- Email: [email protected]
Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3 Jelena Dragojević, Petra Marić, Jovica Lončar, Marta Popović, Ivan Mihaljević, Tvrtko Smital
T ⁎
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
A R T I C LE I N FO
A B S T R A C T
Keywords: Oat1 and Oat3 transporters Zebrafish Stable transfectants Environmental contaminants Interaction screening Cytotoxicity
Organic anion transporters (OATs) are transmembrane proteins which belong to SLC22 subfamily. They are responsible for the uptake of various endo- and xenobiotics into the cells of different organs and tissues. Following our previous work on characterization of zebrafish Oat1 and Oat3, in this study we analyzed interaction of various classes of environmental contaminants with these membrane transporters using the transport activity assay with HEK293 Flp-In cell line stably overexpressing zebrafish Oat1 and Oat3, respectively. Based on the initial screening of a series of 36 environmental contaminants on their ability to interact with zebrafish Oat1 and Oat3, the most potent interactors were selected, their IC50 values calculated and type of interaction determined. Finally, to further confirm the type of interaction and initially evaluate their toxic potential, the cytotoxicity assays were performed. Broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs was confirmed and potent interactors among environmental contaminants identified.
1. Introduction After a xenobiotic compound, environmental contaminants included, enters the cell by passive diffusion or through activity of uptake transporters it typically undergoes biotransformation by phase I and II detoxification enzymes, and is finally pumped out of the cell by transmembrane efflux transporters. However, this is not the case for certain compounds such as heavy metals and persistent organic pollutants (POPs), whose lipophilic nature allows them to bioaccumulate in the fatty tissues of living organisms, and therefore are not substrates for active or facilitated transport by membrane transporters (Hjelmborg et al., 2008). The most important organs for metabolization of xenobiotics in vertebrates are liver and kidney, and once a xenobiotic becomes hydrophilic it is typically excreted out via bile and urine (Goodman and Gilman, 1994). Consequently, it has been found that numerous drug transporters actively participate in the disposition of endogenously formed metabolites and xenobiotics, in addition to involvement in drug-drug interactions (DDIs) of various drugs and new molecular entities (Agarwal et al., 2013; Lee et al., 2017). Among the most important drug transporters listed in white papers by the International Transporter Consortium (Giacomini et al., 2010; Tweedie et al., 2013) are organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) that belong to the SLC22A gene family. They
transport chemically and structurally different endogenous and exogenous organic anions from the blood into the cells of diverse human and animal organs. Because of their substantial and strategic localization in organs important for drug disposition they have critical roles in the absorption, distribution, metabolism, and elimination (ADME) of drugs and xenobiotics (Giacomini et al., 2010; Colas et al., 2016). Human OAT1 and OAT3 are predominantly expressed in kidneys, specifically in the basolateral membrane of renal proximal tubule cells where they mediate the uptake of chemicals from the blood (Nishimura and Naito, 2005; Burckhardt and Burckhardt, 2011). Besides their physiological role in maintenance of systemic levels of endogenous substrates (uric acid, signalling molecules etc.), OAT1 and OAT3 are important drug transporters in kidney where they actively secrete drugs into the urine. Human OAT1 and OAT3 share similar substrate preferences, and their reported drug substrates include NSAIDs, penicilin, methotrexate, HIV protease inhibitors and antiviral drugs. Inhibition of these OATs results in impaired elimination of drugs from kidneys (Nigam, 2018). Zebrafish (Danio rerio) is a significant vertebrate model species increasingly used both for biomedical studies and in environmental science. We recently identified seven Oats in zebrafish genome, which are phylogenetically classified into three subfamilies: OAT1/Oat1, OAT2/ Oat2 and OAT3/Oat3 (Mihaljevic et al., 2016). Zebrafish oat1 and oat3
⁎ Corresponding author at: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia. E-mail address: [email protected] (T. Smital).
https://doi.org/10.1016/j.cbpc.2020.108742 Received 4 December 2019; Received in revised form 28 February 2020; Accepted 6 March 2020 Available online 10 March 2020 1532-0456/ © 2020 Elsevier Inc. All rights reserved.
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
2.2. Transport activity assays
genes showed one-to-one orthology to their mammalian orthologs which indicate possible conservation of OAT1 and OAT3 physiological or defense role(s) in vertebrates. Using the conserved synteny analysis of both zebrafish oat genes we also showed that they are in zebrafish located on the same chromosome 21 (Dragojević et al., 2019), similar to human OAT1 and OAT3 which are both located on chromosome 11. The qPCR expression analysis of Oat1 transcript in adult zebrafish tissues showed the highest expression in testes and brain in females, and low expression in gills and liver in both genders. Oat3 showed the highest expression of all Oats in zebrafish, with very high expression in female kidneys, lower expression in male kidneys, testes and intestine, with higher prevalence in females. Additionally, zebrafish Oat1 and Oat3 showed broad ligand selectivity, similar to their mammalian orthologs, and their potential physiological interactors have been identified (Dragojević et al., 2019). Based on these initial insights, in this study we specifically address the interaction of zebrafish Oat1 and Oat3 with environmental contaminants. Considering their tissue expression pattern and the first data on their substrate preferences obtained in our recent studies, we presume that a similar interaction of zebrafish Oat1 and Oat3 with anionic xenobiotics can be expected, including overlapping substrate specificities. Furthermore, considering potential ecotoxicological relevance, we hypothesize that interactions of environmental compounds with Oat1 and Oat3 transporters may be deleterious due to either (1) increased accumulation of potentially toxic xenobiotic substances caused by increased Oat-mediated uptake and/or modulated excretion of these substances, or (2) compromised transport of physiologically relevant Oat1/Oat3 substrates which in turn can cause changes in physiological homeostasis. To address our main goal, we tested the interaction of zebrafish Oat1 and Oat3 stably overexpressed in HEK293 Flp-In cell line with 36 environmental contaminants broadly classified into three groups: industrial chemicals (industrials), pesticides and pharmaceuticals and personal care products (PPCPs) (Murray et al., 2010). Our selection of chemicals was based on three primary criteria: (1) their occurrence in surface waters (Fent et al., 2006; Murray et al., 2010), (2) reported interactions with human or mammalian OATs/Oats (reviewed in Burckhardt and Burckhardt, 2003; Nigam et al., 2015; Srimaroeng et al., 2008), and/or (3) their anionic character. Furthermore, although not all of the compounds tested are found in significant concentrations in the environment, many of them have a considerable bioaccumulation potential (e.g., PFOA, PFOS, nonylphenol, organotins) with reported bioconcentration factors over 10,000 (Giesy et al., 2002; Hoch et al., 2001; Soares et al., 2008; Whalen et al., 1999), which implies their potential ecotoxicological relevance. Compounds that showed potent interaction were further analyzed in order to determine the type of their interaction (substrates versus inhibitors) with zebrafish Oat1 and Oat3. Finally, to further confirm the type of interaction for the most potent interactors and initially evaluate their toxic potential we performed cytotoxicity assays with Oat1- and Oat3-overexpressing cells.
