Per- and polyfluoroalkyl substances exert strong inhibition towards human carboxylesterases

https://doi.org/10.1016/j.envpol.2020.114463Get rights and content

Highlights

  • Longer-chain perfluoroalkyl substances strongly inhibited carboxylesterase.

  • The higher binding affinity caused more severe inhibition.

  • Inhibition kinetics was studied, and in vitro-in vivo extrapolation was performed.

Abstract

PFASs are highly persistent in both natural and living environment, and pose a significant risk for wildlife and human beings. The present study was carried out to determine the inhibitory behaviours of fourteen PFASs on metabolic activity of two major isoforms of carboxylesterases (CES). The probe substrates 2-(2-benzoyl-3-methoxyphenyl) benzothiazole (BMBT) for CES1 and fluorescein diacetate (FD) for CES2 were utilized to determine the inhibitory potentials of PFASs on CES in vitro. The results demonstrated that perfluorododecanoic acid (PFDoA), perfluorotetradecanoic acid (PFTA) and perfluorooctadecanoic acid (PFOcDA) strongly inhibited CES1 and CES2. The half inhibition concentration (IC50) value of PFDoA, PFTA and PFOcDA for CES1 inhibition was 10.6 μM, 13.4 μM and 12.6 μM, respectively. The IC50 for the inhibition of PFDoA, PFTA and PFOcDA towards CES2 were calculated to be 9.56 μM, 17.2 μM and 8.73 μM, respectively. PFDoA, PFTA and PFOcDA exhibited noncompetitive inhibition towards both CES1 and CES2. The inhibition kinetics parameters (Ki) were 27.7 μM, 26.9 μM, 11.9 μM, 4.04 μM, 29.1 μM, 27.4 μM for PFDoA-CES1, PFTA-CES1, PFOcDA-CES1, PFDoA-CES2, PFTA-CES2, PFOcDA-CES2, respectively. In vitro-in vivo extrapolation (IVIVE) predicted that when the plasma concentrations of PFDoA, PFTA and PFOcDA were greater than 2.77 μM, 2.69 μM and 1.19 μM, respectively, it might interfere with the metabolic reaction catalyzed by CES1 in vivo; when the plasma concentrations of PFDoA, PFTA and PFOcDA were greater than 0.40 μM, 2.91 μM, 2.74 μM, it might interfere with the metabolic reaction catalyzed by CES2 in vivo. Molecular docking was used to explore the interactions between PFASs and CES. In conclusion, PFASs were found to cause inhibitory effects on CES in vitro, and this finding would provide an important experimental basis for further in vivo testing of PFASs focused on CES inhibition endpoints.

Introduction

Per- and polyfluoroalkyl substances (PFASs) are a family of synthetic compounds that contain at least one perfluoroalkyl moiety, -CnF2n-. They have been extensively used as highly effective and efficient surface protectors or surfactants in industrial and commercial products, such as Teflon products, carpet, furniture, food packaging, cookware, outdoor gear, cosmetics, pesticides and fire fighting foam (Ritscher et al., 2018). PFASs are highly persistent in both natural and living environment, and pose a significant risk for wildlife and human beings (Jian et al., 2018). Considering their persistence (P), bioaccumulation potential (B), toxicity (T) and long-range transport (LRT) potential, perfluorooctanesulfonic acid (PFOS) and perfluorooctanesulfonyl fluoride (POSF) have been listed under the Stockholm Convention of Persistent Organic Pollutants, and perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), and their precursors are being evaluated for listing (Ritscher et al., 2018).

Studies show that serum concentrations of PFOA and PFOS in the United States, Sweden and Germany have decreased (Liu et al., 2017). Nevertheless, decreases in PFOA and PFOS have predominantly driven reducing legacy PFASs, replaced PFOA and PFOS with shorter chain PFASs as well as new compounds that are hard to determine utilizing standard methods contributed to the proliferation of environmental literature on new PFASs (Sunderland et al., 2019). The toxicological properties of the PFASs alternatives and congeners are of great concern, and remain unclear. In addition, the body load of legacy PFASs in some developing nations may still be high (Liu et al., 2017). Therefore, understanding the structurally relevant toxicological influence is important for assessing the potential health risks of emerging PFASs.

Evidence suggests that PFASs may contribute to lipid-related metabolic disturbances (Steenland et al., 2010). Human studies have shown that PFASs are associated with hypercholesterolemia. Studies in the occupational populations, PFASs-contaminated community cohort, general population of adults and in the children and adolescents have reported positive associations between PFAS concentrations and circulating total cholesterol (TC) (Geiger et al., 2014; Zeng et al., 2015). However, several animal studies suggested that PFASs exposure can induce hypolipidemic effects with lower TC and triglyceride (TG) (Zeng et al., 2015). To date, the exact biochemical and molecular mechanisms of the relationship between PFAS concentrations and lipid regulation have not been clearly established. It is worth noting that the mechanisms by which PFASs affect lipid regulation may differ between humans and animals.

