PFOA and PFOS Disrupt the Generation of Human Pancreatic

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Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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PFOA and PFOS Disrupt the Generation of Human Pancreatic Progenitor Cells Shuyu Liu,†,‡ Nuoya Yin,†,‡ and Francesco Faiola*,†,‡ †

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China ‡ College of Resources and Environment, University of Chinese Academy of Sciences, Beijing, 100049, China S Supporting Information *

ABSTRACT: The two most infamous perfluoroalkyl acids, PFOA and PFOS, are persistent, widespread, bioaccumulative, and associated with many health issues. However, knowledge about their potential toxicity to the developing pancreas or their association with metabolic diseases, such as diabetes, in humans is limited. In this study, we investigated the effects of PFOA and PFOS on the in vitro generation of pancreatic progenitor cells from human embryonic stem cells, a process that mimics in vivo organogenesis. We demonstrated for the first time that these two pollutants significantly affect the early stages of human pancreatic progenitor cell specification, even at very low environmentally and human-relevant doses. Thus, our findings further emphasize the substantial health risks associated with prenatal exposure to PFOA and PFOS.

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pancreas was a target organ.45 Moreover, high doses of PFOA gave rise to focal ductal hyperplasia, which could further lead to dysfunctional sugar metabolism. Nevertheless, all treatments induced oxidative stress in the pancreas.45 Similarly, in rat pancreatic B-cell-derived RIN-m5F cells, PFOA exposure caused oxidative stress via the generation of reactive oxygen species and reactive nitrogen species and impaired mitochondrial functioning.46 Zebrafish continuously exposed to 32 μM PFOS from 3 to 48 hpf had reduced beta cell mass and small islet area.47 Sant and colleagues also found that PFOS exposure perturbed overall pancreatic organogenesis, suggesting a potential risk for type I diabetes.48 In contrast, some studies also demonstrated that PFC exposure might not affect pancreatic functions at all. For instance, 6 months of oral PFOA administration in male cynomolgus monkeys did not result in any notable pancreatic toxicity.49 However, given that animal tests have shown elevated insulin resistance after exposure to PFOA or PFOS,50−52 it is urgently necessary to shed light on the potential pancreatic toxicity of PFOA and PFOS. As Sant et al. noted, pancreatic development is not easy to track in mammals.48 However, pancreatic induction from human embryonic stem cells (hESCs) is a promising model for the study of and development of treatments for diabetes, and it can certainly be utilized for the assessment of the health

INTRODUCTION Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), the two most common perfluorinated compounds (PFCs), are widely distributed in natural environments and accumulate in organisms.1−3 Considering that their halflives in the human body are on the order of years,4−6 it is necessary to study the long-term health impacts of PFOA and PFOS. Several epidemiological studies have already correlated maternal PFOA and PFOS exposure with birth defects, health conditions, and diseases (reviewed in refs 7 and 8). Moreover, because of their high detection rate in human body fluid samples, especially umbilical cord blood,9−18 amniotic fluid,13,18 and breast milk,19−22 PFOA and PFOS have been of concern for their potential toxic effects on embryonic development. Studies addressing exposure to PFOA and PFOS during development have reported perturbations of cardiogenesis and liver specification as well as delayed embryogenesis and defects in offspring.23−34 In addition, PFOS exposure may also cause altered microRNA expression,35 disturbed metabolic responses,36−38 excessive immune responses, and overactivated AhR, ER, and PPAR signaling.39 Nonetheless, although cohort analyses have identified a link between exposure to PFOA and PFOS and the occurrence of diabetes, dysfunctions in sugar metabolism, and insulin secretion,40−44 only a few studies have provided insightful evidence of the potential health impacts of PFOA and PFOS on the pancreas. PFOA has been shown to accumulate in a dosedependent manner in the pancreases of C57Bl/6 mice fed 0.5, 2.5, or 5.0 mg/kg BW/day PFOA for 7 days, implying that the © XXXX American Chemical Society

