Abstract Bisphenol A (BPA) is a chemical compound widely used in manufacturing plastic products. Recent epidemiological studies suggest BPA exposure is positively associated with the incidence of type 2 diabetes mellitus (T2DM), however the mechanisms underlying this link remain unclear. Human islet amyloid polypeptide (hIAPP) is a hormone synthesized and secreted by the pancreatic β-cells. Misfolding of hIAPP into toxic oligomers and mature fibrils can disrupt cell membrane and lead to β-cell death, which is regarded as one of the causative factors of T2DM. To test whether there are any connections between BPA exposure and hIAPP misfolding, we investigated the effects of BPA on hIAPP aggregation using thioflavin-T based fluorescence, transmission electronic microscopy, circular dichroism, dynamic light scattering, size-exclusion chromatography,fluorescence-dye leakage assay in an artificial micelle system and the generation of reactive oxygen species in INS-1 cells. We demonstrated that BPA not only dose-dependently promotes the aggregation of hIAPP and enhances the membrane disruption effects of hIAPP, but also promotes the extent of hIAPP aggregation related oxidative stress. Taken together, our results suggest that BPA exposure increased T2DM risk may involve the exacerbated toxic aggregation of hIAPP.

Citation: Gong H, Zhang X, Cheng B, Sun Y, Li C, Li T, et al. (2013) Bisphenol A Accelerates Toxic Amyloid Formation of Human Islet Amyloid Polypeptide: A Possible Link between Bisphenol A Exposure and Type 2 Diabetes. PLoS ONE 8(1): e54198. https://doi.org/10.1371/journal.pone.0054198 Editor: Angel Nadal, Universidad Miguel Hernández de Elche, Spain Received: October 9, 2012; Accepted: December 11, 2012; Published: January 23, 2013 Copyright: © 2013 Gong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Basic Research Program of China (2009BC918304 & 2012CB524901 to LZ), the Natural Science Foundation of China (Nos. 81222043, 30970607, 81172971 to KH; and Nos. 81100687 & 31271370 to LZ), the Program for New Century Excellent Talents in University (NECT10-0623 to LZ & NECT11-0170 to KH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Diabetes is a panepidemic endocrine disease, with approximately 285 million diagnosed patients worldwide [1]. Non insulin dependent diabetes or type 2 diabetes mellitus (T2DM) accounts for more than 90% of diagnosed diabetes [2]. An important causative factor of T2DM is the misfolding of human islet amyloid polypeptide (hIAPP), which is a 37-residue peptide synthesized and secreted by the pancreatic β-cells (Fig. 1A; [3]). Despite the important physiological functions including glycemic control and regulation of certain hormones [4], hIAPP has a high intrinsic propensity to misfold into toxic oligomers and linear fibrils [5]. During this transition, natively unstructured hIAPP monomers first form β-structure rich oligomers, which further assemble into mature linear fibrils through lateral growth and elongation [6]. The cytotoxicity of hIAPP is generally attributed to the membrane permeabilization ability of hIAPP oligomers and mature fibrils, which cause apoptosis and eventually the onset of diabetes [7]–[10]. Therefore, preventing the formation of toxic hIAPP amyloid has been viewed as a plausible therapeutic approach for T2DM [11]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Structures of hIAPP and BPA. (A) Primary sequence of hIAPP with a disulfide bridge between Cys-2 and Cys-7. (B) Chemical structure of bisphenol A. https://doi.org/10.1371/journal.pone.0054198.g001 Bisphenol A (BPA; Fig. 1B) is a compound widely used in polycarbonate, epoxy resins and other polymer materials for manufacturing plastic utensils. The leach of BPA from plastic products is considered an important environmental issue [12]. Humans are exposed to BPA primarily through oral and inhalation routes [13]. BPA exposure is associated with multiple diseases, such as diseases of the reproductive system, nervous system and sexual dysfunction [14]–[16], as well as increased risk of cancer and heart disease [17], [18]. Although the exact molecular mechanisms of BPA toxicity remain unclear, official policies have been enacted or are being considered in many countries to reduce the BPA exposure worldwide [19]. Recent epidemiological evidence suggests that a concentration dependent correlation exists between BPA exposure and the occurrence of diabetes. BPA levels have been found significantly higher in both diagnosed diabetic and borderline diabetic patients than those of non-diabetic subjects [20]. A strong association between high urinary levels of BPA and diabetes has been identified by studying 3400 residents in China that a 37% increase in the incidence of T2DM being observed in subjects with urinary BPA concentration above 1.43 ng/ml compared with the reference concentration (≤0.47 ng/ml) [21]. In a clinic investigation with 1455 adults, the risk of diabetes in the highest BPA concentration group was 2.43 times higher compared with those in the lowest concentration group [22]. Additionally, severe metabolic disorders of glucose homeostasis and insulin resistance, hallmarkers of T2DM that are directly correlated with impaired pancreatic β-cell function, have been also observed in normal mice exposed to BPA [23], [24]. It is well recognized that environmental factors, including multiple metal ions, polyphenols, fatty acids and certain natural products of small molecule size, can affect the toxic misfolding of hIAPP and may cause diabetes [25]–[29]. We thus hypothesize that BPA exposure may associate with diabetes through promoting the toxic aggregation of hIAPP. To test this hypothesis, the effects of BPA on hIAPP aggregation were investigated in this work.

