Science of the Total Environment 548–549 (2016) 317–324

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Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos? Susanne Keiter a,⁎, Kathleen Burkhardt-Medicke b,e, Peggy Wellner b, Britta Kais a, Harald Färber c, Dirk Skutlarek c, Magnus Engwall d, Thomas Braunbeck a, Steffen H. Keiter d, Till Luckenbach b,⁎ a

Aquatic Ecology and Toxicology Group, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 504, D-69120 Heidelberg, Germany Department Bioanalytical Ecotoxicology, UFZ-Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig, Germany Institute for Hygiene and Public Health, University of Bonn, Sigmund-Freudstr. 25, D-53127 Bonn, Germany d Man-Technology-Environment Research Centre (MTM), Department of Natural Science, University of Örebro, Fakultetsgatan 1, S-701 12 Örebro, Sweden e Institute of Hydrobiology, Dresden University of Technology, D-01062 Dresden, Germany b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• We evaluated the MXR-modifying potential of PFOS in zebrafish embryos. • The uptake and toxicity of two standard abcb4 substrates were measured. • PFOS demonstrated a strong chemosensitizing effect.

a r t i c l e

i n f o

Article history: Received 30 October 2015 Received in revised form 19 December 2015 Accepted 19 December 2015 Available online 21 January 2016 Editor: D. Barcelo Keywords: Abcb4 Chemosensitization Environment-tissue barrier Multixenobiotic resistance (MXR) Perfluorooctane sulfonate P-glycoprotein Zebrafish embryo

a b s t r a c t Earlier studies have shown that perfluorooctane sulfonate (PFOS) increases the toxicity of other chemicals by enhancing their uptake by cells and tissues. The present study aimed at testing whether the underlying mechanism of enhanced uptake of chemicals by zebrafish (Danio rerio) embryos in the presence of PFOS is by interference of this compound with the cellular efflux transporter Abcb4. Modifications of uptake/clearance and toxicity of two Abcb4 substrates, the fluorescent dye rhodamine B (RhB) and vinblastine, by PFOS were evaluated using 24 and 48 h post-fertilization (hpf) embryos. Upon 90 min exposure of 24 hpf embryos to 1 μM RhB and different PFOS concentrations (3–300 μM) accumulation of RhB in zebrafish was increased by up to 11.9-fold compared to controls, whereas RhB increases in verapamil treatments were 1.7-fold. Co-administration of PFOS and vinblastine in exposures from 0 to 48 hpf resulted in higher vinblastine-caused mortalities in zebrafish embryos indicating increased uptake of this compound. Interference of PFOS with zebrafish Abcb4 activity was further studied using recombinant protein obtained with the baculovirus expression system. PFOS lead to a concentrationdependent decrease of the verapamil-stimulated Abcb4 ATPase activity; at higher PFOS concentrations (250, 500 μM), also the basal ATPase activity was lowered indicating PFOS to be an Abcb4 inhibitor. In exposures of 48 hpf embryos to a very high RhB concentration (200 μM), accumulation of RhB in embryo tissue and adsorption to the chorion were increased in the presence of 50 or 100 μM PFOS. In conclusion, the results indicate that PFOS

⁎ Corresponding authors. E-mail addresses: [email protected] (S. Keiter), [email protected] (T. Luckenbach).

http://dx.doi.org/10.1016/j.scitotenv.2015.12.089 0048-9697/© 2015 Elsevier B.V. All rights reserved.