Cloning of zebrafish Oat1 and Oat3 and development of the Oat1 and Oat3 stable transfectants has been described in detail in our previous paper (Dragojević et al., 2019). HEK Flp-In cells stably expressing zebrafish Oat1 or Oat3 and mock-transfected cells were seeded at densities of 5*105 cells/cm2 or 6*105 cells/cm2 (depending on the growth rate) in 96-well plates 48 h before performing the transport assay, in DMEM/FBS medium with final volume of 125 μL per well. DMEM-FBS was removed and cells were pre-incubated in 100 μL of the transport medium (145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM D-glucose and 5 mM HEPES) for 10 min at 37 °C. For determination of transport rates and dose-responses of model fluorescent substrates, 25 μL of concentrated (5×) fluorescent substrates previously identified by Dragojević et al. (2019) (Lucifer yellow (LY) for Oat1 and 6-carboxyfluorescein (6-CF) for Oat3) were added to the pre-incubation medium. Following the incubation (10 min at 37 °C), the cells were washed two times with 125 μL of cold transport medium and lysed with 125 μL of 0.1% sodium dodecyl sulfate (SDS) for 30 min at 37 °C. Cell lysates were transferred to 96-well black microplates and the fluorescence of model substrate determined using the microplate reader (Infinite M200, Tecan, Salzburg, Austria), at 425 nm excitation and 540 nm emission for LY, and 492 nm excitation and 524 nm emission for 6-CF, respectively. In order to calculate the transport rates, fluorescence response of mock-transfected control cells was subtracted from the fluorescence of transfected cells, followed by normalization of the fluorescence responses by calibration curves determined previously for each model substrate and adjusted for the protein content. Calibration curves for model fluorescent substrates were generated in the cell matrix dissolved in the 0.1% SDS. Bradford assay (Bradford, 1976) was used for measuring total protein content. The normalized uptake of fluorescent substrates was finally expressed in nM of substrate per mg of protein per minute. Following determination of transport kinetics for model fluorescent substrates, the inhibition assay based on co-exposure of transfected cells and mock-transfected control with model substrate and potential interactor were performed. The cells were first pre-incubated in transport medium (10 min) followed by 40 s incubation with test compounds and 10 min incubation with the model substrate at concentration that were in the linear part of the dose response curve (50 μM LY, 5 μM 6-CF) as previously determined. Stock solutions of chemicals were prepared in dimethyl sulfoxide (DMSO; maximal concentration 1% of the total volume). The initial interaction assays were performed at only one concentration of the tested compounds set nominally at 100 μM, unless stated otherwise due to water solubility limitations for some of the compounds tested (Table S1, Fig. 1). Detailed dose response experiments were then performed for all interactors that showed over 70% inhibition of the model substrate uptake and the respective IC50 values determined. Compounds with IC50 values in nanomolar and low micromolar range (< 5 μM) were arbitrarily considered to be very strong interactors, those with IC50s of 5–20 μM were designated as strong interactors, Ki of 20–100 μM indicated moderate interaction, whereas substances with determined IC50 values above 100 μM were classified as weak interactors. After the IC50 values were obtained, the type of interaction for the most potent xenobiotic compounds with zebrafish Oat1 and Oat3 was determined by determining the change in Michaelis-Menten kinetics parameters of LY or 6-CF uptake in the presence and absence of a tested xenobiotic. Concentrations of tested xenobiotics corresponded to their IC50 values. If an interacting compound competitively inhibits the uptake of fluorescent model substrate (i.e., it is a substrate), it will increase Km, but will not affect Vmax value. On the contrary, if the inhibition is noncompetitive, Vmax will decrease while Km will not change. Noncompetitive inhibitors bind away from the active site, and can interact with the transporter independently of the presence of the substrate. Another type of interaction is an uncompetitive one, in which
2. Materials and methods 2.1. Chemicals Model fluorescent substrates, environmental contaminants, and other used substances were obtained from Sigma-Aldrich (Taufkirchen, Germany), Alfa Aesar (Ward Hill, MA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless stated otherwise. Names, abbreviations and other relevant data for tested environmental contaminants (including water solubility, maximal concentrations reported in the environment, maximal concentrations determined in blood/ plasma/serum/animal tissue; and related references) are presented in Supplementary Material (Tables S1 and S2).
2
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
3
(caption on next page)
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
Fig. 1. Interaction of zebrafish Oat1 and Oat3 with environmental contaminants. The initial screening presented was performed by exposing transfected cells to 100 μM concentration of tested substances unless stated otherwise (*) due to limited water solubility for some substances (exposure concentration for nonylphenol was 30 μM, 4 μM for chlorpyrifos, 5 μM for erythromycin, 15 μM for Diclofenac, and 8 μM for indomethacin). Data are expressed as mean percentage (%) ± SD from triplicate determinations of LY or 6-CF uptake after co-incubation with each interactor (100 μM) relative to LY/6-CF uptake in the absence of interactor which is set to 100%.
2.4. Data analysis
both Vmax and Km decrease. Uncompetitive inhibitors have a separate binding site, like noncompetitive inhibitors, but they only bind the transporter-substrate complex. Finally, some interactors may show a mixed inhibition, causing a decrease in Vmax, but also an increase in Km. The described approach for distinguishing among substrates and inhibitors of membrane proteins using Michaelis-Menten kinetics determinations is the only currently available approach for examining a large set of compounds for transporters without their structures solved at high resolution. The approach has been successfully used in numerous previous studies published by other research groups (Yamazaki et al., 2005; Gui et al., 2009; Westholm et al., 2009; Kindla et al., 2011), as well as by our group (Popovic et al., 2014; Mihaljević et al., 2017; Lončar and Smital, 2018; Dragojević et al., 2019).
All transport assays were repeated in at least three independent experiments run in triplicates, and data from a typical experiment are shown in related figures. GraphPad Prism 6 software for Windows was used for all calculations. For the initial interaction analyses performed with fixed concentration of tested xenobiotics, independent-samples Student t-test was applied to evaluate statistically significant difference in the uptake of a model substrate (LY or 6-CF) in transfected versus mock-transfected (control) cells. Level of significance was set at P < 0.05. The kinetic parameters, Km and Vmax values were calculated using the Michaelis-Menten Eq. (1):
V= 2.3. Cytotoxicity assays
Vmax × [S ] S + Km
(1)
where V is velocity (nanomoles of substrate per milligram of proteins per minute), Vmax is maximal velocity, [S] is substrate concentration and Km is the Michaelis-Menten constant. For cytotoxicity assays, cell viability was expressed as percentages of viability obtained from triplicate determinations, with mean ± standard deviation (SD) values. Sigmoidal dose-response curves with 95% confidence intervals (CIs) were obtained using non-linear regression model in GraphPad Prism 6 software for Windows. Classical four parameters sigmoidal dose-response curves were used to obtain the response (y) according to the Eq. (2):
Cytotoxicity of Oat1 and Oat3 interactors was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric reduction assay (originally described by Mosmann, 1983). The assay was performed with HEK Flp-In cells stably overexpressing zebrafish Oat1 and Oat3 uptake transporters and HEK Flp-In/mock cells which were used as a control. Viability of the cells was assessed after the exposure of both stably transfected and mock cells to Oat1 and Oat3 interactors for 72 h. HEK Flp-In/Oat1 and Flp-In/Oat3 cells were seeded in 48-well plates with 5 × 105 cells/mL cultivation density and at 250 μL cells/well seeding volume. Following the 24 h incubation, the cells were exposed to serial dilutions of tested chemicals prepared in DMEM medium without FBS. Half of the medium (125 μL) was removed from each well and replaced with 125 μL of sample dilutions in triplicates. HEK Flp-In/Oat1 and Flp-In/Oat3 cells were then exposed for 72 h to various concentrations of tested substances. Afterwards, 200 μL of medium was removed from the wells and for the next 2.5 h cells were incubated with 200 μL/well of MTT reagent prepared in DMEM medium to reach final concentration of 0.5 mg/mL. Formed formazan salts were dissolved in 200 μL/well of 2-propanol and plates were shaken at 350 rpm. Absorbance was measured using microplate reader at 570 nm with reference filter set to 750 nm. Differences between the measurement filter and the reference filter values were calculated in Microsoft Office Excel 2007. Serial dilutions were log transformed and the obtained values normalized to percentages prior to statistical analysis. Interactors of Oat1 and Oat3 transporters, respectively, whose cytotoxic effect was detected by MTT assay were additionally evaluated using the CCK-8 colorimetric test (Cell Counting Kit-8, Sigma-Aldrich) due to higher detection sensitivity of WST-8 water-soluble tetrazolium salt in order to obtain more accurate data. HEK293 Flp-In/Oat1 and Flp-In/Oat3 cells were seeded in 96-well plates with 2 × 105 cells/mL cultivation density and at 100 μL cells/well seeding volume. After 24 h incubation, the cells were exposed to serial dilutions of tested chemicals prepared in DMEM medium without FBS. Concentration of solvent (DMSO) never exceeded 1% of the total volume. Half of the medium (50 μL) was removed and replaced with 50 μL of sample dilutions in duplicates. Concentrations of tested chemicals were in the range from 0.001 to 3 μM. After exposure period of 72 h, 10 μL of CCK-8 solution were added to each well. After 3 h of incubation, absorbance at 450 nm was measured with the microplate reader. Viability was calculated from duplicate determinations, as described for MTT assay.
y = b + (a–b)/(1 + 10((logLC 50– x ) ∗ h)))
(2)
where b is the minimum (bottom) of response, a represents the maximum (top) response, hillslope (h) is slope of the curve, logLC50 is halfway response from bottom to top and x is the logarithm of inhibitor concentration. 3. Results 3.1. Inhibition tests 3.1.1. The interaction screen assay By using the developed transport activity assay, a series of environmental contaminants reported to be xenobiotic interactors of mammalian OAT1/Oat1 and OAT3/Oat3 was initially tested. Using this assay we found that Oat1 and Oat3 showed interaction with a wide range of xenobiotic compounds (Fig. 1), with high level of overlapping interactors. Xenobiotics that showed the strongest interaction with Oat1 were: indomethacin (0.35% LY uptake remaining in comparison to mock-transfected cells), MK-571 (0.35% LY uptake), diclofenac (1.01% LY uptake), furosemide (2.32% LY uptake), tributyltin (TBT) (5.7% LY uptake), ibuprofen (6.57% LY uptake), caffeine (8.49% LY uptake), probenecid (9.17% LY uptake), methotrexate (15.2% LY uptake), perfluorooctanoic acid (PFOA) (16.63% LY uptake), perfluorooctanesulfonic acid (PFOS) (17.18% LY uptake), 2,4-dichlorophenoxyacetic acid (17.76% LY uptake) and tripropyltin (TPrT) (19.73% LY uptake). The most potent Oat3 interactors were: MK-571 (0% 6-CF uptake), probenecid (0.3% 6-CF uptake), diclofenac (0.86% 6-CF uptake), indomethacin (3.22% 6-CF uptake), TBT (4.36% 6-CF uptake), tripropyltin (TPrT) (4.92% 6-CF uptake), trimethyltin (TMT) (5.44% 6-CF uptake), methotrexate (5.65% 6-CF uptake), triethyltin (TET) (7.56% 6CF uptake), ibuprofen (10.6% 6-CF uptake), 2,4-dichlorophenoxyacetic 4
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
Table 1 IC50 values of the most potent zebrafish Oat1 and Oat3 interactors. Each data point represents the mean ± SD from typical experiment out of three independent determinations. Oat1
Table 3 Type of interaction determined for set of zebrafish Oat3 interactors. Uptake kinetics parameters for 6-CF are given as Km (μM), Vmax (nmol 6-CF/mg protein/min), and 95% confidence intervals (c.i.) for each. The mark “S” denotes Oat3 substrates, and the “I” denotes inhibitors. Data are mean ± SD from typical experiment out of three independent determinations.
