Archives of Toxicology

, Volume 92, Issue 1, pp 359–369 | Cite as

Cytotoxicity of novel fluorinated alternatives to long-chain perfluoroalkyl substances to human liver cell line and their binding capacity to human liver fatty acid binding protein

  • Nan Sheng
  • Ruina Cui
  • Jinghua Wang
  • Yong Guo
  • Jianshe Wang
  • Jiayin DaiEmail author
In vitro systems


Although shorter chain homologues and other types of fluorinated chemicals are currently used as alternatives to long-chain perfluoroalkyl substances (PFASs), their safety information remains unclear and urgently needed. Here, the cytotoxicity of several fluorinated alternatives (i.e., 6:2 fluorotelomer carboxylic acid (6:2 FTCA), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), and hexafluoropropylene oxide (HFPO) homologues) to human liver HL-7702 cell line were measured and compared with perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Their binding mode and affinity to human liver fatty acid binding protein (hL-FABP) were also determined. Compared with PFOA and PFOS, 6:2 Cl-PFESA, HFPO trimer acid (HFPO-TA), HFPO tetramer acid (HFPO-TeA), and 6:2 FTSA showed greater toxic effects on cell viabilities. At low exposure doses, these alternatives induced cell proliferation with similar mechanism which was different from that of PFOA and PFOS. Furthermore, binding affinity to hL-FABP decreased in the order of 6:2 FTCA < 6:2 FTSA < HFPO dimer acid (HFPO-DA) < PFOA < PFOS/6:2 Cl-PFESA/HFPO-TA. Due to their distinctive structure, 6:2 Cl-PFESA and HFPO homologues were bound to the hL-FABP inner pocket with unique binding modes and higher binding energy compared with PFOA and PFOS. This research enhances our understanding of the toxicity of PFAS alternatives during usage and provides useful evidence for the development of new alternatives.


Novel fluorinated alternatives Cytotoxicity HL7702 cell lines Protein binding hL-FABP 


Due to their unique properties, perfluoroalkyl substances (PFASs) have been used in various kinds of consumer products for over six decades, ranging from food packaging and clothing to fire-fighting foams (Calafat et al. 2007; Kannan 2011; Park et al. 2016). High usage, along with their physical and chemical characteristics, have resulted in their environmental persistence and biological toxicity, leading to the phasing out of some long-chain PFASs (Fujii et al. 2007; Hoke et al. 2015; Lam et al. 2016; UNEP 2009; Wang et al. 2013). With this abolishment, the alternative use of shorter chain homologues and other types of fluorinated chemicals with advantageous environmental and toxicological attributes has become a global trend (Gordon 2011; Heydebreck et al. 2015; Ritter 2010; Wang et al. 2015). For example, DuPont commercialized Forafac® 1157 (6:2 fluorotelomer sulfonamide alkylbetaine-based production) for use in fire-fighting foams (Hagenaars et al. 2011; Moe et al. 2012; Pabon and Corpart 2002); F-53B (6:2 chlorinated polyfluorinated ether sulfonate, 6:2 Cl-PFESA) and the potassium salt of 6:2 fluorotelomer sulfonic acid (6:2 FTSA) have been applied as alternatives to perfluorooctane sulfonate (PFOS) in metal plating (Wang et al. 2015; Yang et al. 2014); and ADONA (ammonium 4,8-dioxa-3H-perfluorononanoate) and GenX [ammonium salt of hexafluoropropylene oxide dimer acid (HFPO-DA)] have been used as replacements for perfluorooctanoic acid (PFOA) in fluoropolymer resin manufacturing (Dupont 2010; Gordon 2011). In addition, HFPO trimer acid (HFPO-TA), HFPO tetramer acid (HFPO-TeA), and 6:2 fluorotelomer carboxylic acid (6:2 FTCA) have also been used as alternatives for PFOA in China (Wang et al. 2017; Xu et al. 2011).

