ROS-Triggered Autophagy Is Involved in PFOS-Induced Apoptosis of Human Embryo Liver L-02 Cells

Abstract

The liver is the primary target organ for perfluorooctane sulphonate (PFOS), a recently discovered persistent organic pollutant. However, the mechanisms mediating hepatotoxicity remain unclear. Herein, we explored the relationship between reactive oxygen species (ROS) and autophagy and apoptosis induced by PFOS in L-02 cells, which are incubated with different concentrations of PFOS (0, 50, 100, 150, 200, or 250 μmol/L) for 24 or 48 hrs at 37°C. The results indicated that PFOS exposure decreased cell activities, enhanced ROS levels in a concentration-dependent manner, decreased mitochondrial membrane potential (MMP), and induced autophagy and apoptosis. Compared with the control, 200 μmol/L PFOS increased ROS levels; enhanced the expression of Bax, cleaved-caspase-3, and LC3-II; induced autophagy; decreased MMP; and lowered Bcl-2, p62, and Bcl-2/Bax ratio. The antioxidant N-acetyl cysteine (NAC) protected MMP against PFOS-induced changes and diminished apoptosis and autophagy. Compared with 200 μmol/L PFOS treatment, NAC pretreatment reversed the increase in ROS, Bax, and cleaved-caspase-3 protein caused by PFOS, lowered the apoptosis rate increased by PFOS, and increased the levels of MMP and Bcl-2/Bax ratio decreased by PFOS. The autophagy inhibitor 3-methyladenine and chloroquine decreased apoptosis and cleaved-caspase-3 protein level and increased the Bcl-2/Bax ratio. In summary, our results suggest that ROS-triggered autophagy is involved in PFOS-induced apoptosis in L-02 cells.

Figure 1

Effects of PFOS on cell viability in L-02 cells. (a) Cells were treated with PFOS for 24 hrs or 48 hrs with different concentrations of PFOS. (b) Cells were treated with 200 μmol/L PFOS for various time periods. Data are presented as the mean ± S.D. (n = 3). ^∗P < 0.05 compared with the control group.

Figure 2

ROS levels in the intracellular microenvironment induced by PFOS. ^∗P < 0.05 compared with the control group, ^#P < 0.05 between the indicated groups.

Figure 3

Effect of PFOS on MMP in L-02 cells. MMP was measured using Rh123 staining (n = 3). (a) L-02 cells were treated with 200 μmol/L PFOS for 6 hrs, 12 hrs, and 24 hrs. (b) L-02 cells were treated with 200 μmol/L PFOS for 12 hrs, or L-02 cells were pretreated with NAC for 24 hrs and then exposed to 200 μmol/L PFOS for 12 hrs. ^∗P < 0.05 compared with the control group, ^#P < 0.05 between the indicated groups.

Figure 4

Effects of PFOS, NAC, and 3-MA on monodansyl cadaverine- (MDC-) labelled vesicles: (a) DMSO treatment; (b) 200 μmol/L PFOS treatment; (c) NAC treatment; (d) NAC+200 μmol/L PFOS treatment; (e) 3-MA treatment; (f) 3-MA+PFOS treatment.

Figure 5

Effects of NAC and 3-MA on PFOS-induced apoptosis in L-02 cells: (a) DMSO treatment; (b) 200 μmol/L PFOS treatment; (c) NAC treatment; (d) NAC+200 μmol/L PFOS treatment; (e) 3-MA treatment; (f) 3-MA+PFOS treatment; (g) 3-MA+NAC treatment; (h) 3-MA+NAC+200 μmol/L treatment; (i) Percentage of apoptotic cells. All data are the mean ± S.D. (n = 3). ^∗P < 0.05 between the indicated groups.

Figure 6

Effect of PFOS on LC3-II/LC3-I and p62 protein levels: (a) LC3-II protein levels; (b) p62 protein levels; (c) representative western blotting lane. Values are represented as the mean ± S.D. (n = 3). ^∗P < 0.05 compared with the control group.

Figure 7

Effect of NAC on the abundance of Bcl-2, Bax, cleaved-caspase-3, and LC3 proteins in L-02 cells following PFOS treatment: (a) relative Bax/Bcl-2 ratio; (b) cleaved-caspase-3 protein (17 kD) level; (c) relative LC3-II/LC3-I ratio; (d) representative western blotting lane. Values were represented as the mean ± S.D. (n = 3). ^∗P < 0.05 between the indicated groups.