Oat3
Compound
IC50 (μM)
Compound
IC50 (μM)
Indomethacin Ibuprofen Diclofenac Tri-n-propyltin 2,4-D PFOS PFOA Chlorpyrifos Trimethyltin Methotrexate MK571
0.05 ± 0.16 1 ± 0.06 4.2 ± 1.03 6.63 ± 1.27 13.62 ± 1.12 16.75 ± 1.14 32.48 ± 1.16 39.04 ± 1.12 43.66 ± 1.09 56.4 ± 1.1 1.41 ± 1.1
Indomethacin Tri-n-propyltin Triethyltin Ibuprofen TRIPHENYLTIN PROBENECID 2,4-D Diclofenac Methotrexate Trimethyltin PFOS
0.03 ± 1.08 0.28 ± 1.2 0.43 ± 1.22 5.98 ± 1.12 6.99 ± 1.19 7.39 ± 1.15 7.56 ± 1.23 8.49 ± 1.32 9.98 ± 1.3 11.5 ± 1.1 43.94 ± 1.1
acid (11.23% 6-CF uptake), PFOA (15.4% 6-CF uptake), triphenyltin (TpheT) (15.94% 6-CF uptake), furosemide (26.37% 6-CF uptake), PFOS (28.84% 6-CF uptake) and PAH (29.07% 6-CF uptake).
3.3. Cytotoxicity assays To further evaluate the type of interaction for selected compounds Table 2 Type of interaction determined for set of zebrafish Oat1 interactors. Uptake kinetics parameters for LY are given as Km (μM), Vmax (nmol LY/mg protein/ min), and 95% confidence intervals (c.i.) for each. The mark “S” denotes Oat1 substrates, the “I” denotes inhibitors, and “M” refers to the mixed type of inhibition. Data are mean ± SD from typical experiment out of three independent determinations. Vmax (LY)
c.i.
Control (LY)
17,2
13.48–20.92
10,07
9.42–10.72
2,4-D Chlorpyrifos PFOA Indomethacin Methotrexate Trimethyltin Ibuprofen
27.4 26.13 36.44 25.09 29.89 16.59 14.66
23.06–31.73 16.57–35,69 29.20–43.67 20.82–30.97 16.62–43.17 9.92–23.26 11.90–17.41
6.73 11.65 11.69 10.25 11.31 4.54 9.21
6.45–7.00 10.56–12.75 10.67–12.51 9.55–10.94 9.39–13.24 3.97–5.11 8,64 - 9,37
Vmax (6-CF)
c.i.
Control (6-CF)
18.65
12.56–24.74
22.53
19.26–25.79
PFOS Probenecid Diclofenac 2,4-D Indomethacin Methotrexate Ibuprofen Trimethyltin Tri-n-propyltin
3.57 26.42 31.29 28.82 28.04 64.38 18.33 12.21 38.56
16.05–31.62 20.46–32.39 25.57–37.01 21.07–36.57 25.74–30.34 50.40–78.36 14.64–22.02 1.84–22.57 26.26–50.86
27.38 44.50 47.77 38.98 22.56 24.45 19.10 14.94 10.93
22.64–32.12 39.46–49.54 43.14–52.41 33.56–44.39 18.76–26.36 20.95–27.95 17.40–20.79 10.67–19.24 1.33–20.53
Type of interaction
S S S S S S I I S
Despite being recognized and described as integral elements of the ADME processes in mammals, Oats have not been comprehensively studied in non-mammalian species. Therefore, in this study we analyzed the interaction of different classes of environmentally relevant contaminants with zebrafish Oat1 and Oat3 uptake transporters. To specifically and reliably identify zebrafish Oat1 and Oat3 interactors, and determine their potency and type of interaction, we used previously developed HEK293 Flp-In cell lines characterized by stable over-expression of Oat1 and Oat3 transporters, respectively. Using this model we optimized a high throughput assay based on determination of transport rate of fluorescent model substrates (LY for Oat1; 6-CF for Oat3) in real time. Using the initial screening assay, interaction of Oat1 and Oat3 with numerous tested environmental contaminants was determined, confirming wide and overlapping substrate preferences of these transporters (Fig. 1). The strongest interaction with Oat1 and Oat3 was observed for NSAIDs indomethacin and ibuprofen, antineoplastic methotrexate, diuretic furosemide, and for industrials PFOA, PFOS, and organotin compounds (TMT, TET, TPrT, TBT). Michaelis-Menten kinetics showed that indomethacin is a substrate, while ibuprofen is an inhibitor of both zebrafish Oat1 and Oat3 (Tables 2 and 3). As already reported, various NSAIDs were capable of interaction with human OAT1 and rat Oat1. Among others, high affinities were reported for ibuprofen and indomethacin, with comparable IC50 values for hOAT1 and rOat1 (Burckhardt and Burckhardt, 2011; VanWert et al., 2010). Indomethacin was shown to be high affinity substrate of human OAT1, while ambiguous results have been obtained for ibuprofen (Burckhardt, 2012). Therefore, although NSAIDs can inhibit transport activity of mammalian OAT1/Oat1, their transport rate is rather low. However, the authors concluded that inhibition of OAT1/Oat1 by these drugs could potentially modulate secretion of other anionic compounds, but
After the IC50 values were obtained, the type of interaction for the strongest interactors with zebrafish Oat1 and Oat3 was determined (Tables 2 and 3). Determination was based on comparison of kinetic parameters of LY or 6-CF uptake in the presence and the absence of different interacting compounds, respectively, where their concentrations were equal to their previously calculated IC50 values.
c.i.
c.i.
4. Discussion
3.2. Determining the type of interaction
Km (LY)
Km (6-CF)
as previously determined using the Michaelis-Menten kinetics experiments, we exposed the FlpIn/HEK293 cells stably transfected with Oat1 or Oat3 and mock-transfected (control) cells to a potent competitive inhibitor TPrT identified in the previous steps of our study. As expected, shifts in related dose-response curves were observed (Fig. 3). Oat1 and Oat3-overexpressing cells were more responsive to toxic effect of TPrT, resulting in lower EC50 values in comparison to mock-transfected cells. The 4.5 and 4.4-fold changes in EC50 values for TPrT were obtained with Oat1 and Oat3-overexpressing cells, respectively. In contrast, when exposed to previously identified noncompetitive inhibitor TMT, no significant difference in EC50 values, or shift in related dose-response curves were observed (Fig. 4).
3.1.2. Dose-response assays Based on results of the initial screening, interactors that showed the highest potency for inhibition of the model substrate uptake were subjected to dose-response analyses and determination of their IC50 values for both Oats. The strongest interaction with Oat1 was determined for ibuprofen, indomethacin and diclofenac. All of these substances showed concentration dependent inhibition of Oat1 mediated LY transport (IC50 = 1 μM, 0.05 μM and 4.2 μM, respectively) (Table 1, Fig. S1). Oat3 showed the strongest dose-response interaction with ibuprofen, indomethacin, MTX and TPrT (IC50 = 5.98 μM, 0.03 μM, 9.98 μM and 0.28 μM, respectively) (Table 1, Fig. S2).