Received: April 9, 2018 Accepted: April 12, 2018 Published: April 12, 2018 A

DOI: 10.1021/acs.estlett.8b00193 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Letters

Figure 1. Generation of pancreatic progenitors from H9 hESCs was compromised by PFOA or PFOS. (A) Expression levels of the two pancreatic progenitor-specific transcription factors, Pdx1 and Sox9, were down-regulated in groups treated with PFOA (left panel) and PFOS (right panel). The average of three independent biological experiments in triplicate is shown and is expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All genes were normalized to Gapdh. (B) Immunofluorescence staining for PDX1 in pancreatic progenitors on day 13. The representative images from three independent biological replicates are shown. DAPI was used to stain the DNA of all the cells.

Stem Cell Bank c/o the WiCell Research Institute and maintained in mTeSR1 medium (STEMCELL Technologies, 85850) on vitronectin (Gibco, A14700)-coated tissue culture dishes. For differentiation, hESCs were dissociated with TrypLE Select Enzyme (Gibco, A1285901) and seeded onto 24-well plates (one well per condition) coated with Matrigel (Corning, 354248) in mTeSR1 medium containing 1X RevitaCell Supplement (Gibco, A2644501). When the cells reached 90% confluence, they were differentiated into pancreatic progenitors using a STEMdiff Pancreatic Progenitor kit (STEMCELL Technologies, 05120), according to the instructions from the manufacturer and treated with 0, 5, or 50 nM PFOA or PFOS throughout the 13-day differentiation process. hESCs were always maintained and differentiated in 5% CO2 at 37 °C. For biological repeats, the differentiation experiments were performed on different days. Alamar Blue-Based Cell Viability Test. hESC-derived pancreatic progenitor cell viability was determined after treatment with different concentrations of PFOS and PFOA by the Alamar Blue assay. Briefly, after three washes with DPBS, the cells were incubated with 10 μM Alamar Blue, and after 2 h of incubation at 37 °C, the fluorescence values were measured with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The assays were performed in 96-well

impacts of chemicals on both pancreatic development and pancreatic cells. In this study, we derived human pancreatic progenitor cells from hESCs to study the effects of PFOA and PFOS exposure on the early specification of the pancreas. The changes in expression of lineage-specific markers demonstrated that even at doses as low as 5 nM, PFOA and PFOS had deleterious effects on the generation of pancreatic progenitors. To the best of our knowledge, these findings represent the first evaluation of the toxic effects of PFOA and PFOS on pancreatic development and the health risks associated with prenatal and neonatal exposure to PFOA and PFOS based on an in vitro human system and serve as a significant corroboration of the results of early epidemiological studies.

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MATERIALS AND METHODS Chemicals. PFOA and PFOS were purchased from SigmaAldrich (PFOA: 171468; PFOS: 77282). Stock solutions were generated by dissolving PFOA and PFOS in DMSO to achieve a final concentration of 100 mM. The DMSO concentration in the cell culture medium was always 0.005% for both the chemical treatment and control groups. Maintenance and Pancreatic Differentiation of hESCs. Human H1 and H9 ESCs were purchased from the National B

DOI: 10.1021/acs.estlett.8b00193 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

Letter

Environmental Science & Technology Letters

Figure 2. PFOA and PFOS treatments reduced the expression of early markers of pancreatic progenitor specification in H9 hESCs. (A) Expression levels of hepatocyte nuclear factors upstream of Pdx1 were suppressed in samples treated with PFOA (left panel) and PFOS (right panel). (B) Endodermal marker genes were down-regulated in samples treated with PFOA (left panel) and PFOS (right panel). For all genes in (A) and (B), the expression in the treated groups was normalized to that of the DMSO solvent control (0 nM), which was set as 1. The averages of three independent experiments in triplicate are shown and are expressed as the means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All genes were normalized to Gapdh.