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acts as inhibitor of zebrafish Abcb4; however, the exceptionally large PFOS-caused effect amplitude of RhB accumulation in the 1 μM RhB experiments and the clear PFOS effects in the experiments with 200 μM RhB suggest that an additional mechanism appears to be responsible for the potential of PFOS to enhance uptake of Abcb4 substrates. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Anthropogenic chemicals are usually present as mixtures in the environment. The bioactive potentials of single components can add up in chemical mixtures, which can thus cause toxic stress in organisms at levels exceeding the combined action predicted from the effects of single compounds. With respect to chemical interactions, the importance of mixture toxicity approaches has gained increasing awareness, since single component assessments run the risk of underestimating the true hazard potential (Backhaus and Faust, 2012). Indeed, numerous studies have demonstrated the potential of environmental contaminants to produce synergistic toxic effects when combined with other compounds both in vivo (e.g., Caldwell et al., 2011; Norgaard and Cedergreen, 2010; Xu et al., 2011) and in vitro (e.g., Hu et al., 2003; Jernbro et al., 2007). One mechanism behind the combined action of chemical mixtures is so-called “chemosensitization”, which refers to the inhibition of cellular efflux transporter proteins that act as a biochemical barrier against cellular uptake of toxic compounds by certain chemicals (Kurelec, 1997). These efflux transporters act on a wide range of chemical compounds, thus conferring so called “multixenobiotic resistance” (MXR) of organisms against chemicals dissolved in water (Bard, 2000; Kurelec, 1992). Chemosensitization may be a common effect of anthropogenic pollutants and can even be of relevance for compounds that, according to conventional environmental risk standards, are considered as innocuous (Epel et al., 2008; Kurelec, 1997). Perfluorinated Alkyl Substances (PFAS) represent a group of amphiphilic surface-active chemicals of different carbon chain lengths, which have been ubiquitously detected in aquatic systems on a global scale (Giesy and Kannan, 2001; Stock et al., 2007; Taniyasu et al., 2003; Yeung et al., 2009). PFOS and PFOS-related chemicals have attracted most attention as these compounds commonly constitute a high proportion of environmental contaminations with PFAS (Jernbro et al., 2007; Martin et al., 2004). For example, measured PFOS concentrations in water samples of European rivers ranged between 2 and 193 ng/L, and for river water from Japan between 5 and 10 ng/L (Kwadijk et al., 2010; Pistocchi and Loos, 2009; Senthilkumar et al., 2007; Skutlarek et al., 2006). Various studies have demonstrated that PFOS can modulate the toxicity of other chemicals. Hu et al. (2003) showed in vitro that in the presence of PFOS the chemicals 2,3,7,8-tetrachlorodibenzo-p-dioxin or 17β-estradiol occupied more of the respective intracellular receptors, indicating that PFOS caused more of those molecules to enter the cell. Along these lines, Jernbro et al. (2007) observed increased genotoxicity of cyclophosphamide in vitro when co-administered with PFOS. Moreover, PFOS increased algal growth inhibition by pentachlorophenol, whereas algal growth inhibition of atrazine and diuron was depressed by PFOS (Liu et al., 2009). The mechanisms underlying the synergistic actions of combinations of PFOS with other compounds are still unknown; however, the membrane fluidity-altering potential of PFOS (Hu et al., 2003; Liao et al., 2014; Liu et al., 2008; Matyszewska et al., 2008) has been hypothesized to increase the cell membrane permeability, thus leading to an increased cellular accessibility to other compounds (Hu et al., 2003). Alternatively, the toxicity-enhancing effect of PFAS could be due to chemosensitization, i.e., efflux transporter inhibition. Evidence for this PFAS effect came from a study by Stevenson and co-workers (2006), who demonstrated that when rhodamine B (RhB), a fluorescent dye kept out of cells by efflux transporters, is co-administered with certain PFAS, accumulation of this dye increases in gill tissues of the marine mollusk Mytilus californianus, indicating inhibited efflux transporter activity. In recent years, it has become increasingly clear that environmental

pollutants of different origin and structure can disturb the MXR defense mechanism allowing previously exported chemicals to enter the cell and accumulate (Epel et al., 2008). Environmental pollutants with such chemosensitizing behavior have been suggested to be top-ranked among hazardous chemicals given their potential to inhibit basic biological defense systems (Smital and Kurelec, 1998). In the present study, PFOS was studied for chemosensitizing effects on efflux transporters in embryos of the zebrafish (Danio rerio), which show efflux transporter activity mediated by the Abcb4 transporter (Fischer et al., 2013). Zebrafish Abcb4 is a P-glycoprotein (P-gp) that similarly to human ABCB1 confers multidrug or multixenobiotic resistance (MDR or MXR) by keeping a wide range of toxic compounds out of the cell (Fischer et al., 2013). Chemosensitizers affecting Abcb4 activity thus lead to increased uptake of chemicals by zebrafish embryos that are usually kept out by this transporter resulting in higher sensitivity of the embryos to the toxic impact of these chemicals (Fischer et al., 2013). Using the fluorescent dye rhodamine B (RhB) as proxy for efflux transporter activity in zebrafish embryos, PFOS was studied for effects on accumulation of this dye in embryo tissue. The compound was further tested for modifying embryo toxicity of vinblastine, a toxic efflux transporter substrate, and for efflux transporter interaction using a specific assay with recombinant zebrafish Abcb4. Moreover, to determine the inhibitory effect of PFOS on efflux transporter in zebrafish embryos the uptake of 200 μM RhB in the presence of PFOS was measured. RhB at this concentration is above the level that can be effectively eliminated by transporter activity, and thus efflux transporter inhibition should not have an effect on the overall RhB uptake. 2. Material and methods 2.1. Chemicals Ascorbic acid was from Applichem (Darmstadt, Germany). Perfluorooctanesulfonic acid potassium salt (PFOS; CAS no. 2795-39-3; ≥98% purity), vinblastine sulfate salt (CAS no. 143-67-9; ≥96% purity), verapamil hydrochloride (CAS Number 152-11-4; ≥99% purity), rhodamine B (RhB; CAS no. 81-88-9) and all other reagents used in the experiments were purchased from Sigma-Aldrich (Deisenhofen, Germany). Stock solutions of RhB were freshly prepared in double-distilled water and kept dark. Stock solutions of PFOS, verapamil and vinblastine were prepared in dimethyl sulfoxide (DMSO); the DMSO concentration in the final test solutions did not exceed 0.1%. Concentrations of PFOS in the experiments were kept below the solubility reported for freshwater of 370 mg/L (≈690 μM) (EPA, 2012). 2.2. Maintenance of zebrafish and egg production Maintenance of adult zebrafish and egg production were according to standard protocols (e.g., Kimmel et al., 1995; Kimmel et al., 1988; Lammer et al., 2009; Spence et al., 2006; Wixon, 2000). Fish maintenance and breeding at the University of Heidelberg and at the UFZHelmholtz Centre for Environmental Research were authorized by local authorities under registration nos. 35-9185.64/BH Braunbeck and 75-9185.64, respectively, and were based on the guidelines on the protection of experimental animals by the Council of Europe, Directive 2010-63-EU, which allows zebrafish embryos to be used up to the moment of independent feeding (approximately 5 days after fertilization). Experiments with zebrafish embryos were performed in reconstituted water as specified in ISO 7346-1 (ISO, 1996; 294.0 mg/L CaCl2 × 2