Interactor
Interactor
Type of interaction
M S S S S I I
5
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
A
control chlorpyrifos
transport rate (nmol/mg protein/min)
transport rate (nmol/mg protein/min)
15
B
15
10
5
Control Ibuprofen
10
5
0
0 0
100
100
0
300
200
200
300
LY ( M)
LY ( M)
C control 2,4-D
transport rate (nmol/mg protein/min)
15
10
5
0
0
100
200
300
LY ( M) Fig. 2. Representative dose-response curves showing different types of interaction with Oat1. A) chlorpyrifos as a competitive inhibitor, B) ibuprofen as a noncompetitive inhibitor, C) 2,4-D showing mixed type of inhibition. Each data point represents the mean ± SD from typical experiment out of three independent determinations. Dotted lines represent confidence intervals.
because furosemide interacts with very strong affinity with zebrafish Oat1 (IC50 = 2.5 μM) and with strong affinity with zebrafish Oat3 (IC50 = 26.4 μM), while it showed intermediate affinity towards hOAT1 (IC50 between 10 and 100 mM) and very strong affinity for hOAT3 (IC50 1.7–7.3 μM). Several xenobiotics from the group of industrials also showed strong affinity towards zebrafish Oat1 and Oat3. PFOA, a toxic environmental contaminant showed very strong interaction with zebrafish Oat1 and Oat3 and was confirmed as a substrate of zebrafish Oat1 (Table 2). PFOA is also a substrate of hOAT1 and hOAT3 (Nakagawa et al., 2007). Organotins are extensively used in various industrial processes. Their yearly consumption reaches 40,000–80,000 t which makes this group of chemicals the most extensively used organometallic chemicals in the world (Cole et al., 2015). Consequently, they are shown to be present in aquatic environments in nanomolar concentrations. In our previous study (Mihaljević et al., 2017) we showed that organotins are strong interactors of zebrafish uptake transporter Oct1, with IC50 values for organotins DBT, TPrT and TBT determined in low μM range. In addition, data from this study showed that TPheT, TBT, TPrT, TET, TMT are very strong interactors of zebrafish Oat1 and Oat3 (Fig. 1). TPrT was identified as a substrate (IC50 = 0.3 μM, Fig. 4, Table 3), while TMT was inhibitor of zebrafish Oat3 (Table 3). In the final part of our study we performed cytotoxicity assays with selected interactors to further confirm that previously identified substances indeed are, or are not transported into the cell via Oat1 and Oat3 transporters. We were unable to determine the cytotoxicity response for all identified interactors due to weak toxicity (they showed low cytotoxicity at very high concentrations) and/or lower solubility in DMEM used as solvent in our experiments. Yet, as was expected cytotoxicity determinations performed using either the CCK-8 or MTT
only if free plasma concentration of these substances is close to their IC50 values determined in vitro. Therefore, we hypothesize that xenobiotics like NSAIDs could also affect homeostasis of endogenous substrates of zebrafish Oat1 and Oat3, like bilirubin, deoxycholic acid, αketoglutarate, pregnenolone sulfate, estrone-3-sulfate, corticosterone (Dragojević et al., 2019). Our data showed that antineoplastic methotrexate is also a substrate of both zebrafish Oat transporters (Tables 2 and 3). In comparison, human OAT3 is shown to mediate a high-affinity transport of methotrexate (Km 5–10.9 μM). Methotrexate is a chemotherapy agent used to treat specific types of cancer like choriocarcinomas, acute lymphoblastic leukemia, and some nonneoplastic diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus and dermatomyositis (Balis et al., 1998; Ohno et al., 1993; Zonneveld et al., 1996; Kipen et al., 1997; Itoh et al., 1999). As methotrexate can cause severe toxic effects, like bone marrow suppression and intestinal epithelial damage (Iqbal et al., 1993; Nakamaru et al., 1997), understanding its pharmacokinetics is of high relevance. Methotrexate is in humans eliminated by urine in its original form, both by glomerular filtration and tubular secretion. It was found that the use of methotrexate with acidic drugs, such as NSAIDs and b-lactam antibiotics, causes significant suppression of bone marrow, and it seemed to be linked to, or caused by, competitive inhibition of the transport of organic anions (Cha et al., 2001). A strong interaction with zebrafish Oat1 and Oat3 was observed for loop diuretic furosemide. Furosemide was shown to be a substrate of hOAT1 and hOAT3, while it was inhibitor of hOAT2. To reach their target transporters from the luminal space, these substances have to be transported in the proximal tubules (Burckhardt and Burckhardt, 2011). Our data show that zebrafish Oat1 and Oat3, just like human OAT1 and OAT3, could be transporters responsible for this secretion, 6
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
120
A oat1 mock
100
80
% viability
% viability
60 40 EC50
0 10 -4
oat1 0.2226
oat3 mock
100
80
20
B
120
60 40 20
mock 1.002
0 10 -4
10 2
10 0
10 -2
oat3 0.2264
EC50
mock 1.002
10 -2
10 2
10 0
log c (TPrT)
log c (TPrT)
Fig. 3. Modulation of TPrT cytotoxicity due to the overexpression of the zebrafish Oat1 (A) and Oat3 (B) in FlpIn/HEK293 cells, as determined using the CKK-8 assay. The modulation is seen as a shift of the dose-response curve to the left (lower EC50 in comparison with the mock-transfected cells). For the purpose of EC50 calculations, data were fitted to the sigmoidal four-parameter dose-response model (variable slope) in GraphPad Prism version 6. Each data point represents the mean ± SD from typical experiment out of three independent determinations.
A
120
120
oat1 mock
100 % viability
% viability
100 80 60 40 20 0 10 -2
oat1 3.334
EC50
B
60 40 20
mock 3.496
0 10 0
10 2
oat3 mock
80
10 -4
10 4
EC50
10 -2
10 0
10 2
oat3 2.690
mock 3.806
10 4
log c (TMT)
log c (TMT)
Fig. 4. TMT cytotoxicity in the zebrafish Oat1- (A) and Oat3-overexpressed (B) FlpIn/HEK293 cells, as determined using the MTT assay. No difference was observed in Oat1- or Oat3-overexpressed FlpIn/HEK293 cells in comparison to mock-transfected cells (similar EC50 values were obtained). For the purpose of EC50 calculations, data were fitted to the sigmoidal four-parameter dose-response model (variable slope) in GraphPad Prism version 6. Each data point represents the mean ± SD from typical experiment out of three independent determinations.
interests or personal relationships that could have appeared to influence the work reported in this paper.
assays resulted in higher susceptibility of overexpressing cells to toxic effect of TPrT, identified in this study as Oat1 and Oat3 substrate, in comparison to mock-transfected cells. TPrT showed EC50 value of 0.22 μM for Oat1 and 0.23 μM for Oat3 (Fig. 2). On the contrary, no significant difference in sensitivity of Oat1 and Oat3-overexpressed cells compared to mock-transfected cells was determined upon exposure to TMT, characterized as a non-competitive inhibitor, suggesting that it is not transported into the cell (Fig. 3). Therefore this approach offered a reliable verification of our determination of the type of interaction performed using Michaelis-Menten kinetics.
Acknowledgments This research described here was financially supported by Project No. 4806 granted by the Croatian National Science Foundation, the SCOPES programme joint research project granted by the Swiss National Science Foundation (SNSF) (Grant No. SCOPES IZ73ZO_152274/1), and by the project STIM – REI, Contract Number: KK.01.1.1.01.0003, a project funded by the European Union through the European Regional Development Fund – the Operational Programme Competitiveness and Cohesion 2014-2020 (KK.01.1.1.01).
