Replacement per- and polyfluoroalkyl substances (PFAS) are potent modulators of lipogenic and drug metabolizing gene expression signatures in primary human hepatocytes

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental toxicants, and some, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys. It was hypothesized that perfluorosulfonic acids (C4, 6, 8), perfluorocarboxylic acids (C4-14), perfluoro(2-methyl-3-oxahexanoic) acid (HFPO-DA), 1H, 1H, 2H, 2H-perfluorooctanesulfonic acid (6:2 FTS) along with 3 PFOS precursors could induce expression of lipid metabolism genes and lipid deposition in human hepatocytes. Five-donor pooled cryopreserved human hepatocytes were cultured and treated with 0.1% DMSO vehicle or various PFAS (0.25 to 25 μM) in media. After a 48-h treatment, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured using a Quantigene Plex assay. After 72-h treatments, hepatocytes were stained with Nile Red dye to quantify intracellular lipids. Overall, PFAS were transcriptionally active at 25 μM. In this model, lipid accumulation was not observed with C8-C12 treatments. Shorter chain PFAS (C4-C5), 6:2 FTS, and PFOS precursor, metFOSA, induced significant liver lipid accumulation, and gene activation at lower concentrations than legacy PFAS. In summary short chain PFAS and other alternative PFAS were more potent gene inducers, and potential health effects of replacement PFAS should be critically evaluated in humans.

Keywords: Human hepatocytes; Lipids; PFAS; Perfluorinated compounds.

Fig. 1.. Legacy PFAA exposure modulates gene expression changes in cryopreserved human hepatocytes.

Human hepatocytes were treated with PFOA, PFHxS, and PFOS at concentrations of 0.25-25 μM. Cell lysate was processed, and gene expression was analyzed using a custom QuantiGene bead plex assay and analyzed using BioPlex 200 System according to manufacturer’s protocols. Fluorescence intensity was normalized to β-actin and fold change was calculated compared to the vehicle control. Clofibrate (CLO, 250 μM) and rosiglitazone (ROSI, 5 μM) treatments were used as positive controls. Red indicates gene induction and green indicates gene repression. Fold change was calculated and analyzed using an ANOVA followed by Dunnett’s test compared to the DMSO treated cells. * indicates P < 0.05. All colors represent means; N = 3-4.

Fig. 2.. Short chain PFAA exposure modulates gene expression changes in cryopreserved human hepatocytes.

Human hepatocytes were treated with short chain length PFAA (C4-C7, and C4s) at concentrations of 0.25-25 μM. Cell lysate was processed, and gene expression was analyzed using a custom QuantiGene bead plex assay and analyzed using BioPlex 200 System according to manufacturer’s protocols. Fluorescence intensity was normalized to β-actin and fold change was calculated compared to the vehicle control. Clofibrate (CLO, 250 μM) and rosiglitazone (ROSI, 5 μM) treatments were used as positive controls. Red indicates gene induction and green indicates gene repression. Fold change was calculated and analyzed using an ANOVA followed by Dunnett’s test compared to the DMSO treated cells. * indicates P < 0.05. All colors represent means; N = 3-4.

Fig. 3.. Long chain carboxylic acid PFAA exposure modulates gene expression changes in cryopreserved human hepatocytes.

Human hepatocytes were treated with long chain PFAA (C9-C14)at concentrations of 0.25-25 μM. Cell lysate was processed, and gene expression was analyzed using a custom QuantiGene bead plex assay and analyzed using BioPlex 200 System according to manufacturer’s protocols. Fluorescence intensity was normalized to β-actin and fold change was calculated compared to the vehicle control. Clofibrate (CLO, 250 μM) and rosiglitazone (ROSI, 5 μM) treatments were used as positive controls. Red indicates gene induction and green indicates gene repression. Fold change was calculated and analyzed using an ANOVA followed by Dunnett’s test compared to the DMSO treated cells. * indicates P < 0.05. All colors represent means; N = 3-4.

Fig. 4.. Alternative and precursor PFAS exposure modulates gene expression changes in cryopreserved human hepatocytes.