Recently, however, controversy regarding the safety of these chemicals has emerged as information on their properties, environmental effects, and toxicities remains limited. Amid these growing concerns, recent studies have detected these alternatives in environmental and wildlife samples, and even in human serum (Heydebreck et al. 2015; Wang et al. 2015). For example, Cl-PFESAs have been detected in river water, sludge, wild fish, occupational workers, and general populations (Pan et al. 2017; Ruan et al. 2015; Shi et al. 2015); 6:2 FTSA has been detected in river water and sludge (Lin et al. 2016; Ruan et al. 2015); and HFPO-DA has been recorded in river waters of China, USA, and Europe (Heydebreck et al. 2015; Wang et al. 2013). We also detected HFPO-TA in river and fish samples in earlier research (unpublished data). Despite the above monitoring data, only a few studies on the toxicological effects of these alternatives have been published in recent years. Such studies have shown 6:2 Cl-PFESA to have a similar LC50 as that of PFOS in zebrafish (Shi et al. 2017; Wang et al. 2013), with exposure inducing cardiac toxicity and reducing erythrocyte numbers via the Wnt signaling pathway in zebrafish embryos during development (Shi et al. 2017). Furthermore, 6:2 FTSA, as reported in our previous study, could induce a bit weak hepatotoxicity in mice compared with PFOS, while 6:2 FTCA exposure brought little adverse effects under the same experimental condition (Sheng et al. 2016b); whereas, HFPO-DA and ADONA, as PPARα agonists, have been shown to cause liver damage in rodents following exposure (Gordon 2011; Wang et al. 2017).

Extensive investigations of the toxicities of PFAS legacies to the liver have shown strong activation of peroxisome proliferator-activated receptor α (PPARα) in rodents, but weak activation in humans (Bjork et al. 2011; Vanden Heuvel et al. 2006); hepatic inflammation induced by legacy exposure (Quist et al. 2015); and strong protein-binding capacity with serum albumin and fatty acid binding proteins (Chen and Guo 2009; Sheng et al. 2016a). Liver fatty acid binding protein (L-FABP), as a member of the intracellular lipid-binding protein superfamily, is involved in fatty acid metabolism (Atshaves et al. 2010; Bernlohr et al. 1997). Due to their similar structure, PFAS legacies exhibit effective binding capacity to human L-FABP (hL-FABP), which can lead to the entrance of PFASs into hepatocytes (Luebker et al. 2002; Sheng et al. 2016a; Zhang et al. 2013b). However, as not all PFAS alternatives have similar structure to legacies, whether their protein-binding affinities are stronger or weaker remain to be explored.

In the present study, a normal human liver cell line was exposed to two PFAS legacies (PFOA and PFOS) and six potential alternatives (6:2 Cl-PFESA, 6:2 FTCA, 6:2 FTSA, HFPO-DA, HFPO-TA, and HFPO-TeA) for 24 h. The cytotoxicities of these chemicals were assessed with regard to cell viability and their effects on the cell cycle. In addition, the binding capacity and binding mode of the alternatives to hL-FABP were measured and compared with the legacies. This study will contribute to the currently limited toxicity data of the potential PFAS alternatives.

Materials and methods


The PFOA (CAS No. 335-67-1), PFOS (CAS No. 2795-39-3), dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), and 8-anilino-1-naphthalenesulfonic acid (1,8-ANS) were purchased from Sigma-Aldrich (MO, USA). The 6:2 FTCA (CAS No. 53826-12-3), 6:2 FTSA (CAS No. 59587-39-2), 6:2 Cl-PFESA (CAS No. 73606-19-6), HFPO-DA (CAS No. 62037-80-3), HFPO-TA (CAS No. 13252-14-7), and HFPO-TeA (CAS No. 65294-16-8) were kind gifts from Dr. Yong Guo (Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China). The cell cycle kits, neutral red kits, and bicinchoninic acid (BCA) kits were purchased from Beyotime Biotechnology (Shanghai, China). Isopropyl-β-d-thiogalactopyranoside (IPTG) was obtained from Amresco (OH, USA). The BL21 (DE3) strain of E. coli was bought from TransGen Biotech (Beijing, China). The stock solution of each PFAS was dissolved in DMSO to a final concentration of 50 mM.

Cell culture

The human liver HL-7702 cells (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China) were cultured in RPMI-1640 medium containing 10% fetal bovine serum under standard conditions (5% CO2 at 37 °C). For cell viability assay, cells were seeded in 96-well plates at initial densities of 1 × 104 cells/well. For cell cycle analysis, cells were seeded in 6-well plates at initial densities of 4 × 105 cells/well.

