also has been reported that neuromelanin loss in PD patients can result in increased calcium signaling due to the loss of a crucial calcium storage site, and ultimately leads to the production of reactive oxygen species (ROS) and cell death within the substantia nigra (Knörle, 2018). ATR is reported to have the ability to activate the calcium signaling pathway and enhance the rate of androgenesis caused by hCG in the Leydig cells of rats (Pogrmic-Majkic et al., 2016). Meanwhile, SIM exposure could affect cytoplasmic Ca²⁺ homeostasis, thereby increasing ROS levels and interfering with the ability of mouse oocytes to mature (Shang et al., 2021). Therefore, we proposed that the development of PD may be attributed largely to the impairment of the calcium signaling pathway resulting from exposure to triazine herbicides.

The Ras signaling pathway is the first well-established MAPK pathway that plays critical roles in cellular differentiation, development, survival, and proliferation (Zhao and Luo, 2022). Abnormalities in the Ras-MAPK signaling pathway are critical in the process of dopaminergic neuron loss in PD (Nataraj et al., 2017). Perlecan-conjugated laminin 511/521-E8 fragment treatment enhanced the maturation and neurite extension of the grafted dopamine progenitors by activating Ras signaling, which could be an efficient cell replacement therapy for PD (Adachi et al., 2022). It has been reported that ATR could promote the development of mammary tumors in human c-Ha-Ras proto-oncogene transgenic rats (Fukamachi et al., 2004). Moreover, our previous study suggested that ATR induced hippocampal neurotoxicity by affecting the Ras-MAPK signaling pathway in rats (Li et al., 2019). To our knowledge, no studies have shown that SIM exposure can affect the Ras signaling pathway, but given the similarity in chemical structure, we hypothesized that combined ATR and SIM exposure may contribute to PD pathogenesis through the Ras signaling pathway.

The top 10 hub targets were calculated by using six methods in cytoHubba and screening eight intersecting hub targets. Then, through molecular docking, we finally predicted three targets with a high binding potential to ATR and SIM: CDK2, CYCS, and PGR. CDK2 is mainly known for its pivotal role in cell cycle progression, including DNA synthesis, G1/S phase transition, and G2 progression modulation (Zhang et al., 2022). Within the mitochondria, the hemeprotein CYCS serves as a key player in both cellular respiration and apoptosis (Kalpage et al., 2019). Studies confirm that apoptosis due to abnormal expression of CDK2 and CYCS is the main cause of dopaminergic neuron deficiency in PD (Yu et al., 2005; Wang et al., 2007). ATR has been reported to have the ability to halt the cell cycle of neural stem cells at the G1 phase and hinder the transition from the G1 to the S phase by reducing the expression levels of CDK2 genes (Shan et al., 2021). Meanwhile, transcriptome sequencing analysis demonstrated that exposure to SIM induced reproductive toxicity due to a significant decrease in the expression of genes related to the cell cycle in Xenopus laevis (Sai et al., 2015). Notably, there is no evidence that ATR or SIM exposure can lead to abnormal expression or release of CYCS. Our molecular docking results showed that ATR and SIM bind strongly to CDK2 and CYCS, and we also found that exposure to ATR and SIM alone or in combination can significantly affect the mRNA expression of CDK2 and CYCS in SH-SY5Y cells. Our results suggests that CDK2 and CYCS are the key targets of Parkinson's disease induced by combined exposure to ATR and SIM.

PGR is a transcription factor belonging to the nuclear receptor superfamily, which plays a vital role in the intracellular signaling of progesterone and is involved in a multitude of cell biological activities (Lee et al., 2022). It has been reported that PGR plays a neuroprotective and regenerative role in PD (Zhao et al., 2020). Moreover, the significance of PGR expression during the developmental stage of the mesocortical dopamine pathway is crucial for intricate cognitive behavior in adulthood (Willing and Wagner, 2016). ATR and SIM have been reported to cause estrogen-like effects, which could lead to decreased PGR binding capacity in the cytoplasm (Connor et al., 1996). In addition, evidence suggested that ATR can affect the mRNA expression level of PGR and enhance the occurrence of androgenesis induced by hCG in Leydig cells of rats (Fa et al., 2013). Integrating the molecular docking and qPCR results, we hypothesized that combined ATR and SIM exposure may induce PD pathogenesis by interfering with the expression or function of PGR.

5 Limitations

The study has some limitations currently. First, the network pharmacology analysis relies on the timeliness of the database, and the update of the data could make the study more accurate. Secondly, our study only explored the molecular mechanisms of combined ATR and SIM exposure leading to PD, while other triazine herbicides, such as propazine, remain in the environment, so more complex combined exposures need to be taken into account. Finally, the present study was based on a bioinformatics and in vitro strategies to investigate the mechanism of PD caused by combined ATR and SIM exposure, and our results need to be confirmed by further in vivo studies.

6 Conclusion

Our study used several bioinformatics methods to explore the molecular mechanisms underlying PD induced by simultaneous exposure to ATR and SIM. Our analysis identified the calcium and Ras signaling pathways as potentially significant, with CDK2, CYCS, and PGR emerging as key targets. Our study represents an initial exploration of the potential link between triazine herbicides and PD, producing results that provide valuable insights for future research. Further in vivo and in vitro investigations are warranted to confirm our findings.

CRediT authorship contribution statement

Jianan Li: Funding acquisition, Conceptualization, Investigation, Methodology, Project administration and Roles/Writing - original draft. Haoran Bi: Data curation, Formal analysis, Resources; Software, Supervision, Validation, Visualization and Writing - review & editing.

Funding

This study was supported by the China Postdoctoral Science Foundation (2022M712679) and the National Natural Science Foundation of China (82203994).