5. Conclusions In this study we provide the first data on zebrafish Oat1 and Oat3 interaction with environmental contaminants. We confirmed broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs. Moreover, data obtained using both in vitro transport activity determinations and cytotoxicity assays imply that some of the identified interactors could be of environmental and/or human health concern, making them candidates for more specific follow-up in vivo studies, preferably using zebrafish gene knockouts. Additionally, interaction of Oat1 and Oat3 with pharmaceuticals suggests they could be potential sites of drug-drug interactions.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2020.108742. References Agarwal, S., Chinn, L., Zhang, L., 2013. An overview of transporter information in package inserts of recently approved new molecular entities. Pharm. Res. 30, 899–910. Balis, F.M., Holcenberg, J.S., Poplack, D.G., Ge, J., Sather, H.N., Murphy, R.F., Ames, M.M., Waskerwitz, M.J., Tubergen, D.G., Zimm, S., Gilchrist, G.S., Bleyer, W.A., 1998. Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children's
Declaration of competing interest The authors declare that they have no known competing financial 7
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
626–638. Mihaljević, I., Popović, M., Zaja, R., Maraković, N., Šinko, G., Smital, T., 2017. Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics. Aquat. Toxicol. 187, 18–28. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Murray, K.E., Thomas, S.M., Bodour, A., 2010. Prioritizing research for trace pollutants and emerging contaminants in the freshwater environment. Environ. Pollut. 158, 3462–3471. Nakagawa, H., Hirata, T., Terada, T., Jutabha, P., Miura, D., Harada, K.H., Inoue, K., Anzai, N., Endou, H., Inui, K-i., Kanai, Y., Koizumi, A., 2007. Roles of organic anion transporters in the renal excretion of perfluorooctanoic acid. Basic Clin. Pharmacol. Toxicol. 103, 1–8. Nakamaru, M., Masubuchi, Y., Narimatsu, S., Awazu, S., Horie, T., 1997. Evaluation of damaged small intestine of mouse following methotrexate administration. Cancer Chemother. Pharmacol. 41 (2), 98–102. Nigam, S.K., 2018. The SLC22 transporter family: a paradigm for the impact of drug transporters on metabolic pathways, signaling, and disease. Annu. Rev. Pharmacol. Toxicol. 58, 663–687. Nigam, S.K., Bush, K.T., Martovetsky, G., Ahn, S.-Y., Liu, H.C., Richard, E., Bhatnagar, V., Wu, W., 2015. The organic anion transporter (Oat) family: a systems biology perspective. Physiol. Rev. 95, 83–123. Nishimura, M., Naito, S., 2005. Tissue specific mRNA expression profiles of human ATP binding cassette and solute carrier transporter families. Drug Metab. Pharmacokinet. 20, 452–477. Ohno, Y., Yamauchi, T., Ueda, T., Aizawa, T., Kawakami, S., Tachibana, Y., Kawai, T., Nakagawa, K., Tsuchiya, S., 1993. A case of testicular choriocarcinoma achieving pathological complete response by “COMPE” chemotherapy, consisting of cisplatin, vincristine, methotrexate, peplomycin, and etoposide. Hinyokika Kiyo 39 (2), 183–187. Popovic, M., Zaja, R., Fent, K., Smital, T., 2014. Interaction of environmental contaminants with zebrafish uptake transporter Oatp1d1 (Slco1d1). Toxicol. Appl. Pharmacol. 280, 149–158. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008. Nonylphenol in the environment: A critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ. Int. 34, 1033–1049. Srimaroeng, C., Perry, J.L., Pritchard, J.B., 2008. Physiology, structure, and regulation of the cloned organic anion transporters. Xenobiotica 38 (7–8), 889–935. Tweedie, D., Polli, J.W., Berglund, E.G., Huang, S.M., Zhang, L., Poirier, A., Chu, X., Feng, B., 2013. International Transporter Consortium.Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium. Clin. Pharmacol. Ther. 94, 113–125. VanWert, A.L., Gionfriddo, M.R., Sweet, D.H., 2010. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm. Drug Dispos. 31 (1), 1–71. Westholm, D.E., Salo, D.R., Viken, K.J., Rumbley, J.N., Anderson, G.W., 2009. The blood–brain barrier thyroxine transporter organic anion-transporting polypeptide 1c1 displays atypical transport kinetics. Endocrinology 150, 5153–5162. Whalen, M.M., Loganathan, B.G., Kannan, K., 1999. Immunotoxicity of Environmentally Relevant Concentrations of Butyltins on Human Natural Killer Cells in Vitro. Environ. Res. 81, 108–116 Section A. Yamazaki, M., Li, B., Louie, S.W., Pudvah, N.T., Stocco, R., Wong, W., Abramovitz, M., Demartis, A., Laufer, R., Hochman, J.H., Prueksaritanont, T., Lin, J.H., 2005. Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport. Xenobiotica 35, 737–753. Zonneveld, I.M., Bakker, W.K., Dijkstra, P.F., Bos, J.D., van Soesbergen, R.M., Dinant, H.J., 1996. Methotrexate osteopathy in long-term, low-dose methotrexate treatment for psoriasis and rheumatoid arthritis. Arch. Dermatol. 132 (2), 184–187.
cancer group and pediatric oncology branch study. Blood 92, 3569–3577. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burckhardt, G., 2012. Drug transport by organic anion transporters (OATs). Pharmacol. Ther. 136, 106–130. Burckhardt, B.C., Burckhardt, G., 2003. Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev. Physiol. Biochem. Pharmacol. 146, 95–158. Burckhardt, G., Burckhardt, B.C., 2011. In vitro and in vivo evidence of the importance of organic 504 anion transporters (OATs) in drug therapy. In: Drug Transporters, Edited by Fromm, M. F. and Kim, 505 R. B. vol. 2011. Springer, Berlin, pp. 29–104. Cha, S.H., Sekine, T., Fukushima, J.I., Kanai, Y., Kobayashi, Y., Goya, T., Endou, H., 2001. Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol. 59, 1277–1286. Colas, C., Ung, P.M., Schlessinger, A., 2016. SLC transporters: structure, function, and drug discovery. Med. Chem. Commun. 7, 1069–1081. Cole, R.F., Mills, G.A., Parker, R., Bolam, T., Birchenough, A., Kröger, S., Fones, G.R., 2015. Trends in the analysis and monitoring of organotins in the aquatic environment. Trends Environ. Analyt. Chem. 8, 1–11. Dragojević, J., Mihaljević, I., Popović, M., Smital, T., 2019. Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds. Comp. Biochem. Physiol. B 236 (1–11). Fent, K., Weston, A.A., Caminada, D., 2006. Ecotoxicology of human pharmaceuticals. Aquat. Toxicol. 76, 122–159. Giacomini, K.M., Huang, S.M., Tweedie, D.J., Benet, L.Z., Brouwer, K.L., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., et al., 2010. International transporter consortium. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215–236. Giesy, J.P., Kannan, K., 2002. Perfluorochemical Surfactants in the Environment. Environ. Sci. Technol. 36 (7), 146A–152A. Goodman & Gilman's The Pharmacological Basis of Therapeutics by. Joel Griffith Hardman, Lee E. Limbird, Alfred G. Gilman, 1994. 10th Ed. Gui, C., Wahlgren, B., Lushington, G.H., Hagenbuch, B., 2009. Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17beta-glucuronide transport. Pharmacol. Res. 60, 50–56. Hjelmborg, P.S., Andreassen, T.K., Bonefeld-Jørgensen, E.C., 2008. Cellular uptake of lipoproteins and persistent organic compounds - an update and new data. Environ. Res. 108, 192–198. Hoch, M., 2001. Organotin compounds in the environment — an overview. Appl. Geochem. 16 (7–8), 719–743. Iqbal, M.P., Ali, A.A., Alvi, A.A., 1993. Severe bone marrow suppression in a patient with rheumatoid arthritis on methotrexate. J. Pak. Med. Assoc. 43 (12), 262–263. Itoh, T., Mitsuoka, S., Uji, M., Matsushita, H., 1999. Successful combination chemotherapy with low-dose methotrexate and steroids for dermatomyositis complicated by interstitial pneumonitis. J. Jpn Respir. Soc. 37 (8), 636–640. Kindla, J., Mu, F., Mieth, M., Fromm, M.F., 2011. Influence of non-steroidal antiinflammatory drugs on organic anion transporting polypeptide (OATP) 1B1- and OATP1B3-mediated drug transport. Drug Metab. Dispos. 39, 1047–1053. Kipen, Y., Littlejohn, G.O., Morand, E.F., 1997. Methotrexate use in systemic lupus erythematosus. Lupus 6 985–389. Lee, S.C., Arya, V., Yang, X., Volpe, D.A., Zhang, L., 2017. Evaluation of transporters in drug development: current status and contemporary issues. Adv. Drug Deliv. Rev. 116, 100–118. Lončar, J., Smital, T., 2018. Interaction of environmental contaminants with zebrafish (Danio rerio) multidrug and toxin extrusion protein 7 (Mate7/Slc47a7). Aquat. Toxicol. 205, 193–203. Mihaljevic, I., Popovic, M., Zaja, R., Smital, T., 2016. Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio). BMC Genomics 17,
8
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3 Jelena Dragojević, Petra Marić, Jovica Lončar, Marta Popović, Ivan Mihaljević, Tvrtko Smital
T ⁎
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
A R T I C LE I N FO
A B S T R A C T
Keywords: Oat1 and Oat3 transporters Zebrafish Stable transfectants Environmental contaminants Interaction screening Cytotoxicity
Organic anion transporters (OATs) are transmembrane proteins which belong to SLC22 subfamily. They are responsible for the uptake of various endo- and xenobiotics into the cells of different organs and tissues. Following our previous work on characterization of zebrafish Oat1 and Oat3, in this study we analyzed interaction of various classes of environmental contaminants with these membrane transporters using the transport activity assay with HEK293 Flp-In cell line stably overexpressing zebrafish Oat1 and Oat3, respectively. Based on the initial screening of a series of 36 environmental contaminants on their ability to interact with zebrafish Oat1 and Oat3, the most potent interactors were selected, their IC50 values calculated and type of interaction determined. Finally, to further confirm the type of interaction and initially evaluate their toxic potential, the cytotoxicity assays were performed. Broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs was confirmed and potent interactors among environmental contaminants identified.