Cell viability assay

Research about cytotoxicity of PFASs legacies on HepG-2 cell lines showed that the dose–response effect for PFOA or PFOS appeared after 24 h exposure (Florentin et al. 2011; Hu and Hu 2009). Thus, after attachment for 24 h, the HL-7702 cells were treated with different concentrations of chemicals for another 24 h after attachment for 24 h. Before the formal experiments, influential DMSO content in medium after 24 h of culture (under 5%) was identified with a preliminary MTT test. The concentration of each chemical used in cell viability assay were selected based on the concentration setting shown in our previous study about cytotoxicity of PFASs (Hu et al. 2014). The working solutions were obtained by serial dilution with culture medium, with the final concentrations for each PFAS shown in Table S1. Due to its low boiling point and high volatility, the cytotoxicity of HFPO-DA was not detected in this part of experiment as well as in the flow cytometer analysis. After 24 h of exposure, MTT and neutral red assays were performed as per the manufacturer’s instructions.

Flow cytometry of the cell cycle

To investigate effects on cell proliferation, the exposure concentrations for each potential alternative were chosen lower than IC10 values which were obtained by the MTT and neutral red results. Four treated groups (control, low dosage, moderate dosage and high dosage) were set for each PFAS, and the detailed concentrations are shown in Table S2. The HL-7702 cells cultured in 6-well plates were exposed to different alternatives for 24 h after 24 h attachment. Following exposure, half of the cells were collected, washed, filtrated, and stained for flow cytometry, and the other half were collected for gene expression detection. Cells in different cell cycle phases were then analyzed on a flow cytometer (BD FACSCalibur, Becton–Dickinson, USA) and presented as a percentage of total cell number.

Quantitative real-time polymerase chain reaction (qPCR)

Changes of gene expression of the moderate dosage and high dosage treated groups described in flow cytometry assay were measured with qPCR. Based on the manufacturer’s instructions, RNA was extracted and reverse transcribed to cDNA. Real-time PCR was performed with a SYBR Green PCR Master Mix reagent kit (Tiangen, Beijing, China) on a Stratagene Mx3000P q-PCR system (Stratagene, USA). Taking 18S as the internal control, the quantification for each gene was performed according to previous studies (Arocho et al. 2006; Sheng et al. 2016b). The primer sequences are shown in Table S3.

Overexpression and purification of hL-FABP

The full-length human L-FABP gene was amplified from HL-7702 cell cDNA and then cloned in the pET-28a vector at the BamHI and XhoI restriction sites. After confirmation, the resulting plasmid was transformed into the BL21 (DE3) strain of E. coli. Bacterial proteins were collected after IPTG (1 mM) induction for 18 h at 23 °C. Purified hL-FABP was obtained on an AKTA FPLC system (GE Healthcare, USA), with the final purity of more than 98% ascertained by SDS-PAGE. The concentration of the final purified protein was detected using the BCA kits.

Circular dichroism (CD) spectroscopy

Various concentrations of the PFASs were added into the protein solution to a final concentration of 5 μM (protein) and 0, 10, 50, and 100 μM PFAS. The CD spectra were then obtained on a Chirascan Plus spectrometer (Applied Photophysics, Leatherhead, UK) with a quartz cuvette path length of 1 mm, as per our previous study (Sheng et al. 2016a).

Fluorescence displacement assays

With 1,8-ANS as a fluorometric probe, fluorescence displacement experiments were performed as described in earlier studies (Sheng et al. 2016a; Velkov et al. 2005), though with an adjustment in protein concentration (200 nM).

Molecular docking

The configurations of the PFAS legacies and alternatives were optimized using Molecular Operating Environment software (Chemical Computing Group, QC, Canada) until the gradient was <0.0001. The initial atomic coordinates of hL-FABP (PDB ID: 3STK) were obtained from the Protein Data Bank (PDB, With a binding radius of 15 Å, Discovery Studio 2.5 software (Biovia, CA, USA) was used to dock different PFASs into the inner pocket of the protein in 10 poses. The pose with the highest score for each PFAS was chosen as the optimal result.


Origin Pro 8.5 (OriginLab Corporation, MA, USA) was used to fit the J-shaped and S-shaped curves to calculate the inhibitory concentration ICx (−20, 10, and 50) values with the BiPhasic or DoseResp models, respectively. The higher the coefficient of determination (R 2) and the lower the Chi-square, the better was the fit. The CDSSTR algorithm was applied to estimate the content of the protein secondary structures. GraphPad Prism V5.0 (CA, USA) was used to fit the maximal fluorescence emission plot for 1,8-ANS and competition curves for each PFAS, as well as the IC50 values. Cell counting and gene expression data were analyzed using SPSS for Windows 17.0 Software (SPSS, Inc., Chicago, IL, USA). Differences between the control and treatment groups were determined using one-way analysis of variance (ANOVA) followed by the Fisher LSD post hoc test. The p values were adjusted for multiple tests using Bonferroni’s corrections.


