The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines

Abstract

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Keywords: Bixafen; Boscalid; EPR; SDHI; mitochondria; oxygen consumption rate (OCR); superoxide.

Figure 1

Effect of SDHI-fungicides on mitochondrial ETC. Boscalid and Bixafen block the complex II of the ETC, Bixafen also inhibiting complex III. By disrupting mitochondrial function, SDHIs may impact the production of mitochondrial superoxide radical and the oxygen consumption rate.

Figure 2

Oxygen consumption rate of control (DMSO 0.1%) and SDHIs-exposed human cells (1µM for 2 h). Top row: Boscalid-treated cells. (A): HepG2 liver cells, (B): PBMCs, (C): BJ-fibroblasts. Bottom row: Bixafen-treated cells. (D): HepG2 liver cells, (E): PBMCs, (F): BJ-fibroblasts. Bars represent mean ± SEM (%O₂/min). N = 3, (*): p < 0.05, (**): p < 0.01.

Figure 3

Cellular ATP production of HepG2 cells after treatment with DMSO 0.1% (CTR), Boscalid 1 µM (BOS) and Bixafen 1 µM (BIX) for 2 h (A) and 24 h (B). Data were normalized to controls. Bars represent mean ATP production ± SEM (A.U.), N = 3.

Figure 4

Illustration of the evaluation of mitochondrial superoxide assessment using EPR. Top row: typical experiment with exposure of HepG2 cells to Bixafen. (A): Time course evolution of EPR signal intensity of Mito-TEMPO^• produced from Mito-TEMPO-H. (B): EPR signal intensity recorded for control cells and exposed cells 15 min after the start of the EPR experiment. The incubation with PEG-SOD2 allowed the evaluation of the superoxide contribution to the oxidation of the mitochondrial sensor. Bottom row: Mean values of EPR signal intensity at time t = 15 min recorded for 3 independent experiments. (C): Control HepG2 cells. (D): HepG2 cells exposed for 2 h to Bixafen.

Figure 5

Level of mitochondrial superoxide in control (DMSO 0.1%) and SDHIs-exposed human cells (1 µM for 2 h). Signal intensity of Mito-TEMPO^• at time point 15 min (normalized to control). Top row: Boscalid-treated cells. (A): HepG2 liver cells, (B): PBMCs, (C): BJ-fibroblasts. Bottom row: Bixafen-treated cells. (D): HepG2 liver cells, (E): PBMCs, (F): BJ-fibroblasts. Bars represent mean ± SEM. N = 3, (*): p < 0.05.

Figure 6

Total ROS production in HepG2 cells after treatment with DMSO 0.1% (CTR), 1 µM Boscalid (BOS) and 1 µM Bixafen (BIX) for 2 h, measured using H₂DFCDA probe. Data were normalized to controls. Bars represent mean ratio of MFI (mean fluorescence intensity) ± SEM (A.U.), N = 3.

Figure 7

Effect of SDHI exposure on cytotoxicity. Top and middle rows: histograms of percentage of early apoptotic cells/late apoptotic or necrotic cells in control (DMSO 0.1%) and SDHIs-exposed human cells (1 µM for 2 h). Top row: Boscalid-treated cells. (A): HepG2 liver cells, (**): p < 0.01. (B): PBMCs, (C): BJ-fibroblasts. Middle row: Bixafen-treated cells. (D): HepG2 liver cells, (E): PBMCs, (F): BJ-fibroblasts. Bars represent mean ± SEM (% events). N = 3, (*): p < 0.05. Bottom row: Representative Annexin V/PI flow cytometry plots of HepG2 cells. (G): control cells, (H): Boscalid-treated cells, (I): Bixafen-treated cells.

Figure 8

Chemical structures of ¹⁵N-PDT used as a sensor for oximetry (left) and Mito-TEMPO-H used as a sensor for mitochondrial superoxide production (right).