Qianqian Cui

Novel Chlorinated Polyfluorinated Ether Sulfonates and Legacy Per-/Polyfluoroalkyl Substances: Placental Transfer and Relationship with Serum Albumin and Glomerular Filtration Rate

Per- and polyfluoroalkyl substances (PFASs) may cross the placental barrier and lead to fetal exposure. However, little is known about the factors that influence maternal-fetal transfer of these chemicals. PFAS concentrations were analyzed in 100 paired samples of human maternal sera collected in each trimester and cord sera at delivery; these samples were collected in Wuhan, China, 2014. Linear regression was used to estimate associations of transfer efficiencies with factors. Chlorinated polyfluorinated ether sulfonates (Cl-PFAESs, 6:2 and 8:2) were frequently detected (>99%) in maternal and cord sera. A significant decline in PFAS levels during the three trimesters was observed. A U-shape trend for transfer efficiency with increasing chain length was observed for both carboxylates and sulfonates. Higher transfer efficiencies of PFASs were associated with advancing maternal age, higher education, and lower glomerular filtration rate (GFR). Cord serum albumin was a positive factors for higher transfer efficiency (increased 1.1-4.1% per 1g/L albumin), whereas maternal serum albumin tended to reduce transfer efficiency (decreased 2.4-4.3% per 1g/L albumin). Our results suggest that exposure to Cl-PFAESs may be widespread in China. The transfer efficiencies among different PFASs were structure-dependent. Physiological factors (e.g., GFR and serum albumin) were observed for the first time to play critical roles in PFAS placental transfer.

6:2 Chlorinated polyfluorinated ether sulfonate, a PFOS alternative, induces embryotoxicity and disrupts cardiac development in zebrafish embryos

As an alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used as a mist suppressant in Chinese electroplating industries for over 30 years. It has been found in the environment and fish, and one acute assay indicated F-53B was moderately toxic. However, the toxicological information on this compound was incomplete and insufficient for assessment of their environment impact. The object of this study was to examine the developmental toxicity of F-53B using zebrafish embryos. Zebrafish embryos were incubated in 6-well plates with various concentrations of F-53B (1.5, 3, 6, and 12mg/L) from 6 to 132h post fertilization (hpf). Results showed that F-53B exposure induced developmental toxicity, including delayed hatching, increased occurrence of malformations, and reduced survival. Malformations, including pericardial and yolk sac edemas, abnormal spines, bent tails, and uninflated swim bladders, appeared at 84 hpf, and increased with time course and dose. A decrease in survival percentages was noted in the 6 and 12mg/L F-53B-treated groups at 132 hpf. Continuous exposure to 3mg/L F-53B resulted in high accumulation levels in zebrafish embryos, suggesting an inability for embryos to eliminate this compound and a high cumulative risk to fish. We also examined the cardiac function of embryos at specific developmental stages following exposure to different concentrations, and found that F-53B induced cardiac toxicity and reduced heart rate. Even under low F-53B concentration, o-dianisidine staining results showed significant decrease of relative erythrocyte number at 72 hpf before the appearance of observed effects of F-53B on the heart. To elucidate the underlying molecular changes, genes involved in normal cardiac development were analyzed using real-time qPCR in the whole-body of zebrafish embryos. F-53B inhibited the mRNA expression of β-catenin (ctnnb2) and wnt3a. The mRNA levels of β-catenin targeted genes (nkx2.5 and sox9b), which play critical roles in cardiogenesis, were also reduced after exposure. Thus, exposure to F-53B impaired the development of zebrafish embryos and disrupted cardiac development, which might be mediated by effects on the Wnt signaling pathway and decrease of erythrocyte numbers.

Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ.

Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50 μM PFOS for 48 h and 96 h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200 μM) stimulated HL-7702 cell viability at 48 h and 96 h, reduced the G0/G1 percentage, and increased the S+G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

First Report on the Occurrence and Bioaccumulation of Hexafluoropropylene Oxide Trimer Acid: An Emerging Concern

Here, we report on the occurrence of a novel perfluoroalkyl ether carboxylic acid, ammonium perfluoro-2-[(propoxy)propoxy]-1-propanoate (HFPO-TA), in surface water and common carp (Cyprinus carpio) collected from the Xiaoqing River and in residents residing near a fluoropolymer production plant in Huantai County, China. Compared with the levels upstream of the Xiaoqing River, HFPO-TA concentrations (5200-68500 ng/L) were approximately 120-1600-times higher downstream after receiving fluoropolymer plant effluent from a tributary. The riverine discharge of HFPO-TA was estimated to be 4.6 t/yr, accounting for 22% of total PFAS discharge. In the wild common carp collected downstream from the point source, HFPO-TA was detected in the blood (median: 1510 ng/mL), liver (587 ng/g ww), and muscle (118 ng/g ww). The log BCFblood of HFPO-TA (2.18) was significantly higher than that of PFOA (1.93). Detectable levels of HFPO-TA were also found in the sera of residents (median: 2.93 ng/mL). This is the first report on the environmental occurrence and bioaccumulation of this novel chemical. Our results indicate an emerging usage of HFPO-TA in the fluoropolymer manufacturing industry and raise concerns about the toxicity and potential health risks of HFPO-TA to aquatic organisms and humans.

Occurrence and Tissue Distribution of Novel Perfluoroether Carboxylic and Sulfonic Acids and Legacy Per/Polyfluoroalkyl Substances in Black-Spotted Frog (Pelophylax nigromaculatus)

Research on perfluoroalkyl substances (PFASs) continues to grow. However, very little is known about these substances in amphibians. Here we report for the first time on the occurrence, tissue distribution, and bioaccumulation of two novel PFASs, chlorinated polyfluorinated ether sulfonic acid (6:2 Cl-PFESA) and hexafluoropropylene oxide trimer acid (HFPO-TA), in the black-spotted frog (Pelophylax nigromaculatus) from China. Frogs from cities with large-scale fluorochemical industries had significantly greater liver ∑PFAS levels (mean 54.28 ng/g in Changshu; 31.22 ng/g in Huantai) than those from cities without similar industry (9.91 ng/g in Zhoushan; 7.68 ng/g in Quzhou). Females had significantly lower liver PFAS levels than males, and older frogs tended to have lower PFAS levels than younger frogs. Skin, liver, and muscle contributed nearly 80% to the whole body burden of 6:2 Cl-PFESA in males, whereas the female ovary alone accounted for 58.4%. These results suggest substantial maternal transfer of 6:2 Cl-PFESA to eggs, raising concern regarding its developmental toxicity on frogs and other species. The bioaccumulation factor results (6:2 Cl-PFESA > PFOS; HFPO-TA > PFOA) suggest a stronger accumulative potential in the black-spotted frog for these alternative substances compared to their predecessors. Future studies on their toxicity and ecology risk are warranted.

Worldwide Distribution of Novel Perfluoroether Carboxylic and Sulfonic Acids in Surface Water

Driven by increasingly stringent restrictions on long-chain per- and polyfluoroalkyl substances (PFASs), novel fluorinated compounds have emerged on the market. Here we report on the occurrences of several perfluoroalkyl ether carboxylic and sulfonic acids (PFECAs and PFESAs), including hexafluoropropylene oxide dimer and trimer acids (HFPO-DA and HFPO-TA), ammonium 4,8-dioxa-3 H-perfluorononanoate (ADONA), chlorinated polyfluorinated ether sulfonic acid (6:2 Cl-PFESA), and its hydrogen-substituted analogue (6:2 H-PFESA) in surface waters from China ( n = 106), the United States ( n = 12), the United Kingdom ( n = 6), Sweden ( n = 10), Germany ( n = 14), The Netherlands ( n = 6), and Korea ( n = 6). Results showed that HFPO-DA, HFPO-TA, and 6:2 Cl-PFESA (median = 0.95, 0.21, and 0.31 ng/L, respectively) were frequently detected in all countries, indicating ubiquitous dispersal and distribution in global surface waters. The presence of 6:2 H-PFESA was widely detected in China (detection rate > 95%) but not in any other country. Only trace levels of ADONA (0.013-1.5 ng/L) were detected in the Rhine River flowing through Germany. The estimated total riverine mass discharges of HFPO-DA, HFPO-TA, and ΣPFESAs reached 2.6, 6.0, and 4.3 ton/year in five of the major river systems in China. Our results indicated that novel PFECAs and PFESAs might become global contaminants, and future investigations are warranted.