Cytotoxicity of PFOA, PFOS, and potential alternatives

The concentration–response curves (CRC) of the PFAS legacies and alternatives to HL-7702 cells obtained by MTT and neutral red assays are shown in Fig. 1. Like the two legacies (PFOA and PFOS), 6:2 FTCA and 6:2 FTSA showed J-shaped non-monotonic CRCs, whereas 6:2 Cl-PFESA, HFPO-TA, and HFPO-TeA showed S-shaped CRCs. As seen in Fig. 1, PFOA presented the highest stimulatory effect (Em = −56.35%) at 3.69 × 10−4 M followed by 6:2 FTSA (Em = −40.97%, 1.21 × 10−4 M). Although there was no peak in the S-shaped CRCs representing obvious stimulatory effects, HFPO-TA and HFPO-TeA exposure still increased cell numbers at low dosages (ranging from the lowest concentration to 2.71 × 10−4 and 1.62 × 10−4 M, respectively). The fitted CRC models and IC50 values for each chemicals are listed in Table 1, with the IC50 values ranging from 2.65 × 10−4 M (HFPO-TeA) to 1.04 × 10−3 M (6:2 FTCA). The much higher IC50 value for 6:2 FTCA implies a much weaker cytotoxicity compared with that of PFOA and PFOS. Conversely, the other potential alternatives (6:2 FTSA, F-53B, HFPO-TA, and HFPO-TeA) showed similar or lower IC50 values compared with those of PFOA and PFOS, suggesting stronger effects on cell viability.
Fig. 1

J- and S-shaped concentration–response relationships showing the inhibitory effects of individual compounds on a human liver cell line after 24 h of exposure. a Obtained by MTT assay; b obtained by neutral red assay. Filled square refers to the experimental data, solid line is the fitted concentration–response curve (CRC)

Table 1

Concentration–response models of individual compounds showing their inhibitory effect on a human liver cell line after 24 h of exposure by MTT assay



χ 2

R 2

IC 50





7.77 × 10−4





4.17 × 10−4

6:2 FTCA




1.04 × 10−3

6:2 FTSA




3.54 × 10−4

6:2 Cl-PFESA




3.02 × 10−4





3.42 × 10−4





2.65 × 10−4

Effects of potential alternatives on cells cycles

Flow cytometry was used to measure the effects of the potential alternatives on the cell cycles. As shown in Fig. 2, compared with the control groups, 6:2 FTCA, HFPO-TA, and HFPO-TeA showed similar decreased changing trends in G0/G1 and S phase cells, as well as increased changing trends in G2/M phase cells, indicating the promotion of cells from the G1 phase to the S phase and then to the G2 phase for these PFASs. For 6:2 FTSA, 100 and 200 μM treatment showed similar effects as that of 6:2 FTCA, whereas 50 μM treatment significantly increased cell populations at the S phase from 20.01 to 21.27%. Interestingly, for 6:2 Cl-PFESA, cells at the G0/G1 phase increased in all three treatment groups compared with the control, with significance in the 25 and 50 μM groups (from 9.68 to 12 and 11.93%, respectively). This corresponded to a decrease in cells at the G2/M phase, suggesting that 6:2 Cl-PFESA exposure blocked, rather than promoted, cells at the G0/G1 phase.
Fig. 2

Composition of cells at different division phases measured by flow cytometry after 24 h of exposure

Changes of gene expressions after potential alternatives exposure

Under moderated dosage, except cluster of differentiation 36 (Cd36), the expression levels of peroxisome proliferator-activated receptor alpha (Pparα) and its related genes showed no significant change for all five tested potential alternatives. Significant up-regulation of cytochrome P4504A11 (Cyp4a11) and acyl-CoA thioesterase 1 (Acot1) were observed in high-dosage groups of 6:2 Cl-PFESA and HFPO-TeA, fatty acid binding protein 1 (Fabp1) expression level increased only in HFPO-TeA high-dosage group (Fig. 3a).
Fig. 3

