Subchronic Chlorpyrifos Exposure Induces Thyroid Follicular Cell Pyroptosis to Exacerbate Thyroid Toxicity by Modulating Nrf2/Keap1/NF-κB Pathway in Male Mice

Abstract

Purpose: Chlorpyrifos (CPF), a widely used organophosphate pesticide in agriculture, particularly in China, has raised significant environmental and health concerns due to its persistence and bioaccumulation. While CPF-induced toxicity in multiple organ systems has been documented, its long-term impact on thyroid homeostasis and the underlying mechanisms remain poorly understood. This study aimed to investigate the subchronic effects of CPF on thyroid function and elucidate the underlying mechanisms of CPF-induced thyroid toxicity.

Methods: The study utilized 4-week-old male C57BL/6J mice as experimental subjects. These mice were exposed to CPF via intragastric gavage at doses of 3 or 6 mg/kg for a duration of 8 weeks. Throughout the study period, various parameters were assessed, including body weight, serum antioxidant capacity, thyroid endocrine function and structure, apoptosis markers, inflammatory cytokines, and relevant molecular pathways.

Results: The study revealed that CPF exposure resulted in significant systemic toxicity, manifested through reduced body weight and impaired serum antioxidant capacity. Examination of thyroid-specific effects showed disrupted thyroid endocrine function and structure, accompanied by increased apoptosis and elevated inflammatory cytokines. At the molecular level, CPF significantly stimulated thyroid follicle cell pyroptosis by upregulating the expression of Nlrp3, Caspase-1, and Gsdmd. Further mechanistic analysis demonstrated that CPF activated thyroid follicular cell pyroptosis by modulating the Nrf2/Keap1 antioxidative pathway and enhancing phosphorylation of p65 via NF-κB signaling.

Conclusion: This comprehensive investigation provides novel insights into the mechanisms of CPF-induced thyroid toxicity. The findings demonstrate that CPF exposure compromises thyroid homeostasis through the induction of follicular cell pyroptosis and modulation of the Nrf2/Keap1/NF-κB signaling axis, highlighting the potential health risks associated with CPF exposure and its impact on thyroid function.

Keywords: Nrf2/Keap1/NF-κB pathway; chlorpyrifos; pyroptosis; thyroid follicular cell; thyroid toxicity.

Graphical abstract

Figure 1

Effects of CPF exposure on body weight and serum antioxidant capacity in mice. (A) Effects of CPF exposure on body weight after 8 weeks of CPF treatment. (B–E) The amount of GSH-Px activity (B), T-AOC (C), SOD (D), and MDA (E) in the serum of mice after 8 weeks of CPF exposure. Data are presented as mean ± SEM. Each experiment was performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01 (vs vehicle group), ^#P <0.05 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 2

Effects of CPF exposure on thyroid hormone levels and histological structure. (A) Serum levels of thyroid hormones, including fT₃, fT₄, and TSH, were measured after 8 weeks of CPF exposure. (B and C) Representative histological images of thyroid tissues stained with HE (B) and PAS (C). Red arrows in (B) indicate typical inflammatory infiltration in thyroid. Black arrows in (C) indicate structural changes such as follicular cells and reduced colloid content. (D) Quantitative analysis of thyroid follicular characteristics, including follicle diameter, follicle number (per 0.2 mm²), and epithelial thickness. The diameter of the follicle was calculated by averaging the length of the line through the center of the follicle in four directions (0°, 45°, 90°, 135°). DF = (a1+a2+a3+a4)/4. Data are shown as mean ± SEM. All experiments were performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01 (vs vehicle group), ^#P <0.05 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 3

CPF exposure induces apoptosis in thyroid follicular cells. (A) Representative IF images showing Caspase-3 (green) expression in thyroid tissues after 8 weeks of CPF exposure. DAPI (blue) was used to counterstain nuclei. White arrowheads indicate Caspase-3-positive cells. (B) Quantitative analysis of Caspase-3-positive cells in thyroid tissues. (C) Representative TUNEL staining images of thyroid tissues. TUNEL-positive cells are indicated by white arrowheads. (D) Quantification of TUNEL-positive cells in thyroid tissues, expressed as the percentage of total cells. Data are presented as mean ± SEM. All experiments were performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **P < 0.01 (vs vehicle group), ^##P <0.01 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 4

CPF exposure promotes inflammatory cytokine production in thyroid tissues. (A) Representative IF images showing IL-1β (green) expression in thyroid tissues. DAPI (blue) was used to counterstain nuclei. White arrowheads indicate IL-1β-positive cells. (B) Quantitative analysis of IL-1β expression in thyroid tissues. (C) Representative IHC images of IL-18 expression (brown) in thyroid tissues. Red arrowheads indicate IL-18-positive cells. (D) Quantitative analysis of IL-18 expression in thyroid tissues. (E) Representative IHC images of TNF-α expression (brown) in thyroid tissues. Red arrowheads indicate TNF-α-positive cells. (F) Quantitative analysis of TNF-α expression in thyroid tissues. Data are presented as mean ± SEM. All experiments were performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **P < 0.01 (vs vehicle group), ^##P <0.01 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 5

CPF exposure activates thyroid follicular cell pyroptosis in mice. (A–C) Representative IF images showing the expression of pyroptosis-related proteins (Green), including Nlrp3, Caspase-1, and Gsdmd, in thyroid tissues. DAPI (blue) was used to counterstain nuclei. White arrowheads indicate positive cells for the respective proteins. (D–F) Quantitative analysis of the fluorescence intensity of Nlrp3, Caspase-1, and Gsdmd in thyroid tissues. Data are presented as mean ± SEM. All experiments were performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **P < 0.01 (vs vehicle group), ^##P <0.01 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 6

CPF exposure disrupts the Nrf2/Keap1 antioxidant pathway and activates NF-κB signaling in thyroid tissues. (A and B) Representative IF images (A) and quantitative analysis (B) of Nrf2, Keap1, and HO-1 expression in thyroid tissues. (C and D) Representative IF images (C) and quantitative analysis (D) of p-p65 expression in thyroid tissues. DAPI (blue) was used to counterstain nuclei. White arrowheads indicate positive cells for the respective proteins. Data are presented as mean ± SEM. All experiments were performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **P < 0.01 (vs vehicle group), ^#P <0.05, ^##P <0.01 (vs 3 mg/kg CPF group), n = 6 per group.

Figure 7

A schematic diagram illustrating how CPF exposure induces thyroid follicular cell pyroptosis and exacerbates thyrotoxicity by modulating the Nrf2/Keap1/NF-κB pathway.