6:2 fluorotelomer carboxylic acid (6:2 FTCA) exposure induces developmental toxicity and inhibits the formation of erythrocytes during zebrafish embryogenesis

Saturated fluorotelomer carboxylic acids (FTCAs) are intermediates in the degradation of fluorotelomer alcohols (FTOHs) to perfluorinated carboxylic acids (PFCAs). Recent studies have detected FTCAs in precipitation, surface waters, and wildlife, but few studies have focused on their toxicity. In this study, zebrafish embryos were exposed to different concentrations of 6:2 FTCA (0, 4, 8, and 12mg/L) from 6 to 120h post-fertilization (hpf) to investigate its developmental toxicity. Results showed that 6:2 FTCA exposure decreased the hatching and survival percentages, reduced the heart rate, and increased the malformation of zebrafish embryos. The median lethal concentration of 6:2 FTCA was 7.33mg/L at 120 hpf, which was lower than that of perfluorooctanoic acid (PFOA), thus indicating higher toxicity for zebrafish. The most common developmental malformation was pericardial edema, which appeared in the 8 and 12mg/L 6:2 FTCA-exposed embryos from 60 hpf. Using o-dianisidine staining, we found that the hemoglobin content in embryos was reduced in a concentration-dependent manner after 6:2 FTCA exposure at 72 hpf. Based on quantitative real-time polymerase chain reaction (q-RT-PCR) and whole-mount in situ hybridization, the transcriptional levels of hemoglobin markers (hbae1, hbbe1, and hbae3) were down-regulated at 48 and 72 hpf, even though no observed malformation appeared in zebrafish at 48 hpf. Moreover, 6:2 FTCA exposure decreased the protein level of gata1, a principal early erythrocytic marker, in Tg (gata1:DsRed) transgenic zebrafish at 72 hpf. We analyzed the transcriptional level of other erythrocyte-related genes using q-RT-PCR assay. For heme formation, the transcription of alas2, which encodes the key enzyme for heme biosynthesis, was down-regulated after 6:2 FTCA exposure, whereas the transcription of ho-1, which is related to heme degradation, was up-regulated at 48 and 72 hpf. The transcriptional patterns of gata1 and gata2, which are related to erythroid differentiation, differed. At 48 hpf, the mRNA level of gata2 was significantly increased, whereas that of gata1 exhibited no significant changes in any treatment group. At 72 hpf, the expressions of both were down-regulated in a concentration-dependent manner. Taken together, 6:2 FTCA exposure decreased the erythrocyte number and disrupted erythroid differentiation during zebrafish embryonic development. Our results suggest that 6:2 FTCA can cause developmental toxicity in zebrafish embryos, and that FTCAs exhibit greater toxicity than that of PFCAs.

Subchronic Hepatotoxicity Effects of 6:2 Chlorinated Polyfluorinated Ether Sulfonate (6:2 Cl-PFESA), a Novel Perfluorooctanesulfonate (PFOS) Alternative, on Adult Male Mice