1. Introduction After a xenobiotic compound, environmental contaminants included, enters the cell by passive diffusion or through activity of uptake transporters it typically undergoes biotransformation by phase I and II detoxification enzymes, and is finally pumped out of the cell by transmembrane efflux transporters. However, this is not the case for certain compounds such as heavy metals and persistent organic pollutants (POPs), whose lipophilic nature allows them to bioaccumulate in the fatty tissues of living organisms, and therefore are not substrates for active or facilitated transport by membrane transporters (Hjelmborg et al., 2008). The most important organs for metabolization of xenobiotics in vertebrates are liver and kidney, and once a xenobiotic becomes hydrophilic it is typically excreted out via bile and urine (Goodman and Gilman, 1994). Consequently, it has been found that numerous drug transporters actively participate in the disposition of endogenously formed metabolites and xenobiotics, in addition to involvement in drug-drug interactions (DDIs) of various drugs and new molecular entities (Agarwal et al., 2013; Lee et al., 2017). Among the most important drug transporters listed in white papers by the International Transporter Consortium (Giacomini et al., 2010; Tweedie et al., 2013) are organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) that belong to the SLC22A gene family. They
transport chemically and structurally different endogenous and exogenous organic anions from the blood into the cells of diverse human and animal organs. Because of their substantial and strategic localization in organs important for drug disposition they have critical roles in the absorption, distribution, metabolism, and elimination (ADME) of drugs and xenobiotics (Giacomini et al., 2010; Colas et al., 2016). Human OAT1 and OAT3 are predominantly expressed in kidneys, specifically in the basolateral membrane of renal proximal tubule cells where they mediate the uptake of chemicals from the blood (Nishimura and Naito, 2005; Burckhardt and Burckhardt, 2011). Besides their physiological role in maintenance of systemic levels of endogenous substrates (uric acid, signalling molecules etc.), OAT1 and OAT3 are important drug transporters in kidney where they actively secrete drugs into the urine. Human OAT1 and OAT3 share similar substrate preferences, and their reported drug substrates include NSAIDs, penicilin, methotrexate, HIV protease inhibitors and antiviral drugs. Inhibition of these OATs results in impaired elimination of drugs from kidneys (Nigam, 2018). Zebrafish (Danio rerio) is a significant vertebrate model species increasingly used both for biomedical studies and in environmental science. We recently identified seven Oats in zebrafish genome, which are phylogenetically classified into three subfamilies: OAT1/Oat1, OAT2/ Oat2 and OAT3/Oat3 (Mihaljevic et al., 2016). Zebrafish oat1 and oat3
⁎ Corresponding author at: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia. E-mail address: [email protected] (T. Smital).
https://doi.org/10.1016/j.cbpc.2020.108742 Received 4 December 2019; Received in revised form 28 February 2020; Accepted 6 March 2020 Available online 10 March 2020 1532-0456/ © 2020 Elsevier Inc. All rights reserved.
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
2.2. Transport activity assays
genes showed one-to-one orthology to their mammalian orthologs which indicate possible conservation of OAT1 and OAT3 physiological or defense role(s) in vertebrates. Using the conserved synteny analysis of both zebrafish oat genes we also showed that they are in zebrafish located on the same chromosome 21 (Dragojević et al., 2019), similar to human OAT1 and OAT3 which are both located on chromosome 11. The qPCR expression analysis of Oat1 transcript in adult zebrafish tissues showed the highest expression in testes and brain in females, and low expression in gills and liver in both genders. Oat3 showed the highest expression of all Oats in zebrafish, with very high expression in female kidneys, lower expression in male kidneys, testes and intestine, with higher prevalence in females. Additionally, zebrafish Oat1 and Oat3 showed broad ligand selectivity, similar to their mammalian orthologs, and their potential physiological interactors have been identified (Dragojević et al., 2019). Based on these initial insights, in this study we specifically address the interaction of zebrafish Oat1 and Oat3 with environmental contaminants. Considering their tissue expression pattern and the first data on their substrate preferences obtained in our recent studies, we presume that a similar interaction of zebrafish Oat1 and Oat3 with anionic xenobiotics can be expected, including overlapping substrate specificities. Furthermore, considering potential ecotoxicological relevance, we hypothesize that interactions of environmental compounds with Oat1 and Oat3 transporters may be deleterious due to either (1) increased accumulation of potentially toxic xenobiotic substances caused by increased Oat-mediated uptake and/or modulated excretion of these substances, or (2) compromised transport of physiologically relevant Oat1/Oat3 substrates which in turn can cause changes in physiological homeostasis. To address our main goal, we tested the interaction of zebrafish Oat1 and Oat3 stably overexpressed in HEK293 Flp-In cell line with 36 environmental contaminants broadly classified into three groups: industrial chemicals (industrials), pesticides and pharmaceuticals and personal care products (PPCPs) (Murray et al., 2010). Our selection of chemicals was based on three primary criteria: (1) their occurrence in surface waters (Fent et al., 2006; Murray et al., 2010), (2) reported interactions with human or mammalian OATs/Oats (reviewed in Burckhardt and Burckhardt, 2003; Nigam et al., 2015; Srimaroeng et al., 2008), and/or (3) their anionic character. Furthermore, although not all of the compounds tested are found in significant concentrations in the environment, many of them have a considerable bioaccumulation potential (e.g., PFOA, PFOS, nonylphenol, organotins) with reported bioconcentration factors over 10,000 (Giesy et al., 2002; Hoch et al., 2001; Soares et al., 2008; Whalen et al., 1999), which implies their potential ecotoxicological relevance. Compounds that showed potent interaction were further analyzed in order to determine the type of their interaction (substrates versus inhibitors) with zebrafish Oat1 and Oat3. Finally, to further confirm the type of interaction for the most potent interactors and initially evaluate their toxic potential we performed cytotoxicity assays with Oat1- and Oat3-overexpressing cells.
Cloning of zebrafish Oat1 and Oat3 and development of the Oat1 and Oat3 stable transfectants has been described in detail in our previous paper (Dragojević et al., 2019). HEK Flp-In cells stably expressing zebrafish Oat1 or Oat3 and mock-transfected cells were seeded at densities of 5*105 cells/cm2 or 6*105 cells/cm2 (depending on the growth rate) in 96-well plates 48 h before performing the transport assay, in DMEM/FBS medium with final volume of 125 μL per well. DMEM-FBS was removed and cells were pre-incubated in 100 μL of the transport medium (145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM D-glucose and 5 mM HEPES) for 10 min at 37 °C. For determination of transport rates and dose-responses of model fluorescent substrates, 25 μL of concentrated (5×) fluorescent substrates previously identified by Dragojević et al. (2019) (Lucifer yellow (LY) for Oat1 and 6-carboxyfluorescein (6-CF) for Oat3) were added to the pre-incubation medium. Following the incubation (10 min at 37 °C), the cells were washed two times with 125 μL of cold transport medium and lysed with 125 μL of 0.1% sodium dodecyl sulfate (SDS) for 30 min at 37 °C. Cell lysates were transferred to 96-well black microplates and the fluorescence of model substrate determined using the microplate reader (Infinite M200, Tecan, Salzburg, Austria), at 425 nm excitation and 540 nm emission for LY, and 492 nm excitation and 524 nm emission for 6-CF, respectively. In order to calculate the transport rates, fluorescence response of mock-transfected control cells was subtracted from the fluorescence of transfected cells, followed by normalization of the fluorescence responses by calibration curves determined previously for each model substrate and adjusted for the protein content. Calibration curves for model fluorescent substrates were generated in the cell matrix dissolved in the 0.1% SDS. Bradford assay (Bradford, 1976) was used for measuring total protein content. The normalized uptake of fluorescent substrates was finally expressed in nM of substrate per mg of protein per minute. Following determination of transport kinetics for model fluorescent substrates, the inhibition assay based on co-exposure of transfected cells and mock-transfected control with model substrate and potential interactor were performed. The cells were first pre-incubated in transport medium (10 min) followed by 40 s incubation with test compounds and 10 min incubation with the model substrate at concentration that were in the linear part of the dose response curve (50 μM LY, 5 μM 6-CF) as previously determined. Stock solutions of chemicals were prepared in dimethyl sulfoxide (DMSO; maximal concentration 1% of the total volume). The initial interaction assays were performed at only one concentration of the tested compounds set nominally at 100 μM, unless stated otherwise due to water solubility limitations for some of the compounds tested (Table S1, Fig. 1). Detailed dose response experiments were then performed for all interactors that showed over 70% inhibition of the model substrate uptake and the respective IC50 values determined. Compounds with IC50 values in nanomolar and low micromolar range (< 5 μM) were arbitrarily considered to be very strong interactors, those with IC50s of 5–20 μM were designated as strong interactors, Ki of 20–100 μM indicated moderate interaction, whereas substances with determined IC50 values above 100 μM were classified as weak interactors. After the IC50 values were obtained, the type of interaction for the most potent xenobiotic compounds with zebrafish Oat1 and Oat3 was determined by determining the change in Michaelis-Menten kinetics parameters of LY or 6-CF uptake in the presence and absence of a tested xenobiotic. Concentrations of tested xenobiotics corresponded to their IC50 values. If an interacting compound competitively inhibits the uptake of fluorescent model substrate (i.e., it is a substrate), it will increase Km, but will not affect Vmax value. On the contrary, if the inhibition is noncompetitive, Vmax will decrease while Km will not change. Noncompetitive inhibitors bind away from the active site, and can interact with the transporter independently of the presence of the substrate. Another type of interaction is an uncompetitive one, in which
2. Materials and methods 2.1. Chemicals Model fluorescent substrates, environmental contaminants, and other used substances were obtained from Sigma-Aldrich (Taufkirchen, Germany), Alfa Aesar (Ward Hill, MA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless stated otherwise. Names, abbreviations and other relevant data for tested environmental contaminants (including water solubility, maximal concentrations reported in the environment, maximal concentrations determined in blood/ plasma/serum/animal tissue; and related references) are presented in Supplementary Material (Tables S1 and S2).
