Unravelling the Polytoxicology of Chlorfenapyr on Non-Target HepG2 Cells: The Involvement of Mitochondria-Mediated Programmed Cell Death and DNA Damage

Abstract

Chlorfenapyr (CHL) is a type of insecticide with a wide range of insecticidal activities and unique targets. The extensive use of pesticides has caused an increase in potential risks to the environment and human health. However, the potential toxicity of CHL and its mechanisms of action on humans remain unclear. Therefore, human liver cells (HepG2) were used to investigate the cytotoxic effect and mechanism of toxicity of CHL at the cellular level. The results showed that CHL induced cellular toxicity in HepG2 cells and induced mitochondrial damage associated with reactive oxygen species (ROS) accumulation and mitochondrial calcium overload, ultimately leading to apoptosis and autophagy in HepG2 cells. Typical apoptotic changes occurred, including a decline in the mitochondrial membrane potential, the promotion of Bax/Bcl-2 expression causing the release of cyt-c into the cytosol, the activation of cas-9/-3, and the cleavage of PARP. The autophagic effects included the formation of autophagic vacuoles, accumulation of Beclin-1, transformation of LC3-II, and downregulation of p62. Additionally, DNA damage and cell cycle arrest were detected in CHL-treated cells. These results show that CHL induced cytotoxicity associated with mitochondria-mediated programmed cell death (PCD) and DNA damage in HepG2 cells.

Keywords: Chlorfenapyr; DNA damage; HepG2 cells; apoptosis; autophagy; cell cycle.

Figure 1

Cytoactivity of chlorfenapyr (CHL) against HepG2 cells under various concentrations at various lengths of time. Distinct letters above the columns indicate significant differences at p ≤ 0.05.

Figure 2

Analysis of ROS production and antioxidant enzyme activity in HepG2 cells after treatment with CHL. (A): Analysis of DCF staining by epifluorescence (200×). (B): Analysis of DCF staining by flow cytometry. (C): Quantification of ROS levels. (D): Activity of SOD and CAT enzymes. ** p ≤ 0.01 vs. the negative control.

Figure 3

Analysis of MMP loss in HepG2 cells after treatment with CHL. (A): Analysis of JC-1 staining by flow cytometry. (B): Analysis of Rh123 staining by epifluorescence (200×). (C): Quantification levels of MMP loss. ** p ≤ 0.01 vs. the negative control.

Figure 4

Analysis of cytosolic and mitochondrial Ca²⁺ levels in HepG2 cells after treatment with CHL. (A,C): Analysis of Fluo-3 and Rhod-2 staining by flow cytometry. (B): Quantification levels of Fluo-3 fluorescence. (D): Quantification levels of Rhod-2 fluorescence. ** p ≤ 0.01 vs. the negative control.

Figure 5

Analysis of apoptotic effects of HepG2 cells after treatment with CHL. (A): Analysis of Annexin V/PI staining by flow cytometry. (B): Quantification of apoptotic cells. (C): Expression of apoptotic proteins by Western blot. (D): Quantification levels of apoptotic proteins. (E): Evaluation of caspase-3/9 activity by spectrophotometric analysis. ** p ≤ 0.01 and * p ≤ 0.05 vs. the negative control.

Figure 6

Analysis of autophagic effects of HepG2 cells after treatment with CHL. Observation of autophagy characteristics in HepG2 cells via TEM. (A): Control cells with normal mitochondria in well shape. (B,C): 30 μM (B) or 60 μM (C) CHL-treated cells with autophagic vacuoles (red arrows). (D): Analysis of MDC staining by epifluorescence (200×). (E): Expression of autophagy-associated proteins by Western blot. (F): Quantification levels of autophagy-associated proteins. ** p ≤ 0.01 and * p ≤ 0.05 vs. the negative control.

Figure 7

Analysis of the quantity of mitochondria in HepG2 cells after treatment with CHL. The change trends of Mito-tracker and Lyso-tracker’s fluorescence intensity were detected by epifluorescence (200×).

Figure 8

Analysis of the DNA damage and cell cycle in HepG2 cells after treatment with CHL. (A): Expression of PARP, γH2AX, and OGG1 by Western blot. (B): Quantification levels of γH2AX and OGG1. (C): Analysis of cell cycle by flow cytometry. (D), Proportion of cell cycle phases (G1, S, and G2). ** p ≤ 0.01 and * p ≤ 0.05 vs. the negative control.