The compound 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), an alternative to perfluorooctanesulfonate (PFOS) in the metal-plating industry, has been widely detected in various environmental matrices. However, its hepatotoxicity has yet to be clarified. Here, male mice were exposed to 0.04, 0.2, or 1 mg/kg/day of 6:2 Cl-PFESA for 56 days. Results demonstrated that relative liver weight increased significantly in the 0.2 and 1 mg/kg/day 6:2 Cl-PFESA groups, whereas liver lipid accumulation increased in all 6:2 Cl-PFESA groups. Serum enzyme activities of alanine transaminase and alkaline phosphatase were increased. Serum triglycerides and low-density lipoprotein cholesterol both increased, whereas serum total cholesterol and high-density lipoprotein cholesterol decreased following 6:2 Cl-PFESA exposure. A total of 264 differentially expressed proteins (127 up-regulated and 137 down-regulated), mainly involved in lipid metabolism, xenobiotic metabolism, and ribosome biogenesis, were identified by quantitative proteomics. Bioinformatics analysis highlighted the de-regulation of PPAR and PXR, which may contribute to the hepatotoxicity of 6:2 Cl-PFESA. Additionally, 6:2 Cl-PFESA induced both cell apoptosis and proliferation in the mouse liver. Compared to the overt toxicity of PFOS, 6:2 Cl-PFESA exhibited more-serious hepatotoxicity. Thus, caution should be exercised in the application of 6:2 Cl-PFESA as a replacement alternative to PFOS in industrial areas.

Penetration of PFASs across the blood cerebrospinal fluid barrier and its determinants in humans

Laboratory studies indicate that exposure to perfluoroalkyl and polyfluoroalkyl substances (PFASs) can induce neurobehavioral effects in animals. However, the penetration of PFASs across the brain barrier and its determining factors are yet to be clarified in humans. We studied PFAS levels in 223 matched-pair serum and cerebrospinal fluid (CSF) samples from hospital in-patients using UPLC/MS/MS. Among the 21 target analytes, PFOA, PFOS, and 6:2 Cl-PFESA were dominant in serum, with mean concentrations of 7.4, 6.8, and 6.2 ng/mL, respectively, contributing 79% to the total PFAS burden in serum. In CSF, PFOA, PFOS, and 6:2 Cl-PFESA were again the dominant PFASs, with mean concentrations of 0.078, 0.028, and 0.051 ng/mL contributing 36%, 13%, and 24%, respectively, to the total PFAS burden in CSF. Furthermore, PFAS penetration ( RPFAS, PFASCSF/PFASserum) was positively correlated with the barrier permeability index RAlb (AlbuminCSF/Albuminserum), indicating that barrier integrity was the main determinant of PFAS penetration across the blood-CSF barrier. Positive associations between the RPFAS values of the main PFASs and serum C-reactive protein were observed, implying that inflammation facilitates the penetration of PFASs across the brain barrier.

Elevated concentrations of perfluorohexanesulfonate and other per- and polyfluoroalkyl substances in Baiyangdian Lake (China): Source characterization and exposure assessment

Novel 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) and legacy PFASs, such as perfluorohexane sulfonate (PFHxS), have been used to replace perfluorooctane sulfonate (PFOS), a known persistent organic pollutant. Thus, it is critical to understand these PFOS alternatives regarding their sources and concentrations in the natural environment. In this study, 41 surface water samples as well as edible aquatic organisms were collected from Baiyangdian Lake, the largest freshwater lake in Hebei Province, China. Perfluorooctanoate acid (PFOA) and PFHxS were the predominant PFASs detected in the surface water, reaching concentrations of 8 397.23 ng/L and 1 478.03 ng/L, respectively, with PFHxS accounting for the greatest proportion (∼80.00%) in most water samples. PFHxS (mean: 87.53 ng/g) and PFOS (mean: 35.94 ng/g) were also the most prevalent compounds detected in aquatic organisms. Estimated daily intake (EDI) values of PFOS (16.56 ng/kg bw/d) and PFHxS (16.11 ng/kg bw/d) via aquatic food and drinking water were the highest among PFASs, indicating potential exposure risks to residents. In addition, fish product consumption was the important exposure pathway for residents to PFOA, PFHxS, PFOS, and 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA). This study reports on the highest PFHxS levels ever recorded in surface water, suggesting that further quantification of PFHxS in human serum and assessment of its health risks to local residents are warranted and critical.