Thyroid endocrine disruption effects of perfluoroalkyl phosphinic acids on zebrafish at early development
Graphical abstract
Introduction
Per- and polyfluoroalkyl acids (PFASs) are a class of anthropogenic organic chemicals, and widely used in various industrial formulations and consumer products attributed to their superior surface active properties. Among this family of PFASs, perfluoroalkyl acids (PFAAs), such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been studied extensively due to their high detection frequency, great potential for bioaccumulation and well documented toxicities (Boulanger et al., 2004; Martin et al., 2004; Rankin et al., 2016; Verreault et al., 2006). As a consequence, PFOS and its salts were listed as persistent organic pollutants (POPs) under Annex B of the Stockholm Convention in 2009, and the phase-out of PFOA has been implemented in many regions (Wang et al., 2009). As substituents of PFOS and PFOA, many emerging PFASs, such as perfluoroalkyl phosphinic acids (PFPiAs), are being increasingly manufactured (Wang et al., 2013b). PFPiAs are similar to PFOS in chemical structure, containing a perfluorinated carbon tail attached to phosphinate (R2-P(O)O−) through a carbonphosphorus (CP) bond (Table 1) (Lee et al., 2012; Lee and Mabury, 2017). They are usually used as leveling and wetting agents in household cleaning products and historically used as pesticide formulations in the United States until 2008 (Lee et al., 2012). The annual production volume was 4.5–227 t in 1998 and 2002 in North America (Howard and Muir, 2010). PFPiAs have been found in surface water, sediment, indoor dust, organisms, and humans (Chen et al., 2018b; Guo et al., 2016; Guo et al., 2012; Jin et al., 2015; Lee and Mabury, 2011; Silva et al., 2012; Silva et al., 2016), and 6:6, 6:8 and 8:8 PFPiA were the most detected homologues. The 6:6 and 6:8 PFPiA were detected in over 50% of 50 human sera samples collected from the United States with mean concentrations in the range of 4–38 ng/L (Lee and Mabury, 2011). The mean concentrations of ΣPFPiAs (6:6, 6:8 and 8:8 PFPiA) in dust samples collected from Vancouver was 2.3 ng/g (Silva et al., 2012), and 1.87 ± 2.17 ng/g wet weight in fish, dolphin and bird plasma in North America (Silva et al., 2016).
The increasing detection of PFPiAs in the environment and its similar chemical structure as PFOS prompt interests over the adverse effects of PFPiAs on organisms and humans. A few studies were conducted to try to evaluate the toxicities of PFPiAs in organisms. It was reported that dietary exposure to a mixture of 6:6, 6:8 and 8:8 PFPiAs (around 7.0, 7.7 and 6.3 μg/kg fish/day, respectively) for 30 days significantly reduced the whole body weight and liver growth rate of rainbow trout (Lee et al., 2012). Exposure to Masurf FS-780 (an 80% aqueous solution of a mixture of PFPAs and PFPiAs) was evidenced to cause increased liver weight in mice (Wang et al., 2016). Since these studies were conducted with mixed compounds, it is not clear which ones were responsible for the induced toxicities. Further studies are warranted to understand the toxic potentials of PFPiAs to aquatic organisms in order to evaluate the environmental safety of PFPiAs as emerging contaminants.
Thyroid hormones (THs) play an important role in the development and growth of fish, especially in the early life stage (Kawakami et al., 2008; Walpita et al., 2007). Disruption on THs may result in reduced reproductive fitness through altering behaviors or physiological pathways of fish (Power et al., 2001). The hypothalamic-pituitary-thyroid (HPT) axis controls the thyroid endocrine system and regulates THs synthesis, secretion, transport and metabolism to maintain THs homeostasis (Tu et al., 2016a; Xie et al., 2015). The hypothalamus coordinates HPT axis to secret corticotropin-releasing hormone (CRH), which controls the release of thyroid stimulating hormone (TSH) from the pituitary, which regulates the THs synthesis and release in fish (Shi et al., 2009). Thyroxine (T4) is converted to biologically active triiodothyronine (T3) through the regulations of deiodinase enzymes. T3 binds to thyroid hormone receptor (TR) and mediates actions in the target organs (Jin et al., 2011).