Effects of moderate and high doses (in accordance with the concentrations used in cell flow cytometry assay) of different potential alternatives on gene expression. a Pparα and its related genes; b cell cycle related genes. *p < 0.05 and **p < 0.01 indicate significant differences between control and exposure groups, *red and blue indicate increased and decreased cell content, respectively (color figure online)

As shown in Fig. 3b, although no significant up-regulation in DNA-damage-inducible transcript 3 (Ddit3) was observed under moderate dosage in the HFPO-TA and HFPO-TeA groups, a slight increase in expression was detected under high dosage (1.6 and 2.3 times, respectively). Genes that play roles in the cell cycle, such as cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 6 (Cdk6), and MYC proto-oncogene (Myc), were both significantly up-regulated, whereas tumor protein p53 (p53) showed no significant change, indicating a stimulating effect on the cell cycles (Fig. 2b).

Effects of PFOA, PFOS, and potential alternatives on the secondary structure of hL-FABP

The purity of hL-FABP is shown in Fig. S1. The far-UV CD spectra, with a negative minima at 218 nm and shoulder at 195 nm, displayed a typical structure for recombinant hL-FABP with abundant β-sheets (Fig. 4a). By calculation, the recombinant protein consisted of 15.7% α-helix and 54.4% β-sheet (Table S4), similar to that obtained in our previous study (Sheng et al. 2016a). After the addition of different PFASs, the α-helix content of the protein increased slightly, whereas the β-sheet content decreased by different degrees (Fig. 4b, c). The degree of these secondary structure changes depended on the characters of chemicals including length of the backbone, branched chain, and type of functional group. A slight change occurred after the addition of 6:2 FTCA and HFPO-DA, with a 2.58 and 2.27% decrease in β-sheet content, respectively. After the addition of 6:2 Cl-PFESA to the hL-FABP solution, the β-sheet content decreased from 54.4 to 48.98% (9.97%) and the α-helix content increased by 4.11%, approximating the changes induced by PFOS and HFPO-TA. The changes were the most dramatic for HFPO-TeA (12 atoms in the backbone and three –CH3 side chains), with a 16.42% decrease in β-sheet content and a 4.89% increase in α-helix content.
Fig. 4

Secondary structural changes of the hL-FABP protein after binding with PFOA, PFOS, or different PFAS alternatives

Binding affinity of PFASs to hL-FABP

The binding parameters of the PFASs for hL-FABP were obtained by titrating 200 nM hL-FABP with increasing concentrations of ANS. With nonlinear regression analysis (Hill plot) of fluorescence intensity at 470 nm, a stoichiometry of one ANS molecule per hL-FABP binding site (n = 0.93, Table 2) was then identified. Using 1,8-ANS as a fluorescence probe, the binding affinity for each PFAS to hL-FABP was measured with ligand displacement assay. As shown in Table 2, binding affinity decreased in the order of 6:2 FTCA < 6:2 FTSA < HFPO-DA < PFOA < PFOS/6:2 Cl-PFESA/HFPO-TA < HFPO-TeA. The K d values of 6:2 FTCA and 6:2 FTSA (436.55 and 345.54 μM, respectively) were two orders of magnitude higher than those of PFOA and PFOS, indicating weak binding capacity for 6:2 FTCA and 6:2 FTSA compared with that of PFOA and PFOS.
Table 2

R 2, number of binding sites (N), dissociation constant (K d) of 1,8-ANS, IC 50 value, and dissociation constant (K d) of eight compounds with hL-FABP determined fluorometrically by displacement of 1,8-ANS


IC50 (μM)

K d (μM)