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H2O; 123.3 mg/L MgSO4 × 7 H2O; 63.0 mg/L NaHCO3; 5.5 mg/L KCl). Prior to use, the artificial water was aerated to oxygen saturation. 2.3. Standard fish embryo toxicity test (FET) Lethal and teratogenic potentials of PFOS were evaluated in a 48 h range-finding test with the zebrafish embryo according to DIN 38415T6 and ISO 15088 (DIN, 2001; ISO, 2007) as described by Lammer et al. (2009). The range-finding test was conducted at a minimum of three times on separate days with embryos of different batches. Ten eggs per replicate and treatment were evaluated. The standard FET was also conducted to evaluate the lethal toxicity of vinblastine, a vinca alkaloid and substrate of zebrafish Abcb4 (Fischer et al., 2013) in the absence or presence of PFOS. Freshly spawned eggs were incubated in reconstituted water with either vinblastine (0.5–3 μM), PFOS (21 μM) or with different binary mixtures of the two compounds along with controls without chemicals. The PFOS concentration of 21 μM was found to be non-toxic in the rangefinding FET (see above). Artificial water alone served as a negative control. After 48 h of incubation, lethal effects as documented by coagulation or lack of heart beat were recorded in ten embryos per treatment group. Each experiment was repeated four times on separate days with embryos from different batches. 2.4. Rhodamine B dye accumulation and clearance assays with zebrafish embryos 2.4.1. RhB accumulation experiments Accumulation of the fluorescent Abcb4 substrate RhB in the absence or presence of PFOS or the standard transporter inhibitor verapamil was used as a measure of efflux transporter activity in zebrafish embryos. The assay indicates inhibition of efflux transporter activity in zebrafish embryos when uptake of RhB dye by embryo tissue is higher than compared to controls containing RhB, but no test compound. The assay was performed according to Fischer et al. (2013) with slight modifications: For exposures, ten 24 hpf old embryos were incubated in artificial water with 1 μM RhB either together with PFOS (3 μM, 30 μM, 100 μM, 300 μM), verapamil (2.5 μM, 10 μM) or solvent only (DMSO, 0.1%) as control. For each embryo, 100 μl test solution was used. After several washing steps with clean artificial water to remove RhB from the chorion, eggs were transferred to FastPrep tubes (MP Biomedicals) and homogenized in 200 μl lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris HCl, pH 7.4). The homogenates were briefly centrifuged, 130 μL of the supernatant were transferred to a black 96-well microplate (Nunc, Roskilde, Denmark) and the RhB fluorescence was measured at 595 nm (emission) and 530 nm (excitation) in a GENios plus fluorescence plate reader (Tecan, Crailsheim, Germany). In each experiment, triplicates were set up for controls and each treatment. Experiments were repeated at least twice on separate days with embryos from different batches. 2.4.2. RhB clearance experiments Further experiments were performed to assess as to which degree PFOS prevents RhB depuration from embryo tissue after embryos had been exposed to RhB. Thirty 24 hpf zebrafish embryos per control or treatment were incubated in artificial water with RhB (0.5 μM) and PFOS (20–100 μM). After 1 h of incubation, all embryos were washed, and RhB fluorescence was immediately measured in ten embryos (“1 h RhB/no post-incubation”). The remaining 20 embryos were divided into groups of ten and further incubated for 1 h in either clean artificial water [“1 h RhB/post-incubation (clean water)”] or in PFOS [“1 h RhB/post-incubation (PFOS)”] at respective concentrations (20– 100 μM) and, subsequently, RhB fluorescence was measured. Incubations were performed in 24-well plates and embryos were kept in self-made nylon mesh baskets (mesh size 100 μm; inner core diameter 10 mm) with five embryos per basket and overall six baskets per