Materials and Methods Materials Synthetic hIAPP (1–37) was obtained from Genscript Inc. (Piscataway, NJ, USA). Bisphenol A was obtained from Aladdin-reagent (Shanghai, China). Carboxyfluorescein, thioflavin-T (ThT), 2-Oleoyl-1-palmitoyl-sn-glycerol-3-phospho-rac (1-glycerol) sodium salt (POPG) and hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich (St. Louis, USA). INS-1 cells were obtained from the China Center for Type Culture Collection (CCTCC). All other chemicals were of the highest grade available. hIAPP sample preparation For all experiments, hIAPP was freshly dissolved in HFIP and vigorously sonicated for 2 min to homogenize the sample. After a short-spin, the solution was diluted to desired concentration in 25 mM sodium phosphate buffer (pH 7.4) containing 50 mM NaCl, and a final HFIP concentration of 1%. Freshly prepared BPA stock solution was then immediately added to desired concentrations,thoroughly mixed and ready for further analysis. The whole preparation process is strictly limited to 5 min. Far-UV circular dichroism (CD) and data analysis CD spectra were obtained with a JASCO-810 spectropolarimeter at 25°C under a constant flow of N 2 . Freshly dissolved hIAPP was diluted to a final concentration of 15 µM,. Spectra were obtained from 260 to 190 nm with a 2 nm bandwidth, 1 s response time, 50 nm/min scanning speed and a 1 mm pathlength. Each sample was measured at least three times and the spectra were averaged to give the final result. Spectra of PBS buffer containing corresponding concentrations of BPA were measured as the baselines. The final spectra were obtained by subtracting corresponding baseline spectrum from sample spectrum, which were further converted to mean residue ellipticity [θ] and were analyzed with the software CDPro using the CONTINLL algorithm as previously described [30]. Amyloid formation and thioflavin-T (ThT) fluorescence assays Freshly prepared hIAPP solution (15 µM) was incubated at 25°C for amyloid formation in the presence of different molar ratios of BPA. ThT fluorescence assays were preformed on a Hitachi FL-2700 fluorometer to detect the formation of amyloid at designated time points. The final assay solution contains 25 mM PBS (pH 7.4), 50 mM NaCl and 20 µM thioflavin-T [27]. ThT fluorescence was recorded at 482 nm with an excitation wavelength of 450 nm. PBS buffer containing different concentrations of BPA were measured as the controls. All of the experiments were performed at least three times, and the lag times were calculated as we previously described [31]. Transmission electronic microscopy (TEM) The TEM was performed as previously described [32]. Briefly, 5 µl of sample was applied onto a 300-mesh Formvar-carbon coated copper grid. Excess solvent was removed carefully and stained by dropwise addition of 1% freshly prepared uranyl formate followed by air drying. Images were observed under a transmission microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 100 kV. Dye leakage assays POPG was dissolved in chloroform at a concentration of 10 mg/mL. Chloroform was then removed under a stream of N 2 , and samples were dried under vacuum to remove residual chloroform. Multilamellar vesicles were made by mixing dry POPG films with 25 mM PBS (pH 7.4) containing 40 mM carboxyfluorescein. PD-10 columns (Sangon, Shanghai, China) were then used to remove nonencapsulated carboxyfluorescein as previously described [33]. POPG vesicles containing carboxyfluorescein were diluted in 25 mM PBS (pH 7.4) for florescence measurements. hIAPP stock solution was added to POPG vesicles at a final concentration of 1 µM immediately before measurement. The samples were excited at a wavelength of 493 nm, and the emission was detected at 518 nm. The fluorescence signal was recorded for 90 s, POPG vesicles alone were tested as the baseline and the signals of POPG vesicles treated with 0.2% (v/v) Triton X-100 (for complete membrane leakage) were used as the positive control. All measurements were repeated at least three times. Size-exclusion chromatography (SEC) The SEC analysis was performed on a Tosoh TSK GW2000 column (Tokyo, Japan). hIAPP was freshly prepared to a final concentration of 30 µM,mixed with different amounts of BPA, and were immediately injected into a Hitachi L-2000 HPLC system, and the column was eluted with a 20% acetonitrile containing 0.003% TFA at a flow rate of 0.3 ml/min as previously described [34]. Dynamic light scattering (DLS) analysis Dynamic light scattering was performed by using a zeta pals potential analyzer (Brookhaven Instruments, New York, USA). 