R 2


7.47 ± 0.97

0.93 ± 0.03



2.15 ± 0.54

8.03 ± 2.10



1.34 ± 0.21

4.99 ± 0.97


6:2 FTCA

116.88 ± 0.52

436.55 ± 1.92


6:2 FTSA

78.97 ± 0.56

345.54 ± 2.18


6:2 Cl-PFESA

1.14 ± 0.12

4.05 ± 0.42



4.76 ± 0.31

15.36 ± 1.09



1.21 ± 0.37

4.36 ± 1.17



0.98 ± 0.18

3.35 ± 0.46


Binding mode of PFASs to the hL-FABP inner binding pocket

The binding of the PFAS legacies and alternatives to hL-FABP in a 15-Å gorge was predicted with molecular docking. Except for 6:2 FTSA, all PFASs fit well at the binding pocket, indicating direct interactions between these chemicals and hL-FABP. As shown in Fig. 5, PFOA bonded with hL-FABP by the “head-out” mode, in which, the carboxyl head of PFOA will interacted with R122 amino acid residue and N111 amino acid by hydrogen bond and hydrophobic interaction, respectively (Fig. 5; Table 3). The same mode was detected during the binding of the protein with PFOS and 6:2 FTCA (Fig. 5; Table 3). With the oxygen atom in their backbone, 6:2 Cl-PFESA and HFPO homologues exhibited structural distortion, to a certain extent. The tail of the 6:2 Cl-PFESA molecule “folded” upward, resulting in interactions with F59, F72, and M74 amino acid residues. Unlike HFPO-DA and HFPO-TA, the carboxyl head of HFPO-TeA was faced the protein interior and interacted with N61 by hydrogen bond (Fig. 5; Table 3). The absolute values of average docking energies for the predicted interactions (Table 3) increased in the order of HFPO-TeA > 6:2 Cl-PFESA/PFOS > HFPO-DA/HFPO-TA > PFOA > 6:2 FTCA, indicating a rank of binding affinity for these chemicals in approximate accordance with the fluorescence displacement results.
Fig. 5

Proposed binding modes between the hL-FABP protein and PFOA, PFOS, and PFAS alternatives in molecular docking

Table 3

Ebinding values and number of hydrogen bonds (N Hydrogen bond) between hL-FABP and PFOA, PFOS, and different PFAA alternatives



E binding (kJ/Mol)

N Hydrogen bond

Hydrogen bond










Potential alternatives

6:2 FTCA




6:2 FTSA

No binding


No binding

6:2 Cl-PFESA

















To date, various PFAS alternatives have been detected in multiple samples (Chu et al. 2016; Heydebreck et al. 2015; Lam et al. 2016; Ruan et al. 2015; Wang et al. 2016). With incremental usage in the industry, the types of PFAS alternatives identified in environmental and biological samples will increase at increasing concentrations. Recognizing the lack of toxicity information for these alternative chemicals, we explored the cytotoxicity and hL-FABP binding capacity of six potential alternatives and compared them with those of PFOA and PFOS, two well-known PFAS legacies.

CD36 is involved in transport of long-chain fatty acids through the adipocyte membrane (Hajri et al. 2002; Spitsberg et al. 1995), and the significantly up-regulated Cd36 expression level in this study seem to be responsible for the uptake of these alternatives. It has been reported that FABP protein can form a complex with CD36 on the cytoplasmic side of the cell membrane, and the up-regulated gene expression level of FABP 1 after high-dosage treatment of 6:2 Cl-PFESA, HFPO-TA and HFPO-TeA might also have a relationship with the transport of these alternatives. Due to its essential role in fatty acid metabolism in liver, the nuclear receptor Pparα has become the key research subject for the studies on hepatotoxicity of PFAS legacies, and its key role in rodent liver has been reported in many studies (Abbott et al. 2012; Quist et al. 2015; Wang et al. 2014). To date, the species-related activation difference of Pparα for PFAS legacies has been widely accepted. Like PFOA and other legacies, all these potential alternatives tested in this study changed the gene expression levels of Pparα itself in HL7702 cells. Also, as the transcriptional indicators of Pparα activation, the nearly equal expression levels of Cyp4a11 and Acot1 indicating the weak activation of Pparα, was in accordance with previous studies about PFOA and PFOS (Bjork et al. 2011).

Previous studies have investigated the cytotoxicities of PFAS legacies (Florentin et al. 2011; Gimenez-Bastida et al. 2015; Tian et al. 2012), while to our knowledge, only few studies on the cytotoxicity for these alternatives has been published. Using IC50 values provided a direct order of cytotoxicity of PFOA, PFOS, and potential PFAS alternatives. Except for 6:2 FTCA, the other alternatives exhibited equal or stronger adverse effects on cell viability compared with that of PFOA and PFOS, indicating that their toxicities to the liver might not lower. With the increase of backbone atom number, the cytotoxic effects for PFOA (n = 8), HFPO-TA (n = 9), HFPO-TeA (n = 12) became more serious, indicating a potential association of backbone length of one chemical to its cytotoxicity. Although the replacement of hydrogen to fluorine for 6:2 FTCA to PFOA led to a decrease of cytotoxic effects, while for 6:2 FTSA, the decreased cytotoxicity was not observed in this study.