Many studies have indicated that some PFASs are potential thyroid toxicants (Jain, 2013; Lin et al., 2013; Wang et al., 2014; Webster et al., 2016; Webster et al., 2014). PFOS was reported to significantly reduce the total T4 level and interfere the thyroid function in male Sprague-Dawley rats (Yu et al., 2009). PFOS also displayed developmental toxicities by enhancing T3 level and altered the gene expressions along HPT axis in zebrafish (Shi et al., 2008; Shi et al., 2009). Perfluorobutane sulfonate (PFBS) was demonstrated to alter the THs levels and cause thyroidal disturbance in the three generations of marine medaka and hatching delay in F1 larvae, although only the F0 medaka eggs were exposed (Chen et al., 2018a). PFOA, perfluorobutyric acid (PFBA) and perfluorododecanoic acid (PFDoA) displayed disruption effects on thyroid functions of zebrafish embryos (Godfrey et al., 2017; Zhang et al., 2018b). PFASs could also affect T4 homeostasis by binding with human thyroid hormone transport protein transthyretin (TTR) (Weiss et al., 2009). All these studies evidenced that the thyroid disruption was mainly manifested as changes in the levels of THs and transcriptional genes in the HPT axis. PFPiAs usually have longer carbon chain length (≥12 carbon atoms) than PFOA and PFOS. Several studies demonstrated that PFAAs with longer carbon chain length induced stronger toxicities than the shorter ones (Kudo et al., 2001, Kudo et al., 2006). Therefore, it is speculated that PFPiAs may exert strong thyroidal disturbance on fish. However, up to now, there is a large knowledge gap on the toxicities, especially the thyroid endocrine disrupting effects of individual PFPiAs on aquatic organisms, which makes the ecological risk assessment of these compounds difficult.
The zebrafish embryos represent a good model for risk assessment and toxicological investigation of hazardous chemicals (Mu et al., 2018; Zhang et al., 2018a). In developing embryos/larvae, several potential endpoints can be used to assess the functions of the thyroid gand: specific expression in differentiated thyroid follicular cells (e.g., thyroglobulin), markers of activation of the HPT axis (e.g., thyroid-stimulating hormone) and levels of THs (Zhu et al., 2014). Hence, zebrafish embryos/larvae could be used as an ideal model to screen xenobiotics impairing thyroid functions. The HPT axis regulates thyroid hormone dynamics by coordinating their synthesis, secretion, transport and metabolism, and plays an important role in the development and growth, especially in the early life stage of fish (Kawakami et al., 2008).
Therefore, the objective of this study was to investigate the possible disruption effects of PFPiAs on thyroid endocrine system and related development effects on zebrafish at early development. The variations of THs and related genes involved in the HPT axis were investigated. Zebrafish embryos at 6 hpf were exposed to individual solutions of 6:6, 6:8 and 8:8 PFPiA at three concentrations until 168 hpf. The potential disruption mechanisms along the HPT axis elicited by the PFPiAs in zebrafish larvae were explored. It was the first to focus solely on effects of PFPiAs on function of the HPT axis during early development. The results would provide a scientific basis for assessing environmental risks induced by the emerging PFPiAs.
Section snippets
Chemicals
Sodium bis(perfluorohexyl)phosphinate (6:6 PFPiA, with purity > 98%, CAS: 70609-44-8), sodium perfluorohexylperfluorooctylphosphinate (6:8 PFPiA, >98%, CAS: 610800-34-5) and sodium bis(perfluorooctyl)phosphinate (8:8 PFPiA, >98%, CAS: 500776-69-2) were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Stock solutions (10 μg/mL) of the three PFPiAs were prepared in methanol, respectively, and diluted with water referring to the ISO standard (ISO7346-2, 1996) to the desired
Solvent effect
According to the statistics, there was no significant difference between the solvent and blank control for all tested indicators (Table S3). The data for the control shown in all figures (and tables) are the experimental data for the solvent control.
Concentrations of PFPiAs in the exposure solutions
The average measured concentrations of PFPiAs at 0 and 168 hpf are listed in Table S4. In the control group, no target compound was detected. In the treatment groups, the actual concentrations before exposure (at 0 hpf) were 6:6 PFPiA (0.50, 4.93
Conclusions
With increasing application of PFPiAs, a large amount of PFPiA homologues would be discharged to aquatic environment. The results in this study demonstrated that PFPiAs (6:6, 6:8 and 8:8) modulated the expression of a number of genes in the HPT axis and caused THs imbalance, demonstrating a thyroid endocrine disrupting toxicity. Exposure to 8:8 PFPiA resulted in significantly enhanced levels of T4 and T3, reduced body length and heart rate, which could be a result of thyroid endocrine
Acknowledgements
This work was financially supported by National Science Foundation of China (NSFC 21737003, 21577067, 21677081), Ministry of Science and Technology of China (2017ZX07301002), the 111 program of Ministry of Education of China (T2017002), Tianjin Municipal Science and Technology Commission (16PTSYJC00020, 17JCYBJC23200) and Yangtze River scholar program.
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