Human hepatocytes were treated with HFPO-DA, 6:2 FTS, FOSA, metFOSA, and etFOSA at concentrations of 0.25-25 μM. Cell lysate was processed, and gene expression was analyzed using a custom QuantiGene bead plex assay and analyzed using BioPlex 200 System according to manufacturer’s protocols. Fluorescence intensity was normalized to β-actin and fold change was calculated compared to the vehicle control. Clofibrate (CLO, 250 μM) and rosiglitazone (ROSI, 5 μM) treatments were used as positive controls. Red indicates gene induction and green indicates gene repression. Fold change was calculated and analyzed using an ANOVA followed by Dunnett’s test compared to the DMSO treated cells. * indicates P < 0.05. All colors represent means; N = 3-4.

Fig. 5.. Legacy PFAA did not induced liver lipid accumulation.

Human hepatocytes were treated with PFOA, PFHxS, and PFOS (0.25-25 μM) for 72 hours to induce liver lipid accumulation. 1:2 Palmitate and Oleate (0.5 mM, P/O) was included as positive control for lipid accumulation. Representative fluorescent images at 25 μM (A-E) were taken using an EVOS® FL Auto Cell Imaging System. Nile Red fluorescence was measured (excitation 485 nm/emission 535 nm) and normalized to DAPI fluorescence (excitation 358 nm/emission 461 nm). Fold change (F) was calculated compared to the DMSO treated cells. Calculations were done using an ANOVA followed by Dunnett’s test and * indicates P < 0.05. All values are means ± SEM; N = 3-4.

Fig. 6.. Short chain PFAA induce liver lipid accumulation.

Human hepatocytes were treated with short chain PFAA (C4-C7, and C4s) at 0.25-25 μM for 72 hours to induce liver lipid accumulation. 1:2 Palmitate and Oleate (0.5 mM, P/O) was included as positive control for lipid accumulation. Representative fluorescent images at 25 μM (A-G) were taken using an EVOS® FL Auto Cell Imaging System. Nile Red fluorescence was measured (excitation 485 nm/emission 535 nm) and normalized to DAPI fluorescence (excitation 358 nm/emission 461 nm). Fold change (H) was calculated compared to the DMSO treated cells. Calculations were done using an ANOVA followed by Dunnett’s test and * indicates P < 0.05. All values are means ± SEM; N = 3-4.

Fig. 7.. PFTrDA induce liver lipid accumulation.

Human hepatocytes were treated with long chain carboxylic acid PFAA (C9-C14) at 0.25-25 μM for 72 hours to induce liver lipid accumulation. 1:2 Palmitate and Oleate (0.5 mM, P/O) was included as positive control for lipid accumulation. Representative fluorescent images at 25 μM (A-H) were taken using an EVOS® FL Auto Cell Imaging System. Nile Red fluorescence was measured (excitation 485 nm/emission 535 nm) and normalized to DAPI fluorescence (excitation 358 nm/emission 461 nm). Fold change (I) was calculated compared to the DMSO treated cells. Calculations were done using an ANOVA followed by Dunnett’s test and * indicates P < 0.05. All values are means ± SEM; N = 3-4.

Fig. 8.. 6:2 FTS and metFOSA induces liver lipid accumulation.

Human hepatocytes were treated with HFPO-DA, 6:2 FTS, FOSA, metFOSA, and etFOSA at 0.25-25 μM for 72 hours to induce liver lipid accumulation. 1:2 Palmitate and Oleate (0.5 mM, P/O) was included as positive control for lipid accumulation. Representative fluorescent images at 25 μM (A-G) were taken using an EVOS® FL Auto Cell Imaging System. Nile Red fluorescence was measured (excitation 485 nm/emission 535 nm) and normalized to DAPI fluorescence (excitation 358 nm/emission 461 nm). Fold change (H) was calculated compared to the DMSO treated cells. Calculations were done using an ANOVA followed by Dunnett’s test and * indicates P < 0.05. All values are means ± SEM; N = 3-4.