Evaluation of the Effect of Perfluorohexane Sulfonate on the Proliferation of Human Liver Cells

Abstract

Perfluorohexane sulfonate (PFHxS) is a widely detected replacement for legacy long-chain perfluoroalkyl substances (PFAS) in the environment and human blood samples. Its potential toxicity led to its recent classification as a globally regulated persistent organic pollutant. Although animal studies have shown a positive association between PFHxS levels and hepatic steatosis and hepatocellular hypertrophy, the link with liver toxicity, including end-stage liver cancer, remains inconclusive. In this study, we examined the effects of PFHxS on the proliferation of Hep3B (human hepatocellular carcinoma) and SK-Hep1 (human liver sinusoidal endothelial cells). Cells were exposed to different PFHxS concentrations for 24-48 h to assess viability and 12-14 days to measure colony formation. The viability of both cell lines increased at PFHxS concentrations <200 μM, decreased at >400 μM, and was highest at 50 μM. Colony formation increased at <300 μM and decreased at 500 μM PFHxS. Consistent with the effect on cell proliferation, PFHxS increased the expression of proliferating cell nuclear antigen (PCNA) and cell-cycle molecules (CDK2, CDK4, cyclin E, and cyclin D1). In summary, PFHxS exhibited a biphasic effect on liver cell proliferation, promoting survival and proliferation at lower concentrations and being cytotoxic at higher concentrations. This suggests that PFHxS, especially at lower concentrations, might be associated with HCC development and progression.

Keywords: cell-cycle progression; colony formation; human hepatocellular carcinoma; liver cancer; perfluorohexane sulfonate.

Figure 1

The effects of PFHxS on the viability of HCC and LSEC. Hep3B (A) and SK-Hep1 (B) cells were treated with the indicated concentrations of PFHxS for 24 and 48 h. Cell viability was measured using an MTT assay. Data represent the mean ± SEM (n = 3–4) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. 24 h control; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. 48 h control).

Figure 2

The effects of PFHxS on colony formation of HCC and LSEC. Hep3B and SK-Hep1 cells were treated with the indicated concentrations of PFHxS for 12–14 days. The colonies were stained with crystal violet and representative photos of the colonies are presented (A). The absorbance of resolved dyes was measured at 570 nm (B). Data represent the mean ± SEM (n = 6–12; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control).

Figure 3

The effects of PFHxS on cell-cycle signaling molecules in HCC and LSEC. Hep3B (A) and SK-Hep1 (B) cells were treated with the indicated concentrations of PFHxS for 48 h. The protein levels of CDKs, cyclins, and PCNA were detected by Western blotting. The band intensities were measured and presented in the bar graphs. Data represent the mean ± SEM (n = 3) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control).