30 µM hIAPP was measured in a 200 µl cuvette incubated at 37°C with a scattering angle of 90°. The starting time for the very first sample scan was marked as time zero. All of the samples were scanned for three times (4 min/scan) and the mean particle size was recorded and analyzed by the multimodal size distribution (MSD) software. MTT cell toxicity assay Pancreatic INS-1 cells were cultured in 1640 medium containing 10% FBS, 1% sodiumpyruvat, 1% penicillin-streptomycin solution and 50 µM β-mecaptoethanol. And cells were plated in 96-well plates at a density of 5×104 cells/well and incubated at 37°C in 5% CO 2 atmosphere for 24 h. The medium was then replaced with fresh medium containing hIAPP (5 µM) and varied amounts of BPA for 24 h further incubation. Cells treated with BPA or PBS were used as the controls. For MTT assay, cells were co-incubated with 10 µl MTT (5 mg/ml) per well for 4 h. 100 µl formazan buffer was then added to each well and the absorbance was measured at 570 nm [35]. Synergistic effects were analyzed by calculating coefficient of drug interaction (CDI) [36]. Measurement of reactive oxygen species (ROS) INS-1 cells were seeded into 6-well plates and treated with or without 10 µM hIAPP and different ratios of BPA for 12 h. The harvested cells were washed by PBS and incubated in 1640 medium containing 10 µM carboxy-H 2 DCFDA (Beyotime, Shanghai, China) for 20 min at 37°C. The cells were then washed twice with PBS and the levels of ROS were detected by a flow cytometer (Beckman, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm [37]. Statistical analysis The Kruskal-Wallis test and the Mann-Whitney test were used to evaluate statistical significance. All results were expressed as the mean ± SD. Difference was considered statistically significant at P<0.05.

Discussion hIAPP has a strong tendency to form toxic oligomers and fibrils that lead to pancreatic β-cell apoptosis and eventually the onset of T2DM [47]. It is clear from our results that BPA promotes hIAPP aggregation in a dose-dependent manner, which is supported by the significantly accelerated aggregation lag time as well as the enhanced fluorescence intensity that reflects the orderly β-structures formed (Fig. 3B). CD data further confirms the accelerated transition of hIAPP from unordered structure to β-structure in the presence of BPA (Fig. S2). The helical intermediates are thought to play a role in hIAPP aggregation [48], [49], the transition from helix to β-sheet structure may cause the reduction of helical structures as what we observed (Fig. S2). It is well established that the toxic hIAPP oligomers can disrupt the islet β-cell membrane and lead to permeabilization. In MTT study, we observed the cytotoxicity of hIAPP on INS-1 cells rose sharply with the addition of BPA (Fig. 7). The observed strong cytotoxicity by BPA alone also agree with previous studies which suggested BPA itself also disrupt the cell function through stimulating the estrogen-receptor and several other apoptosis-related pathways [50]–[53]. It is interesting to note that the increased toxic effect of hIAPP in combination with BPA was only observed at high BPA to hIAPP ratios but not at the lower ratios. The CDI was calculated to explore the potential BPA and hIAPP interaction, and it was found that the two compounds showed a synergistic exacerbation of cototoxicity especially at high molar ratios of BPA. Further analysis was conducted to distinguish the direct molecular toxic effects of BPA on live cells from its interaction with hIAPP. Dye leakage assays were performed to monitor exclusively the membrane disruption property of hIAPP in the presence of BPA. The hIAPP oligomers have been proven to bind and penetrate membranes more efficiently than monomers and are regarded as an important causative factor to β-cell death [47]. Serious dye leakage was observed in the hIAPP group and was dose-dependently enhanced in the presence of BPA (Fig. 8). It is interesting to note that no evident hIAPP aggregation was identified within a short incubation time at a low concentration (1 µM) as suggested by the ThT and CD results (data not shown), whereas hIAPP at this concentration immediately caused significant membrane disruption in dye leakage assays. This may be explained by a recent report that hIAPP form oligomers much faster in the presence of membrane structures [54]. These data suggested that BPA significantly increases the ability of hIAPP to disrupt membranes. Oxidative stress induced cytotoxicity was another mechanism underlying amyloid-related β-cell apoptosis