Hexafluoropropylene oxide dimer acid (GenX) exposure induces apoptosis in HepG2 cells

Abstract

Hexafluoropropylene oxide dimer acid, also known as GenX, is a poly- and perfluoroalkyl substance (PFAS). PFASs are nonvolatile synthetic substances that can be readily disseminated into the environment during processing and use, making them easy to implement in the soil, drinking water, and air. Compared to other PFASs, GenX has a comparatively short carbon chain length and is expected to have a lower tendency to accumulate in humans; therefore, GenX has recently been used as a substitute to other PFASs. However, the mechanisms underlying GenX action and intoxication in humans remains unclear. In this study, the apoptotic capacity of GenX in human liver cells was investigated. When representative human-derived liver cells (HepG2 cells) were treated with GenX for 12 h, cell viability was reduced, and apoptosis was greatly increased. In addition, GenX increased the generation of intracellular reactive oxygen species (ROS), indicating the induction of oxidative stress in a dose-dependent manner. GenX treatment increased the expression of major apoptosis-related genes relative to the untreated control group. This research indicates that GenX causes apoptosis through ROS mediation in HepG2 cells, which may expand our knowledge of the molecular and toxicological mechanisms of GenX.

Keywords: Apoptosis; Hexafluoropropylene oxide dimer acid; Liver cells; Oxidative stress; Poly- and perfluoroalkyl substance.

Graphical abstract

Figure 1

Morphological alterations of HepG2 cells after hexafluoropropylene oxide dimer acid (GenX) exposure. HepG2 cells were treated to the indicated concentrations of PFOA and GenX for 12 h (A) and 48 h (B). Scale bar, 400 μm. All experiments were performed in triplicate, and morphological alterations in cells were observed using an inverted microscope under the 100x magnification (IX73P2F; Olympus Optical, Tokyo, Japan).

Figure 2

Cell viability of HepG2 cells after GenX exposure. HepG2 cells were treated with the indicated concentrations of PFOA and GenX for 12 h (A) and 48 h (B). Values are presented as the mean ± SD (n = 3). Significant differences (p < 0.05), determined using by Tukey's post hoc test, are represented by different letters (a–d) above the bars.

Figure 3

ROS production after GenX exposure in HepG2 cells. HepG2 cells were exposed to the indicated concentrations of PFOA and GenX for 12 h and 48 h. The DCF fluorescence intensity (% of the control) represents the relative ROS level. All DCF fluorescence values are compared to the control values (control = 100). Values are presented as mean ± SD (n = 3). Significant differences (p < 0.05), determined using by Tukey's post hoc test, are represented by different letters (a–d) above the bars.

Figure 4

Apoptotic rates in HepG2 cells after GenX exposure. HepG2 cells were exposed to the indicated concentrations of PFOA and GenX for 12 h. (A): Control, (B): 40 μM PFOA, (C): 40 μM GenX, (D): 100 μM GenX, (E): 250 μM GenX, (F) Apoptotic rate (%): sum of Q1-LR and Q1-UR quadrants. Flow cytometry contour plots of HepG2 cells labelled with annexin V-fluorescein isothiocyanate (FITC) fluorescence vs propidium iodide (PI) fluorescence. Q1-UL: the upper left quadrant represents necrosis; Q1-UR: the upper right quadrant represents late apoptosis; Q1-LL: the lower left quadrant represents the distribution of living cells; Q1-LR: the lower right quadrant represents early apoptosis. Values are presented as mean ± SD (n = 3). Significant differences (p < 0.05), determined using by Tukey's post hoc test, are represented by different letters (a–d) above the bars.

Figure 5

Levels of mRNA expression after GenX exposure in HepG2 cells. HepG2 cells were exposed to the indicated concentrations of PFOA and GenX for 12 h. All of the data are compared to the control values (control = 1). Values are presented as mean ± SD (n = 3). Significant differences (p < 0.05), determined using by Tukey's post hoc test, are represented by different letters (a–d) above the bars.

Figure 6

Effects of PFOA and GenX exposure on expression of apoptosis-related proteins in HepG2 cells. HepG2 cells were incubated with the indicated concentrations of PFOA and GenX for 12 h. CON: control; P 40: PFOA 40 μM; G 40: GenX 40 μM; G 100: GenX 250 μM; G 250: GenX 500 μM; All of the data are compared to the control values (control = 1). Values are presented as mean ± SD (n = 3). Significant differences (p < 0.05), determined using by Tukey's post hoc test, are represented by different letters (a–d) above the bars.