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treatment. For washes or changes of exposure solutions, baskets were transferred to new wells containing the respective solutions. This setup facilitated the quick transfer of embryos to new solutions. Embryos were sonicated in 200 μl hypotonic lysis buffer (2 × 10 s) in 1.5 ml Eppendorf tubes. The sonicates were briefly centrifuged and the RhB fluorescence was measured in 150 μl supernatant as above. The experiment was performed twice on separate days with embryos from different batches. Dye accumulation in the treatments was quantified as fold increase over controls. In RhB accumulation experiments the amount of RhB per embryo was quantified based on a standard curve (Fig. S1). 2.4.3. Test for PFOS effect on RhB fluorescence In order to investigate the auto-fluorescence of PFOS and its potential influence on the fluorescence intensity of RhB, fluorescence of artificial water with PFOS (100 and 1000 μM) was measured at gain 95 both in the absence and presence of RhB (1 μM; no embryos included). Results indicated no influence of PFOS on the fluorescence intensity of RhB (Fig. S2). 2.5. Zebrafish Abcb4 ATPase activity test Specific interactions of PFOS with zebrafish Abcb4 were determined with an in vitro assay using recombinant zebrafish Abcb4 protein generated with the baculovirus expression system (Fischer et al., 2013). As human ABCB1 (Chen et al., 1986), zebrafish Abcb4 is an ATPase that uses the energy liberated by the cleavage of ATP to ADP and Pi for transporting its substrates against a concentration gradient across cell membranes. In the assays applied here, the ATPase activity of recombinant zebrafish Abcb4 was used as indication for interaction of the test compound with the transporter protein using the amount of generated Pi in the experiment as measure for the Abcb4 ATPase activity as first described by Sarkadi et al. (1992) for ABCB1. Interaction of PFOS with zebrafish Abcb4 was studied in experimental series with Abcb4 protein with non-stimulated (“basal”) and with verapamil-stimulated ATPase activities, which can provide an indication of the interaction of the compound with the transporter protein as substrate (i.e., a compound that is transported by the protein) and/or as inhibitor (i.e., a compound that disrupts transporter function). For achieving a stimulated state of the Abcb4 ATPase, showing activity close to its maximum, verapamil was applied at 40 μM in the experiments according to Fischer et al. (2013). Stimulation of the basal transporter ATPase activity is indicated by increased Pi levels in the test; it is often observed for transporter substrates (Sarkadi et al., 1992). In contrast, inhibition of the basal and/or stimulated ATPase activities indicates a compound's property as transporter inhibitor and is indicated by decreasing Pi levels (von Richter et al., 2009). Production of recombinant Abcb4 with the baculovirus expression system and performance of the ATPase assay were according to Fischer et al. (2013). Briefly, abcb4 baculovirus was produced with the Bac-to-Bac Baculovirus Expression System (Invitrogen). To produce recombinant Abcb4, 200 ml of Sf (Spodoptera frugiperda) 9 cell suspension with 2 × 106 cells/ml were infected with abcb4 baculoviral particles at a multiplicity of infection (MOI) of 2.5. Cells were incubated for 72 h, then harvested, disrupted by nitrogen cavitation, and cell membranes were pelleted by centrifugation and ultracentrifugation steps. The pellet containing the membrane fractions was dissolved in ice cold TMEP buffer (Germann, 1998; Sarkadi et al., 1992), homogenized using syringes, and total protein was determined with the BCA™ Protein Assay Kit (Thermo Scientific). ATPase assays were performed in 96-well plates with 20 μg of total protein per well. Liberated Pi was quantified colorimetrically by measuring the absorption at 750 nm using a detection reagent with 7.5% ascorbic acid, 8.75 mM ammoniummolybdate tetrahydrate and 3.75 mM zinc acetate. The Abcb4 ATPase activity is ortho-vanadate-sensitive and test series were performed with and without sodium ortho-vanadate to determine the Abcb4 ATPase activity

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within the total ATPase activity in the membrane preparations. Abcb4 ATPase activities in each test were determined by subtracting the values from tests with ortho-vanadate from those from tests without orthovanadate (Sarkadi et al., 1992). 2.6. Detection of RhB in zebrafish embryos with epi-fluorescence and brightfield microscopy Epi-fluorescence and brightfield microscopy served to visualize PFOS effects on accumulation and tissue distribution of RhB in zebrafish embryos. This approach enabled to visually differentiate compartmentspecific accumulations of the dye. Fluorescence images of RhB accumulation in zebrafish embryos were taken with a AZ100 Multizoom microscope (Nikon, New York, USA). To ensure identical imaging conditions, camera and microscope settings were kept constant at all times. For brightfield microscopic images, a CKX41 inverted microscope (Olympus, Hamburg, Germany) equipped with a digital Olympus C5060 camera and the digitizing software Analysis 5.0 (Soft Imaging Systems, Olympus) was used. Embryos at 48 hpf were incubated for 1 h in 200 μM RhB with PFOS (50 and 100 μM). Reconstituted water with RhB (200 μM) with or without DMSO (0.05%) served as negative and solvent controls, respectively. After incubation, excess dye was removed by washes of embryos in clean water. To allow a clear view of the RhB accumulation in the embryo as well as in the chorion, embryos were photographed prior to and after removal of the chorion with forceps. In order to investigate the RhB uptake in the absence of the chorion, dechorionated embryos were re-incubated for 1 h and thereafter photographed. 2.7. Data analysis and statistics Normal distribution of data was assumed, and parametric statistics were used for data analyses. Mean values and standard deviations (SD) or standard errors (SEM) were determined from replicate values. Differences between treatments and controls were evaluated with one-way analysis of variance (ANOVA), followed by Dunnett's test. For FET data, Student's t-test was applied to compare mortalities of embryos exposed to vinblastine and PFOS to those in treatments with vinblastine at the respective concentrations only. Differences were considered significant at p b 0.05. Statistics were performed using SigmaStat 3.5 (Systat-Jandel Scientific, Erkrath, Germany) or JMP 10.0.0 (SAS Institute GmbH, Germany). For data from the Abcb4 ATPase assays, regressions for concentration response relationships of ATPase activity stimulation and of inhibition of stimulated ATPase activity were determined using the four parameter logistic equation (equation 1). VðcÞ ¼ min þ

max  min 1 þ 10ðlogEC50cÞp

Fig. 1. Accumulation of RhB in 24 hpf zebrafish embryos from controls (RhB + DMSO) as well as from PFOS and verapamil treatments in RhB dye accumulation assays. Data above each bar represent fold changes in RhB tissue contents from controls. *: p b 0.05 (Dunnett's test with solvent control as reference). Data are given as means ± S.D. (PFOS: n = 3; verapamil: n = 4).