2
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
3
(caption on next page)
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
Fig. 1. Interaction of zebrafish Oat1 and Oat3 with environmental contaminants. The initial screening presented was performed by exposing transfected cells to 100 μM concentration of tested substances unless stated otherwise (*) due to limited water solubility for some substances (exposure concentration for nonylphenol was 30 μM, 4 μM for chlorpyrifos, 5 μM for erythromycin, 15 μM for Diclofenac, and 8 μM for indomethacin). Data are expressed as mean percentage (%) ± SD from triplicate determinations of LY or 6-CF uptake after co-incubation with each interactor (100 μM) relative to LY/6-CF uptake in the absence of interactor which is set to 100%.
2.4. Data analysis
both Vmax and Km decrease. Uncompetitive inhibitors have a separate binding site, like noncompetitive inhibitors, but they only bind the transporter-substrate complex. Finally, some interactors may show a mixed inhibition, causing a decrease in Vmax, but also an increase in Km. The described approach for distinguishing among substrates and inhibitors of membrane proteins using Michaelis-Menten kinetics determinations is the only currently available approach for examining a large set of compounds for transporters without their structures solved at high resolution. The approach has been successfully used in numerous previous studies published by other research groups (Yamazaki et al., 2005; Gui et al., 2009; Westholm et al., 2009; Kindla et al., 2011), as well as by our group (Popovic et al., 2014; Mihaljević et al., 2017; Lončar and Smital, 2018; Dragojević et al., 2019).
All transport assays were repeated in at least three independent experiments run in triplicates, and data from a typical experiment are shown in related figures. GraphPad Prism 6 software for Windows was used for all calculations. For the initial interaction analyses performed with fixed concentration of tested xenobiotics, independent-samples Student t-test was applied to evaluate statistically significant difference in the uptake of a model substrate (LY or 6-CF) in transfected versus mock-transfected (control) cells. Level of significance was set at P < 0.05. The kinetic parameters, Km and Vmax values were calculated using the Michaelis-Menten Eq. (1):
V= 2.3. Cytotoxicity assays
Vmax × [S ] S + Km
(1)
where V is velocity (nanomoles of substrate per milligram of proteins per minute), Vmax is maximal velocity, [S] is substrate concentration and Km is the Michaelis-Menten constant. For cytotoxicity assays, cell viability was expressed as percentages of viability obtained from triplicate determinations, with mean ± standard deviation (SD) values. Sigmoidal dose-response curves with 95% confidence intervals (CIs) were obtained using non-linear regression model in GraphPad Prism 6 software for Windows. Classical four parameters sigmoidal dose-response curves were used to obtain the response (y) according to the Eq. (2):
Cytotoxicity of Oat1 and Oat3 interactors was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric reduction assay (originally described by Mosmann, 1983). The assay was performed with HEK Flp-In cells stably overexpressing zebrafish Oat1 and Oat3 uptake transporters and HEK Flp-In/mock cells which were used as a control. Viability of the cells was assessed after the exposure of both stably transfected and mock cells to Oat1 and Oat3 interactors for 72 h. HEK Flp-In/Oat1 and Flp-In/Oat3 cells were seeded in 48-well plates with 5 × 105 cells/mL cultivation density and at 250 μL cells/well seeding volume. Following the 24 h incubation, the cells were exposed to serial dilutions of tested chemicals prepared in DMEM medium without FBS. Half of the medium (125 μL) was removed from each well and replaced with 125 μL of sample dilutions in triplicates. HEK Flp-In/Oat1 and Flp-In/Oat3 cells were then exposed for 72 h to various concentrations of tested substances. Afterwards, 200 μL of medium was removed from the wells and for the next 2.5 h cells were incubated with 200 μL/well of MTT reagent prepared in DMEM medium to reach final concentration of 0.5 mg/mL. Formed formazan salts were dissolved in 200 μL/well of 2-propanol and plates were shaken at 350 rpm. Absorbance was measured using microplate reader at 570 nm with reference filter set to 750 nm. Differences between the measurement filter and the reference filter values were calculated in Microsoft Office Excel 2007. Serial dilutions were log transformed and the obtained values normalized to percentages prior to statistical analysis. Interactors of Oat1 and Oat3 transporters, respectively, whose cytotoxic effect was detected by MTT assay were additionally evaluated using the CCK-8 colorimetric test (Cell Counting Kit-8, Sigma-Aldrich) due to higher detection sensitivity of WST-8 water-soluble tetrazolium salt in order to obtain more accurate data. HEK293 Flp-In/Oat1 and Flp-In/Oat3 cells were seeded in 96-well plates with 2 × 105 cells/mL cultivation density and at 100 μL cells/well seeding volume. After 24 h incubation, the cells were exposed to serial dilutions of tested chemicals prepared in DMEM medium without FBS. Concentration of solvent (DMSO) never exceeded 1% of the total volume. Half of the medium (50 μL) was removed and replaced with 50 μL of sample dilutions in duplicates. Concentrations of tested chemicals were in the range from 0.001 to 3 μM. After exposure period of 72 h, 10 μL of CCK-8 solution were added to each well. After 3 h of incubation, absorbance at 450 nm was measured with the microplate reader. Viability was calculated from duplicate determinations, as described for MTT assay.
y = b + (a–b)/(1 + 10((logLC 50– x ) ∗ h)))
(2)
where b is the minimum (bottom) of response, a represents the maximum (top) response, hillslope (h) is slope of the curve, logLC50 is halfway response from bottom to top and x is the logarithm of inhibitor concentration. 3. Results 3.1. Inhibition tests 3.1.1. The interaction screen assay By using the developed transport activity assay, a series of environmental contaminants reported to be xenobiotic interactors of mammalian OAT1/Oat1 and OAT3/Oat3 was initially tested. Using this assay we found that Oat1 and Oat3 showed interaction with a wide range of xenobiotic compounds (Fig. 1), with high level of overlapping interactors. Xenobiotics that showed the strongest interaction with Oat1 were: indomethacin (0.35% LY uptake remaining in comparison to mock-transfected cells), MK-571 (0.35% LY uptake), diclofenac (1.01% LY uptake), furosemide (2.32% LY uptake), tributyltin (TBT) (5.7% LY uptake), ibuprofen (6.57% LY uptake), caffeine (8.49% LY uptake), probenecid (9.17% LY uptake), methotrexate (15.2% LY uptake), perfluorooctanoic acid (PFOA) (16.63% LY uptake), perfluorooctanesulfonic acid (PFOS) (17.18% LY uptake), 2,4-dichlorophenoxyacetic acid (17.76% LY uptake) and tripropyltin (TPrT) (19.73% LY uptake). The most potent Oat3 interactors were: MK-571 (0% 6-CF uptake), probenecid (0.3% 6-CF uptake), diclofenac (0.86% 6-CF uptake), indomethacin (3.22% 6-CF uptake), TBT (4.36% 6-CF uptake), tripropyltin (TPrT) (4.92% 6-CF uptake), trimethyltin (TMT) (5.44% 6-CF uptake), methotrexate (5.65% 6-CF uptake), triethyltin (TET) (7.56% 6CF uptake), ibuprofen (10.6% 6-CF uptake), 2,4-dichlorophenoxyacetic 4
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
Table 1 IC50 values of the most potent zebrafish Oat1 and Oat3 interactors. Each data point represents the mean ± SD from typical experiment out of three independent determinations. Oat1
Table 3 Type of interaction determined for set of zebrafish Oat3 interactors. Uptake kinetics parameters for 6-CF are given as Km (μM), Vmax (nmol 6-CF/mg protein/min), and 95% confidence intervals (c.i.) for each. The mark “S” denotes Oat3 substrates, and the “I” denotes inhibitors. Data are mean ± SD from typical experiment out of three independent determinations.
Oat3
Compound
IC50 (μM)
Compound
IC50 (μM)
Indomethacin Ibuprofen Diclofenac Tri-n-propyltin 2,4-D PFOS PFOA Chlorpyrifos Trimethyltin Methotrexate MK571
0.05 ± 0.16 1 ± 0.06 4.2 ± 1.03 6.63 ± 1.27 13.62 ± 1.12 16.75 ± 1.14 32.48 ± 1.16 39.04 ± 1.12 43.66 ± 1.09 56.4 ± 1.1 1.41 ± 1.1
Indomethacin Tri-n-propyltin Triethyltin Ibuprofen TRIPHENYLTIN PROBENECID 2,4-D Diclofenac Methotrexate Trimethyltin PFOS
0.03 ± 1.08 0.28 ± 1.2 0.43 ± 1.22 5.98 ± 1.12 6.99 ± 1.19 7.39 ± 1.15 7.56 ± 1.23 8.49 ± 1.32 9.98 ± 1.3 11.5 ± 1.1 43.94 ± 1.1
acid (11.23% 6-CF uptake), PFOA (15.4% 6-CF uptake), triphenyltin (TpheT) (15.94% 6-CF uptake), furosemide (26.37% 6-CF uptake), PFOS (28.84% 6-CF uptake) and PAH (29.07% 6-CF uptake).