As reported in our previous studies, exposure to low doses of PFOA or PFOS stimulated HL-7702 cell proliferation by promoting cells from the G1 to S phase, whereas high doses resulted in the opposite effect on cell number (Cui et al. 2015; Hu et al. 2014; Zhang et al. 2016). The MTT results of PFOA and PFOS obtained in the present study showed the same phenomenon, with an obvious stimulatory effect at low concentrations. Although 6:2 FTCA and 6:2 FTSA demonstrated similar CRCs to those of PFOA and PFOS due to their similar structures, their exposure not only promoted cells to transform from the G1 to S phase, as observed with the two legacies, but also promoted the transition of cells from the S to G2 phase. The same trend for HFPO-TA and HFPO-TeA also explained the increase in cell number at low doses. Noticeably, low doses of 6:2 Cl-PFESA induced cell arrest in the G0/G1 phase, together with an increase in cells in the G2/M phase, which might be responsible for the decrease in cell number in the S phase.

Up-regulation in Ddit3 gene expression can result in general cellular damage and activation of the unfolded protein response (Bjork et al. 2011; Bjork and Wallace 2009). For cells exposed to 240 μM HFPO-TA or 140 μM HFPO-TeA, the increase in the Ddit3 level implied a certain degree of cytotoxicity, which was in agreement with the slight cell inhibition effects measured by MTT assay in this study. Cyclin E/Cdk2 complexes play important roles during G1 progression and the onset of DNA synthesis and will reach maximum amounts in the late G1 and early S phases (Cui et al. 2015; Dulic et al. 1992; Lees et al. 1992). After exposure with alternatives, the increased Cdk2 and Cdk6 levels, which are important for G0 cells, suggested a stimulation of cells from the G0 to G1 then S phase. As reported in previous studies, exposure to PFOS at low dosages did not induce significant changes in p53 levels but did decrease the p21 waf1/cip1 expression, which could lead to increased myc (Cui et al. 2015; Obaya et al. 1999). In the present study, the up-regulation of myc and unchanged p53 levels of all treated cells were in accordance with that of PFOS, indicating possible inhibition of the p53 signaling pathway.

As the most broadly distributed mammalian FABP expressed in the liver, L-FABP plays an essential role in the nucleo-cytoplasmic shuttling of activator ligands for nuclear receptors, including PPAR activators, pregnane X receptors, and liver X receptors (Hostetler et al. 2009; Wolfrum et al. 2001). Via binding with L-FABP, fatty acid and other ligands can be transported to the nucleus to realize their regulatory functions (Schroeder et al. 2001; Zimmerman and Veerkamp 2002). FABPs could bind to the thrombospondin (TSP)-binding domain of CD36 and has a subsequent effect on cell proliferation (Spitsberg et al. 1995; Zimmerman and Veerkamp 2002). It has been reported that L-FABP modulates the mitogenesis of hepatoma cells via activating two classes of carcinogenic peroxisome proliferators (Sorof 1994; Zimmerman and Veerkamp 2002). A potentially positive relationship between L-FABP content and proliferation rate of hepatoma cells has been observed (Keler and Sorof 1993; Zimmerman and Veerkamp 2002). In this study, the increased Fabp 1 expression levels might relate with the proliferation of HL7702 cells to a certain extent.

Due to their similarity in structure to endogenous fatty acids, PFASs, especially long-chain PFASs (n > 8), can bind to hL-FABP and be transported into the cell nucleus to activate nuclear receptors (Sheng et al. 2016a; Vanden Heuvel et al. 2006; Zhang et al. 2013a). With the addition of different PFASs into the protein solution, hL-FABP will change its secondary structure to varying degrees to bind the chemical. As reported previously, for PFASs with carbon lengths ranging from 4 to 12, β-sheet content decreases with increased carbon length, whereas α-helix content increases (Sheng et al. 2016a; Zhang et al. 2013a). In the present study, the degree of these changes depended not only on the length of the backbone, but also on the branched chain and functional group. Similar to PFAS legacies, the longer the backbone lengths, the greater was the change in protein secondary structure during binding. For chemicals with the same backbone length, atom types and branches determined the changes in protein structure. With two fluorine atoms replaced by hydrogen atoms, binding with 6:2 FTCA and 6:2 FTSA led to only slight changes compared with that for PFOA and PFOS; the branched chains of HFPO-TA resulted in more severe changes in the protein structure than that for 6:2 FTSA and PFOS with the same backbone length (n = 9). Thus, the most significant changes in the protein were observed during the binding of hL-FABP with HFPO-TeA.