PFOS treatments, RhB levels were statistically significantly (p b 0.05) higher than in solvent controls (Fig. 1). Verapamil, used as a reference inhibitor, significantly enhanced RhB accumulation in zebrafish embryos by up to 1.7-fold compared to control levels (Fig. 1).

3.1.2. PFOS effects on RhB clearance PFOS effects on RhB efflux in zebrafish embryos were further investigated by determining depuration of RhB in zebrafish embryos with PFOS present (Table 1). Upon 1 h exposure to RhB in combination with different PFOS concentrations, RhB tissue levels in embryos were either directly measured (“1 h RhB/no post-incubation”) or embryos were transferred either to artificial water without chemicals [(“1 h RhB/1 h post-incubation (clean water)”] or to water containing PFOS at the respective concentration as in the exposure with RhB [“1 h RhB/ 1 h post-incubation (PFOS)”] and RhB tissue levels were measured after further 1 h incubations. Embryos from “1 h RhB/1 h postincubation (PFOS)” treatments displayed RhB tissue levels equal to or higher than RhB levels of embryos measured directly after the 1 h RhB/PFOS treatment (Table 1). In contrast, after 1 h post-incubation in pure artificial water [(“1 h RhB/1 h post-incubation (clean water)”], all zebrafish embryos previously exposed to PFOS displayed RhB fluorescence reduced by 35–70% (20 and 100 μM PFOS, respectively). This indicates a clear PFOS effect on RhB depuration in the embryo tissue.

ð1Þ

V(c) is the enzyme activity at a certain concentration min is the minimal enzyme activity max is the maximal enzyme activity c is the concentration of the tested compound p is the HILL slope EC50 is the concentration at half-maximal enzyme activity. 3. Results

Table 1 Fold increases in RhB fluorescence intensities in 24 hpf zebrafish exposed to 0.5 μM RhB and different concentrations of PFOS versus controls (RhB only). Data are given for embryos immediately after treatments with RhB and PFOS (1 h RhB/no post-incubation) and for embryos after 1 h post-incubation either in water with PFOS at respective concentrations or in clean water. Data are given as means ± S.D. of two experiments (10 eggs per treatment). PFOS (μM)

3.1. Effects of PFOS on rhodamine B (RhB) accumulation and clearance in zebrafish embryos 3.1.1. PFOS effects on RhB accumulation RhB fluorescence intensity in zebrafish embryos increased in the presence of PFOS in a concentration-dependent manner (Fig. 1). Maximum RhB levels were reached at 100 μM PFOS (11.9-fold increase vs. control) and were similar at 300 μM PFOS. In 30, 100 and 300 μM

20 1 h RhB/no post-incubation 1 h RhB/1 h post-incubation (PFOS) 1 h RhB/1 h post-incubation (clean water)

40

60

80

100

3.8 ± 3.2 3.2 ± 1.5 5.8 ± 1.8 8.1 ± 0.3 9.4 ± 0.2 4.5 ± 2.4 4.2 ± 0.1 6.7 ± 3.7 8.5 ± 2.3 9.7 ± 1.9 2.5 ± 1.1 1.5 ± 0.2 1.7 ± 0.3 3.7 ± 0.4 2.6 ± 0.9

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3.2. Effects of PFOS on the embryo toxicity of the Abcb4 substrate vinblastine In order to investigate the role of Abcb4 transporter inhibition by PFOS as mechanism behind its apparent chemosensitizing effect as indicated by the RhB accumulation assays (see above), PFOS effects on the toxic sensitivity of zebrafish embryos to vinblastine were determined. Vinblastine is a toxic substrate of zebrafish Abcb4, and its toxicity for zebrafish embryos is enhanced in the presence of efflux transporter inhibitors (Fischer et al., 2013). Mortality of zebrafish embryos at 48 hpf exposed to vinblastine in the absence or presence of PFOS at a nontoxic concentration (21 μM) is illustrated in Fig. 2. Despite little mortality in the 21 μM PFOS exposure group (2.5 ± 5%), the presence of PFOS significantly increased the lethal potential of vinblastine at 1–2.5 μM causing an up to five-fold elevated mortality when compared with treatments containing vinblastine only. No mortality was observed in the negative control group throughout the 48 h exposure period (Fig. 2).

3.3. PFOS effects on zebrafish Abcb4 ATPase activity The ATPase activity assay with recombinant zebrafish Abcb4 was performed to determine whether PFOS stimulates the basal Abcb4 activity acting as Abcb4 substrate or whether PFOS inhibits the verapamil-stimulated Abcb4 ATPase activity. The mean basal Abcb4 ATPase activity was 7.3 nmol Pi/min/mg protein (basal activity, bA), while the mean Abcb4 ATPase activity stimulated with verapamil was 17.1 nmol Pi/min/mg protein (stimulated activity, sA; Fig. 3). The PFOS concentrations applied were in the range of 3.9 to 500 μM. In the tests for stimulation, Abcb4 ATPase activities were similar to baseline ATPase activities with PFOS concentrations up to 125 μM; at 250 and 500 μM PFOS, ATPase activities were significantly below baseline (p b 0.05) indicating inhibition of the basal Abcb4 ATPase activity (Fig. 3). The absence of stimulation of the basal Abcb4 ATPase activity by PFOS indicates that the compound does not act as Abcb4 substrate. With increasing PFOS concentrations, the verapamil-stimulated Abcb4 ATPase activity progressively decreased reaching values below the baseline Abcb4 ATPase activity at 250 and 500 μM PFOS (Fig. 3). PFOS effects on the verapamil-stimulated ATPase activities were significant at PFOS concentrations at and above 15.6 μM (Fig. 3). The EC50 value for PFOS resulting from regression analysis (equation 1; constrains: min = 2.8, max = 17.1) was 68.5 μM.