Mammalian carboxylesterases (CES) are phase I metabolic enzymes. Generally, the “CES” is employed for human carboxylesterases, and “Ces” is employed for mouse and rat carboxylesterases. The majority of human CES are classified into the CES1 or CES2 gene family sharing 47% amino acid sequence identity. CES1 is mainly expressed in liver and also distributed in the heart, kidney, macrophages and adipose tissue. CES2 is abundant in the intestine and low in liver. Metabolic substrates for CES include chemicals containing thioester, amide, and ester bonds. CES1 prefers to hydrolyze the substrates containing large acyl group and small alcohol group, and CES2 prefers to hydrolyze the substrates containing small acyl group and large alcohol group (Lei et al., 2017; Lian et al., 2018). CES have been broadly studied for their roles in xenobiotic metabolism (e.g., (pro) drug metabolism) (Laizure et al., 2013). In addition, carboxylesterases also metabolize endogenous lipids. The important role of CES as lipases in energy homeostasis and metabolic disorders has gained a great deal of research interest. CES1 shows hydrolase activity toward TG (Lian et al., 2018), cholesteryl ester (CE) (Crow et al., 2010), endocannabinoid 2-arachidonoylglycerol (2-AG) and prostaglandin glyceryl esters (PG-Gs) (Xie et al., 2010). CES2 was demonstrated to possess TG, diacylglycerol and diacylglycerol (DG) hydrolase activity (Ruby et al., 2017).

As mentioned above, both PFASs and CES are related to lipids in the body, PFASs can interfere with lipid metabolism, and CES can catalyze the hydrolysis of lipid. An animal experiment showed that PFOA increased the protein expression and activity of Ces1 and 2 (Wen et al., 2019), which is consistent with a decrease in lipid content in animals caused by PFOA (Zeng et al., 2015). But the impact of PFASs on human CES has not been reported. Thus, the present study investigated the inhibitory behaviours of fourteen PFASs towards the activity of two main human CES (CES1 and CES2) in vitro.

Section snippets

Chemicals and reagents

PFOA, perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoA), perfluorotetradecanoic acid (PFTA), perfluorooctadecanoic acid (PFOcDA), perfluorohexanoic acid (PFHxA), PFOS, perfluorohexyl iodide (PFHxI), perfluorooctyl iodide (PFOI), 1H,1H,2H,2H-Nonafluoro-1-hexanol (4:2 FTOH), 1H,1H-Undecafluoro-1-hexanol (5:1 FTOH), 1H,1H,2H,2H-Perfluorooctan-1-ol (6:2 FTOH), 1H,1H-Perfluoro-1-octanol (7:1 FTOH), 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctane-1-sulphonic Acid (6:2 FTS) was obtained as

Inhibition potential of PFASs towards CES

The residual activity of CES1 and CES2 at 100 μM of PFASs was shown in Fig. 1. The activity of CES1 was inhibited by 99.3%, 97.0%, 93.2% at 100 μM of PFDoA, PFTA and PFOcDA, respectively. CES2 was inhibited by 100 μM of PFDoA, PFTA and PFOcDA for 90.3%, 87.9% and 83.8%, respectively.

IC50 for the inhibition of PFASs on CES

Given the stronger inhibition of PFDoA, PFTA and PFOcDA towards CES, the IC50 value for the inhibition of PFDoA, PFTA and PFOcDA towards CES were determined (Fig. 2). The IC50 value for the inhibition of PFDoA, PFTA

Discussion

PFASs are a series of environmental pollutants that have received attention as they may have an adverse impact on wildlife and human health (Kato et al., 2015). Studies have shown that the toxicity of PFASs includes hepatotoxicity, reproductive and developmental toxicity, endocrine disrupting effects, neurotoxicity and immunotoxicity (Liu et al., 2017). PFASs can also cause changes in metabolic enzymes. For instance, PFOS exposure in rats and cultured hepatocytes of Atlantic salmon was found to

Conclusion

Overall, we gained the inhibition information of fourteen PFASs (PFHxA, PFOA, PFDA, PFDoA, PFTA, PFOcDA, PFOS, PFHxI, PFOI, 4:2 FTOH, 6:2 FTOH, 5:1 FTOH, 7:1 FTOH and 6:2 FTS) on the activity of CES (CES1 and CES2). All polyfluoroalkyl substances exhibited weak inhibition towards CES. In the perfluoroalkyl substances, PFDoA, PFTA and PFOcDA strongly inhibited CES. The IC50, inhibition kinetics type and Ki for the inhibition of PFDoA, PFTA and PFOcDA towards CES, and threshold for inducing

CRediT authorship contribution statement

Yong-Zhe Liu: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft. Li-Hua Pan: Methodology. Yu Bai: Investigation, Methodology. Kun Yang: Formal analysis. Pei-Pei Dong: Formal analysis. Zhong-Ze Fang: Conceptualization, Funding acquisition, Project administration, Writing - review & editing.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the project for the National Key Research and Development Program of China (2019YFA0802302, 2019YFA0802300), State Key Project on Infectious Diseases of China (2018ZX10723204), and National Natural Science Foundation of China (81602850).

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