Fipronil Triggers Immunotoxicity Through Reactive Oxygen Species-Driven Mitochondrial Apoptosis in Thymocytes

Abstract

Fipronil (FPN), a widely used pesticide, is associated with significant immunotoxic effects, particularly impacting thymocyte survival and immune homeostasis. This study explores the mechanistic pathways underlying FPN-induced apoptosis and oxidative stress. Short-term FPN exposure (1-10 mg/kg) notably suppressed the expression of both anti-apoptotic (Bcl-2, Bcl-6, Mcl-1) and pro-apoptotic (Bnip3, Bim) genes in thymic tissues in vivo. Additionally, in isolated primary thymocytes, FPN directly decreased the expression of Bcl-2, Bcl-6, Mcl-1, and Bnip3 expression, coupled with a significant increase in pro-apoptotic Bim expression in a dose-dependent manner. FPN treatment directly led to elevated reactive oxygen species (ROS), lipid peroxidation, mitochondrial membrane depolarization, reduced cellular metabolic activity, and depleted intracellular calcium and glutathione (GSH) levels, indicating mitochondrial dysfunction and oxidative stress. Annexin V/PI staining confirmed that FPN induced late-stage apoptosis and necrosis in primary thymocytes. These findings elucidate the immunotoxic effects of FPN on thymocytes, highlighting its detrimental impact on immune system integrity, thymic development, and T cell maturation through oxidative damage and mitochondrial-mediated apoptosis.

Keywords: BCL-2 family; apoptosis; fipronil; glutathione; immunotoxicity; lipid peroxidation; mitochondrial membrane potential; reactive oxygen species.

Figure 1

Protocol for fipronil (FPN) administration.

Figure 2

Fipronil significantly decreased mRNA expression of the Bcl-2 family of thymus in vivo. The total mRNA harvested from different thymus treatment groups was extracted to detect the mRNA expression by qPCR. The expression level of Hprt was used as the control for semi-quantification. Results were expressed as the mean ± SEM pooled from four independent experiments with technological duplication in each group (N = 20/group). * p < 0.05 was significant compared to the VH group.

Figure 3

Fipronil significantly decreased mRNA expression of the Bcl-2 family ex vivo. Total RNA was extracted from primary thymocytes following ConA stimulation to quantify mRNA expression by qPCR. The expression level of Hprt was used as the control for semi-quantification. Results were expressed as the mean ± SEM pooled from four independent experiments with technological duplication in each group (N = 20/group). * p < 0.05 was significant compared to the VH group.

Figure 4

Fipronil significantly decreased mRNA expression of the Bcl-2 family in vitro. The total mRNA harvested from different treatment groups was extracted to detect the mRNA expression by qPCR. The expression level of Hprt was used as the control for semi-quantification. Results were expressed as the mean ± SEM pooled from four independent experiments with technological duplication in each group. * p < 0.05 was significant compared to the VH group.

Figure 5

Fipronil exhibited a reduction of primary thymocyte viability and IL-2 production stimulated by PMA/Iono. The primary thymocytes (5 × 10⁶ cells/mL) were treated with 0.05% DMSO (VH) or FPN in different concentrations and stimulated with PMA/Iono (PMA/Iono: 80 nM/1 μM) for 24 h. (A) The viability of thymocytes was measured by MTT assay. (B) The level of IL-2 in the supernatants was measured by ELISA. Data were expressed as the mean ± SEM of quadruplicate cultures and representative of four independent experiments. * p < 0.05 was significant compared to the VH without the NAC group. # p < 0.05 was significant compared to the VH with the NAC group.

Figure 6

Fipronil inducted of thymocyte apoptosis in vitro. The apoptosis indicator was measured by Annexin V/PI staining. (A–C) The representative dot plot shows either the VH group and 50 μM FPN with or without NAC treatment at different time points. (D) Statistical data represent the sum of Annexin V⁺/PI⁺ and Annexin V⁺/PI⁻ populations. The primary thymocytes (5 × 10⁶ cells/mL) were treated with 0.05% DMSO (VH) or FPN in different concentrations for 2, 6, and 18 h. Data were expressed as the mean ± SEM of quadruplicate cultures and representative of four independent experiments. * p < 0.05 was significant compared to the VH group of each time point.

Figure 7

Effects of FPN on mitochondrial membrane potential, intracellular calcium levels, and GSH levels in primary thymocytes. Primary thymocytes (5 × 10⁶ cells/mL) were treated with 0.05% DMSO (VH) or FPN at different concentrations for 2, 6, and 18 h. (A) Mitochondrial membrane potential was assessed using JC-1 staining, (B) intracellular calcium levels were measured using Fluo-4 AM staining, and (C) intracellular GSH levels were determined using CMF-DA staining. Data are expressed as the mean ± SEM of quadruplicate cultures and are representative of four independent experiments. * p < 0.05 compared to the VH group of each time point. # p < 0.05 compared to the VH with the NAC group.

Figure 8

Induction of ROS and LPO levels by FPN. Primary thymocytes (5 × 106 cells/mL) were treated with 0.05% DMSO (VH) or FPN at different concentrations for 0.5, 2, and 6 h. ROS levels were measured using H₂-DCFDA staining, while LPO was assessed using C11-Bodipy581/591 staining. Data were expressed as the mean ± SEM of quadruplicate cultures and representative of four independent experiments. * p < 0.05 was significant compared to the VH group of each time point.