Fig. 2. Relative mortality of 48 hpf zebrafish (Danio rerio) embryos after 48 h exposure to vinblastine alone and in combination with PFOS (21 μM). Asterisks indicate significantly increased mortality in binary mixtures compared to single exposures to vinblastine (p b 0.05) according to Student's t-test. Data are given as means ± S.D. of four experiments (10 eggs per treatment).

Fig. 3. Effects of PFOS on ATPase activities of recombinant zebrafish Abcb4. PFOS was tested for stimulation (Δ) of the basal Abcb4 ATPas e and inhibition of the verapamilstimulated Abcb4 ATPase activities (○). Values are given as means ± SEM from three independent experiments. Means and SEM ranges of the baseline (basal activity, bA) and with 40 μM verapamil stimulated ATPase activities (stimulated activity, sA) are indicated as horizontal dotted lines. Statistically significant effects on ATPase stimulation or inhibition of stimulated ATPase activities were identified by comparison of data with baseline and verapamil-stimulated ATPase activities, respectively, and are indicated by asterisks (*: p b 0.05) below the baseline line (different from baseline activity) or above the line of verapamil stimulation (different from the verapamil-stimulated activity).

3.4. Effects of PFOS on RhB accumulation in zebrafish embryos upon exposure to 200 μM RhB In tests for determining cellular efflux transporter activity fluorescent indicator dyes such as RhB and calcein-am are generally used in the low μM range (Essodaigui et al., 1998; Neyfakh, 1988); at higher concentrations, the transporter capacity is overwhelmed and dye accumulates inside cells despite transporter activity (Neyfakh, 1988). Effects of PFOS on accumulation of RhB in zebrafish embryos were investigated upon exposure of the embryos to a high RhB concentration (200 μM) at which efflux activity in the zebrafish embryo is supposedly overwhelmed; the RhB concentration chosen for these experiments was also about 10-fold above the RhB concentration that was previously found to inhibit the verapamil-stimulated Abcb4 ATPase activity (Fischer et al., 2013). The degree of RhB accumulation in zebrafish embryos at this RhB concentration should thus not be influenced by efflux transporter activity. RhB accumulation in embryos from different treatments was examined with fluorescence microscopy (Fig. 4). RhB staining of tissues upon exposure of embryos to this high RhB concentration was strong and also detectable with bright-field microscopy (Fig. S3). The experiments showed that PFOS clearly enhanced the RhB levels in embryos exposed to 200 μM RhB; the PFOS effect was more pronounced at 100 μM than at 50 μM (Fig. 4, Fig. S3). Fluorescence intensities of both chorion and embryo tissue were higher in PFOS treatments compared to controls indicating that, with PFOS present, adsorption of RhB to the chorion and accumulation of RhB in the embryo tissue were increased. Enhanced adsorption of RhB to the chorion in PFOS treatments can be seen in the images of intact eggs (Figs. 4A–D); removal of the chorion reveals that fluorescence levels of the embryo tissue were also increased in the PFOS treatments (Figs. 4E–H). Accumulation of RhB was increased in all embryos from the treatments continued upon removal of the chorion from the embryos after a first exposure (Fig. 4I–L), as compared to embryos photographed without chorion directly after the first exposure (Fig. 4E–H); however, also in those embryos RhB fluorescence was enhanced in PFOS treatments (Figs. 4K, L) compared to controls (Fig. 4I, J). This can be seen as indication that differences in accumulation of RhB in embryo tissue due to PFOS did not depend on the presence of the chorion. Whereas RhB mainly occurred in the yolk of embryos from controls (Fig. 4E, F, I, J),

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Fig. 4. Epi-fluorescence images of RhB accumulation in 48 hpf zebrafish (Danio rerio) embryos. Zebrafish embryos were incubated with 200 μM RhB alone (A, E, I) and in combination with 0.05% DMSO (B, F, J), 50 μM PFOS (C, G, K) or 100 μM PFOS (D, H, L). Embryos were incubated for 1 h (A–D), when the chorion was removed mechanically to provide a clear view of RhB accumulation in the embryo (E–H). In order to investigate RhB accumulation in the absence of the chorion, non-exposed dechorionated embryos were incubated for 1 h in the same exposure solutions described above (I–L). Exposure times in (A-H) and (I–L) were 42 and 14 milliseconds, respectively.