and liver. Later, pancreatic organogenesis first deviates away from the anterior foregut that will give rise to the lungs53 and then diverges from the region that will form the liver in the posterior foregut.54 Therefore, the pancreas, liver, and lungs are closely related during organogenesis. Given that PFOA suppressed pulmonary development32 and both PFOA and PFOS inhibited hepatic specification,28,55−57 we suspected that the influence of those two chemicals on the pancreas might occur as early as the formation of progenitor cells. PFOA and PFOS levels in human umbilical cord blood, breast milk, and amniotic fluid vary significantly among populations.9−19,21,22,58 To better represent the exposure conditions in utero, we chose two PFOA and PFOS concentrations (5 and 50 nM) for our differentiation assays. Previous studies suggested that PFOA and PFOS may inhibit the process of pancreatic organogenesis45,47,48 but did not elucidate the exact time window during which PFOA and PFOS might exert pancreatic developmental toxicity. Moreover, since both PFOA and PFOS have long half-lives in the human body,4−6 it is possible that embryos are continuously exposed to PFOA and PFOS from the maternal umbilical cord blood. Taking all the above into consideration, we initiated the chemical treatment with the induction of differentiation and continued it for 13 days until pancreatic progenitor cells were generated (Figure S1A). To determine the effects of PFOA and PFOS on the efficiency of the generation of pancreatic progenitor cells, we monitored the expression of the pancreatic master regulator PDX1, which is the transcription factor required for pancreatic development.59 PFOA and PFOS significantly suppressed the expression of Pdx1 during the induction procedure at both the mRNA (Figure 1A) and protein (Figure 1B) levels. Since Pdx1 is also expressed in the tissues adjacent to the pancreatic buds,

plates; each biological repeat was performed in sextuplicate on different days. qRT-PCR analysis. Total RNA was extracted with the TRIzol reagent (Life Technologies, 15596018). Reverse transcription was performed by using TAKARA RT MasterMix (RR036A), and qPCR was performed with TAKARA SYBR Green PreMix (RR042A) on a QuantStudio 6 Flex System (Life Technologies, USA). Each qPCR assay was performed in triplicate. Gapdh and β-actin were used as reference genes. A list of the primer sets used is provided in Table S1. Immunofluorescence. Cells were fixed for 15 min at room temperature with 4% paraformaldehyde in DPBS, washed with DPBS, and blocked for 1 h with 5% goat serum (Solarbio, SL038) in DPBS containing 0.1% Triton X-100 (Amresco, 0694−1L). The antibodies were diluted in DPBS containing 1% BSA and 0.1% Triton X-100. The cells were incubated with primary antibodies overnight at 4 °C and then with the secondary antibodies for 1 h at room temperature. The antibodies used included the following: rabbit anti-PDX1 (1:400, Cell Signaling Technology, 5679), rabbit anti-SOX9 (1:400, Cell Signaling Technology, 82630), and Alexa 488conjugated secondary antibody (1:1000, Cell Signaling Technology, 4340S). Statistical Analysis. Standard deviation (SD) was used to determine the statistical significance. Mean differences were tested for statistical significance by nonparametric one-way ANOVA.

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RESULTS AND DISCUSSION

PFOA and PFOS Significantly Impaired the Specification of Pancreatic Progenitor Cells in Vitro at Low, Human-Relevant Doses. During embryogenesis, the pancreas originates from the definitive endoderm, as do the lungs C