besides direct membrane disruption. Amyloid formation has been reported to associate with ROS generation [46]. hIAPP oligomers may form pores on membrane and lead to permeabilization of lipid bilayers [55]. The generalized increase in membrane permeability results in intracellular calcium elevation which disrupts mitochondrial function and finally increases ROS generation [56], [57]. In this study, ROS accumulation is also observed in INS-1 cells treated with hIAPP. The ROS levels rose significantly in the presence of BPA and hIAPP, while BPA by itself has little effect on ROS levels (Fig. 8B), suggesting BPA has a synergistic effect on the ROS production related to hIAPP amyloid formation. A summary of possible molecular scheme is provided for the toxic effects of BPA on the formation of hIAPP amyloid which further result in cell damage (Fig. 9). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 9. A schematic representation of hIAPP aggregation pathway. BPA may promote the oligomerization of hIAPP and form pores on membrane which disrupt the membrane and cause the leakage of cellular contents, moreover, permeabilization of lipid bilayers may cause the elevation of intracellular calcium levels and lead to the generation of ROS. https://doi.org/10.1371/journal.pone.0054198.g009 The influence on human health of BPA exposure is regarded as an accumulative process because of its widespread penetration in daily life [58]. The BPA tolerable intake has been set as 50 µg/kg/day [58], but adverse effects at lower BPA concentrations in animal studies have been demonstrated, which may lead to the requirement of a new risk assessment for BPA [59]. BPA concentrations in human blood (serum and plasma) are in the range of 0.3–4.4 ng/ml (1.3–19.4 nM) in developed countries [13]. In contrast, physiological circulating concentrations of hIAPP are below 10 pM in fasted non-diabetic people and rise up to over 20 pM after a meal [60], suggesting the physiological ratio of BPA to hIAPP may actually be much higher than those used in the present study. Moreover, since BPA exposure is a continuous and accumulative process, it is logical to expect long-term BPA exposure may be accompanied with accelerated hIAPP amyloid formation and β-cell apoptosis, and eventually a higher risk of T2DM. In a recent report, BPA at near physiological concentration also showed direct toxicity [61]. Due to the detection sensitivity limitation of existing biophysical technologies, in the present study, we tested the interaction between hIAPP and BPA at much higher concentrations in vitro. Therefore the system we used may be considered as a model that simulates physiological interactions at accelerating rates. Future biophysical study with novel experimental methods which can tackle hIAPP and BPA interaction at physiological conditions will be important. In summary, our data provide the evidence that BPA exposure concentration-dependently accelerates the toxic amyloid formation, exacerbates the toxic membrane disruption of hIAPP and promotes the levels of toxic ROS generated by hIAPP in vitro. Our study suggest that in addition to direct biological effect, long-term BPA exposure may also have adverse effects on hIAPP amyloid formation that eventually contribute to the onset of T2DM. The results may provide a new angle on how BPA exposure influences the risk of diabetes from hIAPP aggregation related pathogenesis. Moreover, since BPA also possess other important biological effects including estrogen-like function, it will be interesting to explore the effect of BPA exposure on physiological functions of hIAPP, for example, hIAPP secretion and hIAPP related insulin resistance. In addition to hIAPP, a great variety of amyloidogenic proteins such as amyloid β peptide and α-synuclein, are known to form extracellular amyloid deposits that induce human diseases including Alzheimer's disease and Parkinson's disease [62]. It will be of future interest to study how BPA exposure may affect the misfolding of those amyloidogenic proteins.

Acknowledgments The authors are grateful to the Huazhong University of Science and Technology Analytical and Testing Center for support. The authors wish to thank Dr. Mitchell Sullivan (University of Queensland) and Mr. Justus Grave (Univeristy of Marburg) for help with the editing. The authors thank two anonymous reviewers for their insightful scientific suggestions.

Author Contributions Conceived and designed the experiments: HG LZ KH. Performed the experiments: HG XZ BC YS CL TL. Analyzed the data: HG LZ KH. Wrote the paper: HG LZ KH.