embryos from PFOS treatments showed beyond increased RhB levels in the yolk also RhB accumulation in the other tissues (Fig. 4G, H, K, L). Likewise, the brightfield microscopy images indicated enhanced accumulation of RhB in embryos from PFOS treatments (Fig. S3). Photographs of the chorions show higher adsorption of RhB to the chorions from embryos from PFOS treatments (Fig. S3E–H). 4. Discussion Based on previous observations that PFOS, among other PFAS, enhances chemical uptake by cells and tissues (Ahrens and Bundschuh, 2014; Ding and Peijnenburg, 2013), this study aimed at determining whether PFOS acts as a chemosensitizer inhibiting cellular efflux transporter activity in zebrafish embryos. PFOS enhanced the accumulation and inhibited the clearance of RhB in zebrafish embryos. Moreover, the mortality by vinblastine for zebrafish embryos was clearly increased when vinblastine was coadministered with PFOS. Both RhB and vinblastine are substrates of MXR transporters and have earlier been used as model compounds in studies on MXR in aquatic organisms including fish (Epel et al., 2008; Luckenbach et al., 2014). Increased accumulation/reduced clearance of RhB in cells in the presence of a test compound are seen as indication for disrupted efflux activity by inhibitory action of the test compound on respective transporter proteins. Likewise, enhanced toxicity of vinblastine is a result of higher levels of the compound being able to enter the cells due to chemical inhibition of transporter efflux activity. The relationship of an increase of sensitivity to vinblastine toxicity with higher levels of internal vinblastine levels as a consequence of disruption of Abcb4 transporter activity has previously been shown for zebrafish embryos (Fischer et al., 2013). The strong effects of PFOS on RhB accumulation and clearance, respectively, as well as the increased embryo toxicity of vinblastine can thus be interpreted as a result from efflux transporter inhibition. Indeed, the ATPase assay with recombinant zebrafish Abcb4 confirmed interference of PFOS with Abcb4 function. Both basal and verapamil-stimulated

transporter ATPase activities were strongly reduced by PFOS indicating action of PFOS as Abcb4 inhibitor. Although these results indicate interference of PFOS with Abcb4 function, the effects of PFOS observed showed striking differences to effects of other compounds tested so far, including model transporter inhibitors such as cyclosporin A, PSC833 and phenanthrene (Fischer et al., 2013). This raises questions about the nature of interference of PFOS with Abcb4 function and, further, whether transporter inhibition entirely explains the PFOS effect on the cellular uptake of other compounds. In this context, it is noticeable that in RhB accumulation assays the effect amplitude of PFOS considerably exceeded that of other compounds acting as Abcb4 inhibitors. In accordance to previous data obtained by Fischer et al. (2013) verapamil-increased RhB uptake by embryos was about 1.7-fold higher compared to the control (Fig. 1). Effects of the known transporter inhibitor cyclosporin A on RhB accumulation in embryos were maximally 3fold (Fischer et al., 2013). In comparison, PFOS caused an RhB increase of 11.9-fold (cf. Fig. 1). PFOS effects on sensitivity of embryos to vinblastine in the toxicity assay were comparable to those of cyclosporin A in an equivalent FET with vinblastine, which showed corresponding increases in mortality at certain vinblastine concentrations when transporter inhibitor was also present (Fischer et al., 2013). However, the cyclosporin A concentration used in these tests (5 μM) was at a level that in the RhB accumulation tests caused effects that were approximately 1/3 below the maximally observed effects (Fischer et al., 2013); in contrast, the PFOS concentration used in the FET here (21 μM) was in a range considerably less potent in RhB accumulation experiments than higher concentrations (Fig. 1). Since PFOS was toxic at those levels it could not be applied at higher concentrations together with vinblastine in the FET. However, if PFOS was not embryotoxic by itself, it should be expected that its enhancing effects on the embryotoxic potential of vinblastine would be more pronounced at higher PFOS concentrations. The high effect amplitude of PFOS may be explained by interference of PFOS also with efflux transporters other than Abcb4. In contrast to PFOS, it might be that other efflux inhibitors, which show a lower effect

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amplitude than PFOS, affect the Abcb4 transporter more specifically and therefore the overall effect is less pronounced because other efflux transporters are not targeted by those inhibitors. In this context, efflux transporter candidates are Abcc and Abcg2 transporters. Abcc transporters, however, can be considered irrelevant since experiments with the specific inhibitor MK571 revealed that members of this ABC transporter subfamily possess only a minor impact on RhB efflux in zebrafish embryos (Fischer et al., 2013). Zebrafish has four Abcg2 paralogs (Annilo et al., 2006; Luckenbach et al., 2014), which are also putatively relevant efflux transporters of RhB; thus, rhodamine 123, which is similar to RhB, was identified as substrate of human ABCG2 (Litman et al., 2001). The role of the Abcg2 paralogs as efflux transporters in zebrafish embryos has so far not been explored. However, considering that human ABCG2 is a multi-specific efflux transporter, it seems unlikely that specifically PFOS, but none of the other compounds tested so far (Fischer et al., 2013) showed an inhibitory effect on relevant zebrafish Abcg2 paralog(s). The second noticeable aspect with respect to the effects of PFOS and other efflux transporter inhibitors regards differences in effective concentrations. Whereas compounds inhibiting Abcb4 efflux activity in zebrafish embryos were active in the low μM concentration range (Fischer et al., 2013), PFOS effects occurred at concentrations one to two orders of magnitude higher. Thus, whereas the maximal effect in the RhB accumulation assay with zebrafish embryos occurred at approximately 10 μM for standard inhibitors and other compounds, maximal RhB accumulation for PFOS was at 100 μM (Fig. 1). In addition, effective PFOS concentrations causing inhibition of the verapamilstimulated Abcb4 ATPase were considerably above those for cyclosporin A, a potent zebrafish Abcb4 inhibitor. Based on effect data for cyclosporin A on the stimulated Abcb4 ATPase activity (Fischer et al., 2013) an EC50 value of 0.2 μM was determined based on a regression analysis using equation 1. In contrast, the EC50 for PFOS was approximately 325fold higher (see above). Verapamil, used as inhibitor in the RhB accumulation assay here, stimulates the Abcb4 ATPase activity indicating its interaction with the transporter as substrate, but it has no ATPase activity-decreasing effect (Fischer et al., 2013). This indicates that the verapamil effect in the RhB accumulation assay is via competitive inhibition. Its exceptionally high effect concentrations together with the high effect potency of PFOS on the Abcb4 ATPase activity suggest that, in contrast to other inhibitors identified so far, PFOS may not affect Abcb4 activity by direct interaction with the protein, but by another still unidentified mechanism. Based on the present results and data from literature, we assume that both PFOS interference with Abcb4 activity and enhanced RhB/vinblastine uptake by embryos in the presence of PFOS may be associated with the detergent-like properties, which lead to its intercalation into cellular membranes resulting in increased membrane fluidity as well as variations in phospholipid and membrane protein components (Hu et al., 2003; Liao et al., 2014; Matyszewska et al., 2007; Yao et al., 2014). ABC efflux transporter protein function crucially depends on the protein's membrane environment (Ferte, 2000; Liao et al., 2014; Romsicki and Sharom, 1999). Along the lines with our findings, inhibition of transporter ATPase activity in cell cultures has been found to be associated with increased cell membrane fluidization caused by the compounds investigated (Regev et al., 1999). Therefore, we assume that comparatively high PFOS concentrations are necessary for modifying membrane fluidity to an extent that Abcb4 activity is disrupted. The RhB accumulation experiments with high RhB exposure concentrations (200 μM) further indicate an effect of PFOS on RhB uptake that does not involve efflux transporter inhibition as main cause. At this high RhB concentration in the medium, it can be expected that cellular efflux transporter activity is overwhelmed and unable to antagonize RhB uptake by the embryo. As a cause for the clear PFOS effects in these experiments (Fig. 4, Fig. S3), Abcb4 inhibition by PFOS can be excluded. PFOS also leads to increased adsorption of RhB to the chorion; however, to the

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best of our knowledge, it is not clear whether this PFOS effect is based on the same mechanism as the increased RhB uptake by the embryo. The increased accumulation of RhB and vinblastine in the embryo may be explained by membrane properties and composition altered by PFOS. This mechanism has previously been proposed for several different PFAS by Hu et al. (2003). In addition, Liao et al. (2014) found that the level of major components of phospholipids varied with concentrations of PFOS, which may result in changes of the membrane permeability. The RhB clearance experiments may provide further indication that the PFOS effects on RhB uptake were mainly by changes of membrane properties upon PFOS incorporation into the membrane. Thus, the effects were strikingly quickly abolished (within 1 h) as soon as the embryos were transferred to clean water (Table 1). In contrast, in similar experiments with bivalve tissue, efflux transporter-inhibiting effects of test compounds lasted for up to 48 h indicating long-term binding to the transporter protein (Luckenbach and Epel, 2005). Interestingly, zebrafish embryos post-incubated in artificial water with PFOS displayed no RhB loss, but the dye content was in the same order of magnitude as prior to the post-incubation. Thus, the RhB clearance experiments in this study suggest: (1) As RhB levels in zebrafish embryos quickly decreased when PFOS was absent, PFOS may rapidly be removed from cellular membranes once absent from the external medium as source. (2) When PFOS is present at a certain level in the cellular membrane, it causes trapping of cationic RhB inside the cells; once PFOS is absent from the membrane RhB diffuses quickly out of the cells. The effect levels of PFOS recorded in this study were four to six orders of magnitude above reported environmental concentrations (Kwadijk et al., 2010; Pistocchi and Loos, 2009; Senthilkumar et al., 2007; Skutlarek et al., 2006). However, PFOS has a strong potential to bioaccumulate and tissue levels reported for fish were in the range of five orders of magnitude above environmental concentrations (Houde et al., 2008). Considering that a large proportion of accumulated PFOS in biota is found in the blood (Houde et al., 2011) it can be assumed that it has access to biological targets and can exert effects. In conclusion, although this study clearly shows that at high concentrations PFOS is a potent inhibitor of the MXR-related zebrafish Abcb4 transporter, chemosensitization is most likely not the only cause for increased accumulation of Abcb4 substrates in the presence of PFOS. Rather, both transporter protein inhibition and enhanced accumulation of substrate compounds in zebrafish embryos are assumed to be associated with intercalation of PFOS into cellular membranes leading to modifications of membrane properties. Other PFAS should be studied with respect to the question whether they exert similar effects. Effect concentrations of PFOS in our tests were above reported environmental levels. However, considering the high bioaccumulative potential of PFOS, studies are needed that address the question to which extent the comparatively high levels accumulated in animal tissue can also exert effects. Acknowledgments The authors wish to thank Stephan Fischer for his demonstration of the transporter activity assay with RhB and with zebrafish embryos and Erik Leist and Nicole Schweiger for their assistance during the experimental work. The first author was supported by the Landesgraduiertenförderung (LGFG), University of Heidelberg, Ministry of Science, Research and the Arts of Baden-Württemberg. The second author was sponsored through the scholarship program of the German Federal Environmental Foundation (DBU). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.12.089.

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