DOI: 10.1021/acs.estlett.8b00193 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Letters such as the antral stomach and rostral intestine,60 we additionally investigated the expression of Sox9, which, together with Pdx1, can discriminate between pancreatic progenitors and other cell types.61 The qRT-PCR analyses shown in Figure 1A demonstrated that the expression of Sox9 was also impaired after exposure to the two chemicals, confirming that the formation of pancreatic precursor cells was delayed by exposure to PFOA or PFOS. PFOA and PFOS Treatment May Disturb Pancreatic Organogenesis at Stages as Early as Definitive Endoderm Specification. To dissect the earliest stages at which PFOA and PFOS exert adverse effects during pancreatic specification, we examined a group of hepatocyte nuclear factors (HNFs), namely, HNF1b, HNF4a, and HNF6, which together orchestrate the proper expression of PDX1 and distinguish the PDX1-positive posterior foregut.62 As depicted in Figure 2A and Figure S3, exposure to PFOA and PFOS repressed the expression of those HNF factors, implying that the alteration of pancreatic development caused by exposure to PFOA and PFOS may be manifested at early stages, before the pancreatic buds are distinctively formed. To further investigate the timing of the action of PFOA and PFOS during the generation of hESC-derived pancreatic progenitors, we inspected the expression of specific definitive endoderm transcription factors, such as Foxa1, Foxa2, Sox7, and Sox17, which are expressed as early as during gastrulation,63 yet are still present in pancreatic progenitors.64 Our data showed that they were down-regulated in PFOA- and PFOS-treated samples compared to control samples treated with DMSO solvent (Figure 2B and Figure S3), suggesting potential adverse effects of PFOA and PFOS not only on pancreas organogenesis at stages as early as definitive endoderm formation but also on all other endoderm lineages. To exclude cytotoxicity as an explanation for the effects observed on pancreatic differentiation, we measured the cell viability of differentiating hESCs treated with PFOA, PFOS, or DMSO solvent for 13 days. No significant differences in cell viability were observed among the different treatment groups (Figures S1B−C). These results imply that prenatal exposure to PFOA and PFOS does not cause cytotoxicity during the in vivo process of embryonic pancreatic development and agree with a previous study based on mouse pancreatic cells.46 To confirm the effects of PFOA and PFOS observed during the pancreatic differentiation of H9 hESCs, we performed the same pancreatic developmental toxicity assays with another hESC cell line, H1. As depicted in Figure S2, the two pollutants decreased the mRNA levels of all measured pancreatic markers (Figure S2A), as well as the protein levels of PDX1 (Figure S2B), as seen during H9 hESC-directed differentiation. Pancreatic progenitor cells generate all kinds of pancreatic cells, including endocrine, exocrine, and ductal cells.53,54 The perturbations of the generation of pancreatic precursors caused by PFOA and PFOS suggest that those two pollutants might compromise the formation of the mature pancreas, which would result in increased risks for the occurrence of type I diabetes. However, only a few studies have addressed the implications of compromised glucose metabolism due to prenatal exposure to those pollutants.50−52 Therefore, our findings argue in favor of a thorough evaluation of the association between maternal PFOS and PFOA exposure and the risk of developing diabetes. In conclusion, our study is, to the best of our knowledge, the first one to address the impacts of PFOA and PFOS on the

development of the human pancreas. We demonstrated that these two pollutants affect the expression of several transcription factors crucial or important for the generation of pancreatic progenitors, and the results of this study are generally in agreement with the conclusions drawn by previous studies in animal models.45,47,48 However, the overall influence of PFOA and PFOS on the formation of mature pancreatic cells, especially B cells, remains unclear. Therefore, follow-up studies should be performed to comprehensively evaluate the toxic effects of PFOA and PFOS, as well as other emerging contaminants, on the development of the pancreas.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.8b00193. Figures S1−S3 and Table S1: Schematic of induction and pollutant treatments, phase-contrast morphology images of pancreatic progenitor cells, H1 hESCs-based toxicity data, and H9 hESC RT-qPCR data normalized to β-actin. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Francesco Faiola. E-mail: [email protected]. Tel/Fax: 86 1062917609. ORCID

Francesco Faiola: 0000-0002-3512-8253 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the following: the National Natural Science Foundation of China (21577166 and 21461142001), the Chinese Academy of Sciences Strategic Priority Research Program (XDB14040301), the Chinese Academy of Sciences Hundred Talent Program (29[2015] 30), and the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-DQC017). We would also like to thank the K. C. Wong Education Foundation.

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REFERENCES

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DOI: 10.1021/acs.estlett.8b00193 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.estlett.8b00193 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX