Nan Sheng

Perfluorooctanoic acid exposure for 28 days affects glucose homeostasis and induces insulin hypersensitivity in mice.

Perfluoroalkyl acids (PFAAs) are widely used in many applications due to their unique physical and chemical characteristics. Because of the increasing prevalence of metabolic syndromes, including obesity, dyslipidemia and insulin resistance, concern has arisen about the roles of environmental pollutants in such diseases. Earlier epidemiologic studies showed a potential association between perfluorooctanoic acid (PFOA) and glucose metabolism, but how PFOA influences glucose homeostasis is still unknown. Here, we report on the modulation of the phosphatidylinositol 3-kinase-serine/threonine protein kinase (PI3K-AKT) signaling pathway in the livers of mice after 28 d of exposure to PFOA. Compared with normal mice, PFOA exposure significantly decreased the expression of the phosphatase and tensin homologue (PTEN) protein and affected the PI3K-AKT signaling pathway in the liver. Tolerance tests further indicated that PFOA exposure induced higher insulin sensitivity and glucose tolerance in mice. Biochemical analysis revealed that PFOA exposure reduced hepatic glycogen synthesis, which might be attributed to gluconeogenesis inhibition. The levels of several circulating proteins were altered after PFOA exposure, including proteins potentially related to diabetes and liver disease. Our results suggest that PFOA affected glucose metabolism and induced insulin hypersensitivity in mice.

Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

Abstract Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans.

Toxic effects of perfluorononanoic acid on the development of Zebrafish (Danio rerio) embryos

Perfluorononanoic acid (PFNA) is a nine-carbon perfluoroalkyl acid widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. There is a concern that this chemical might be a developmental toxicant and teratogen in various ecosystems. In the present study, the toxic effects of PFNA were evaluated in zebrafish (Danio rerio) embryos. One hour post-fertilization embryos were treated with 0, 25, 50, 100, 200, 300, 350, and 400 μmol/L PFNA for 96 hr in 6-well plates. Developmental phenotypes and hatching rates were observed and recorded. Nineteen genes related to oxidative stress and lipid metabolism were examined using Quantitative RT-PCR and confirmed by whole mount in situ hybridization (WISH). Results showed that PFNA delayed the development of zebrafish embryos, reduced the hatching rate, and caused ventricular edema and malformation of the spine. In addition, the amount of reactive oxygen species in the embryo bodies increased significantly after exposure to PFNA compared with that of the control group. The Quantitative RT-PCR and WISH experiments demonstrated that mRNA expression of the lfabp and ucp2 genes increased significantly while that of sod1 and mt-nd1 decreased significantly after PFNA exposure. The mRNA expression levels of gpx1 and mt-atp6 decreased significantly in the high concentration group. However, the mRNA expression levels of both ppara and pparg did not show any significant variation after exposure. These findings suggest that PFNA affected the development of zebrafish embryos at relatively low concentrations.

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.

Zebrafish reproductive toxicity induced by chronic perfluorononanoate exposure

Perfluoroalkyl acids (PFAAs) are a group of anthropogenic compounds that have been widely used in consumer products for over 50 years. One of the most dominant PFAAs is perfluorononanoate (PFNA), a compound detected ubiquitously in aquatic ecosystems. While PFNA is suspected of being an endocrine disruptor, the mechanisms behind PFNA-induced reproductive disorders are poorly understood. The aim of this study was to investigate the reproduction-related effects and possible mechanisms of PFNA on adult zebrafish (Danio rerio) following 180 days of exposure at different concentrations (0.01, 0.1, 1mg/L). PFNA concentration in the gonads of zebrafish was tested by HPLC-MS/MS after chronic exposure to study possible inconsistent accumulation between the genders. The results showed that the accumulation of PFNA in the male gonads was almost one-fold higher than that in the female gonads, indicating a possible higher PFAA gonad burden for male zebrafish. Significant reductions in the male gonadosomatic index (GSI) and female egg production were observed. In addition, the decreased 72h hatching rate displayed an evident dosage effect, indicating that maternal exposure to PFNA might impair offspring developmental success. To investigate how PFNA exposure affects the hypothalamic-pituitary-gonadal-liver axis (HPGL axis), the transcriptional levels of genes were measured by real-time PCR. The disrupted expression of genes, such as ERα, ERβ, FSHR, LHR, StAR, and 17βHSD, indicated the possible interference of PFNA on the HPGL axis function and sex hormone synthesis. Furthermore, testosterone (T) and estradiol (E2) levels in serum and VTG content in the liver were detected to clarify the influences of PFNA on sex hormone levels. Except for the increase in serum estrogen levels, as an estrogen analogue, PFNA also induced the synthesis of biomarker protein vitellogenin (VTG) in the adult male liver. The results of this study indicate that chronic exposure to PFNA can lead to dysfunction in the HPGL axis and sex hormone synthesis and cause adverse effects on fish reproduction.

RNA-sequencing analysis reveals the hepatotoxic mechanism of perfluoroalkyl alternatives, HFPO2 and HFPO4, following exposure in mice.

The toxicological impact of traditional perfluoroalkyl chemicals has led to the elimination and restriction of these substances. However, many novel perfluoroalkyl alternatives remain unregulated and little is known about their potential effects on environmental and human health. Daily administration of two alternative perfluoroalkyl substances, HFPO2 and HFPO4 (1 mg kg−1 body weight), for 28 days resulted in hepatomegaly and hepatic histopathological injury in mice, particularly in the HFPO4 group. We generated and compared high‐throughput RNA‐sequencing data from hepatic tissues in control and treatment group mice to clarify the mechanism of HFPO2 and HFPO4 hepatotoxicity. We identified 146 (101 upregulated, 45 downregulated) and 1295 (716 upregulated, 579 downregulated) hepatic transcripts that exhibited statistically significant changes (fold change ≥2 or ≤0.5, false discovery rate < 0.05) after HFPO2 and HFPO4 treatment, respectively. Among them, 111 (82 upregulated, 29 downregulated) transcripts were changed in both groups, and lipid metabolism associated genes were dominant. Thus, similar to their popular predecessors, HFPO2 and HFPO4 exposure exerted hepatic effects, including hepatomegaly and injury, and altered lipid metabolism gene levels in the liver, though HFPO4 exerted greater hepatotoxicity than HFPO2. The unregulated use of these emerging perfluoroalkyl alternatives may affect environmental and human health, and their biological effects need further exploration. Copyright

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.