We also detected a relationship between backbone length and the distinctive structures of the PFASs and their binding affinities to hL-FABP. Like the influence on protein secondary structure, HFPO-TeA showed the highest binding affinity, whereas 6:2 FTCA showed the lowest, with PFOA and PFOS in the middle of the binding affinity order. For PFASs with carbon numbers ranging from 7 to 11, the binding affinity for hL-FABP increased with the increase in hydrocarbon chain length, as well as their negative effects on cells and rodents (Fang et al. 2012; Hu et al. 2014; Kudo et al. 2006; Zhang et al. 2013a). Here, the higher binding affinities of 6:2 Cl-PFESA and HFPO homologues to hL-FABP compared with those of PFOA and PFOS suggested critical cytotoxicity to HL-7702 cells, and possibly more serious hepatotoxicity. The lower binding affinity for 6:2 FTCA and 6:2 FTSA than PFOA and PFOS might be the results of the replacement of hydrogen by fluorine. Moreover, for PFOA, HFPO-TA and HFPO-TeA, higher binding affinity occurred with longer backbone length. Higher binding affinity of 6:2 Cl-PFESA than PFOS may be the result of the presence of the chlorine atom and insertion of the oxygen atom.

Our previous study showed that PFOA can bind to the two binding pockets of hL-FABP (Sheng et al. 2016a). For the first molecule PFOA, it will bind to the inner pocket with moderate affinity, while for the second molecule, its binding to outer pocket was very weak. As N111 amino acid residue plays key role in the binding of chemicals to the outer pocket of protein, N111 variant, which PFAS can only bind to outer pocket, exhibited inferior binding affinity (Sheng et al. 2016a). The order of interaction energy to the inner binding pocket calculated by molecular docking was similar to that found by fluorescence replacement assay, confirming the relationship between the binding affinities and characters of PFASs. Based on the much weaker binding affinities of 6:2 FTCA and 6:2 FTSA to the protein, it is not surprising that no binding for 6:2 FTSA was observed. As 6:2 FTCA has the same carbon backbone as PFOA, though with two hydrogen atoms replacing two fluorine atoms, it also showed “head-out” binding and the same amino acid residues as that of PFOA and PFOS. Interestingly, with one or more oxygen atoms in the backbones of 6:2 Cl-PFESA and the HFPO homologues, structural distortion occurred during their binding processes, leading to different binding modes, including the “head in” binding for HFPO-TeA, “upward folded” tail binding for 6:2 Cl-PFESA, and the distinctive hydrophobic interaction between F95 and 6:2 Cl-PFESA and between F50 and HFPO-TA and HFPO-TeA.

Taken together, compared with PFOA and PFOS, 6:2 FTCA and 6:2 FTSA showed weaker or equal cytotoxicity and weaker binding affinity, whereas 6:2 Cl-PFESA and HFPO homologues exhibited stronger toxicity and protein-binding affinity due to their unique structure, suggesting possibly greater hepatotoxicity than legacies. The replacement of hydrogen by fluorine might reduce the toxicity of PFAS, while the insertion of the oxygen atom and the increased backbone length seem to be able to induce more serious toxicities. The distinctive cytotoxic mechanism as well as protein binding mode of the studied alternatives are worthy of future research. The cytotoxicity of these chemicals and their potential disruption to fatty acid metabolism cannot be ignored. As such, their usage as alternatives to PFAS legacies should be given greater caution and consideration.



This work was supported by the National Natural Science Foundation of China (21737004, 31320103915 and 21377128) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14040202).

Supplementary material

204_2017_2055_MOESM1_ESM.docx (157 kb)
Supplementary material 1 (DOCX 157 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Nan Sheng
    • 1
    • 2
  • Ruina Cui
    • 1
  • Jinghua Wang
    • 3
  • Yong Guo
    • 4
  • Jianshe Wang
    • 1
  • Jiayin Dai
    • 1
    Email author
  1. 1.Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Research Center of Environmental ScienceZhejiang University of TechnologyHangzhouPeople’s Republic of China
  4. 4.Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic ChemistryChinese Academy of SciencesShanghaiPeople’s Republic of China

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