3.3. Cytotoxicity assays To further evaluate the type of interaction for selected compounds Table 2 Type of interaction determined for set of zebrafish Oat1 interactors. Uptake kinetics parameters for LY are given as Km (μM), Vmax (nmol LY/mg protein/ min), and 95% confidence intervals (c.i.) for each. The mark “S” denotes Oat1 substrates, the “I” denotes inhibitors, and “M” refers to the mixed type of inhibition. Data are mean ± SD from typical experiment out of three independent determinations. Vmax (LY)
c.i.
Control (LY)
17,2
13.48–20.92
10,07
9.42–10.72
2,4-D Chlorpyrifos PFOA Indomethacin Methotrexate Trimethyltin Ibuprofen
27.4 26.13 36.44 25.09 29.89 16.59 14.66
23.06–31.73 16.57–35,69 29.20–43.67 20.82–30.97 16.62–43.17 9.92–23.26 11.90–17.41
6.73 11.65 11.69 10.25 11.31 4.54 9.21
6.45–7.00 10.56–12.75 10.67–12.51 9.55–10.94 9.39–13.24 3.97–5.11 8,64 - 9,37
Vmax (6-CF)
c.i.
Control (6-CF)
18.65
12.56–24.74
22.53
19.26–25.79
PFOS Probenecid Diclofenac 2,4-D Indomethacin Methotrexate Ibuprofen Trimethyltin Tri-n-propyltin
3.57 26.42 31.29 28.82 28.04 64.38 18.33 12.21 38.56
16.05–31.62 20.46–32.39 25.57–37.01 21.07–36.57 25.74–30.34 50.40–78.36 14.64–22.02 1.84–22.57 26.26–50.86
27.38 44.50 47.77 38.98 22.56 24.45 19.10 14.94 10.93
22.64–32.12 39.46–49.54 43.14–52.41 33.56–44.39 18.76–26.36 20.95–27.95 17.40–20.79 10.67–19.24 1.33–20.53
Type of interaction
S S S S S S I I S
Despite being recognized and described as integral elements of the ADME processes in mammals, Oats have not been comprehensively studied in non-mammalian species. Therefore, in this study we analyzed the interaction of different classes of environmentally relevant contaminants with zebrafish Oat1 and Oat3 uptake transporters. To specifically and reliably identify zebrafish Oat1 and Oat3 interactors, and determine their potency and type of interaction, we used previously developed HEK293 Flp-In cell lines characterized by stable over-expression of Oat1 and Oat3 transporters, respectively. Using this model we optimized a high throughput assay based on determination of transport rate of fluorescent model substrates (LY for Oat1; 6-CF for Oat3) in real time. Using the initial screening assay, interaction of Oat1 and Oat3 with numerous tested environmental contaminants was determined, confirming wide and overlapping substrate preferences of these transporters (Fig. 1). The strongest interaction with Oat1 and Oat3 was observed for NSAIDs indomethacin and ibuprofen, antineoplastic methotrexate, diuretic furosemide, and for industrials PFOA, PFOS, and organotin compounds (TMT, TET, TPrT, TBT). Michaelis-Menten kinetics showed that indomethacin is a substrate, while ibuprofen is an inhibitor of both zebrafish Oat1 and Oat3 (Tables 2 and 3). As already reported, various NSAIDs were capable of interaction with human OAT1 and rat Oat1. Among others, high affinities were reported for ibuprofen and indomethacin, with comparable IC50 values for hOAT1 and rOat1 (Burckhardt and Burckhardt, 2011; VanWert et al., 2010). Indomethacin was shown to be high affinity substrate of human OAT1, while ambiguous results have been obtained for ibuprofen (Burckhardt, 2012). Therefore, although NSAIDs can inhibit transport activity of mammalian OAT1/Oat1, their transport rate is rather low. However, the authors concluded that inhibition of OAT1/Oat1 by these drugs could potentially modulate secretion of other anionic compounds, but
After the IC50 values were obtained, the type of interaction for the strongest interactors with zebrafish Oat1 and Oat3 was determined (Tables 2 and 3). Determination was based on comparison of kinetic parameters of LY or 6-CF uptake in the presence and the absence of different interacting compounds, respectively, where their concentrations were equal to their previously calculated IC50 values.
c.i.
c.i.
4. Discussion
3.2. Determining the type of interaction
Km (LY)
Km (6-CF)
as previously determined using the Michaelis-Menten kinetics experiments, we exposed the FlpIn/HEK293 cells stably transfected with Oat1 or Oat3 and mock-transfected (control) cells to a potent competitive inhibitor TPrT identified in the previous steps of our study. As expected, shifts in related dose-response curves were observed (Fig. 3). Oat1 and Oat3-overexpressing cells were more responsive to toxic effect of TPrT, resulting in lower EC50 values in comparison to mock-transfected cells. The 4.5 and 4.4-fold changes in EC50 values for TPrT were obtained with Oat1 and Oat3-overexpressing cells, respectively. In contrast, when exposed to previously identified noncompetitive inhibitor TMT, no significant difference in EC50 values, or shift in related dose-response curves were observed (Fig. 4).
3.1.2. Dose-response assays Based on results of the initial screening, interactors that showed the highest potency for inhibition of the model substrate uptake were subjected to dose-response analyses and determination of their IC50 values for both Oats. The strongest interaction with Oat1 was determined for ibuprofen, indomethacin and diclofenac. All of these substances showed concentration dependent inhibition of Oat1 mediated LY transport (IC50 = 1 μM, 0.05 μM and 4.2 μM, respectively) (Table 1, Fig. S1). Oat3 showed the strongest dose-response interaction with ibuprofen, indomethacin, MTX and TPrT (IC50 = 5.98 μM, 0.03 μM, 9.98 μM and 0.28 μM, respectively) (Table 1, Fig. S2).
Interactor
Interactor
Type of interaction
M S S S S I I
5
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
A
control chlorpyrifos
transport rate (nmol/mg protein/min)
transport rate (nmol/mg protein/min)
15
B
15
10
5
Control Ibuprofen
10
5
0
0 0
100
100
0
300
200
200
300
LY ( M)
LY ( M)
C control 2,4-D
transport rate (nmol/mg protein/min)
15
10
5
0
0
100
200
300
LY ( M) Fig. 2. Representative dose-response curves showing different types of interaction with Oat1. A) chlorpyrifos as a competitive inhibitor, B) ibuprofen as a noncompetitive inhibitor, C) 2,4-D showing mixed type of inhibition. Each data point represents the mean ± SD from typical experiment out of three independent determinations. Dotted lines represent confidence intervals.
because furosemide interacts with very strong affinity with zebrafish Oat1 (IC50 = 2.5 μM) and with strong affinity with zebrafish Oat3 (IC50 = 26.4 μM), while it showed intermediate affinity towards hOAT1 (IC50 between 10 and 100 mM) and very strong affinity for hOAT3 (IC50 1.7–7.3 μM). Several xenobiotics from the group of industrials also showed strong affinity towards zebrafish Oat1 and Oat3. PFOA, a toxic environmental contaminant showed very strong interaction with zebrafish Oat1 and Oat3 and was confirmed as a substrate of zebrafish Oat1 (Table 2). PFOA is also a substrate of hOAT1 and hOAT3 (Nakagawa et al., 2007). Organotins are extensively used in various industrial processes. Their yearly consumption reaches 40,000–80,000 t which makes this group of chemicals the most extensively used organometallic chemicals in the world (Cole et al., 2015). Consequently, they are shown to be present in aquatic environments in nanomolar concentrations. In our previous study (Mihaljević et al., 2017) we showed that organotins are strong interactors of zebrafish uptake transporter Oct1, with IC50 values for organotins DBT, TPrT and TBT determined in low μM range. In addition, data from this study showed that TPheT, TBT, TPrT, TET, TMT are very strong interactors of zebrafish Oat1 and Oat3 (Fig. 1). TPrT was identified as a substrate (IC50 = 0.3 μM, Fig. 4, Table 3), while TMT was inhibitor of zebrafish Oat3 (Table 3). In the final part of our study we performed cytotoxicity assays with selected interactors to further confirm that previously identified substances indeed are, or are not transported into the cell via Oat1 and Oat3 transporters. We were unable to determine the cytotoxicity response for all identified interactors due to weak toxicity (they showed low cytotoxicity at very high concentrations) and/or lower solubility in DMEM used as solvent in our experiments. Yet, as was expected cytotoxicity determinations performed using either the CCK-8 or MTT
only if free plasma concentration of these substances is close to their IC50 values determined in vitro. Therefore, we hypothesize that xenobiotics like NSAIDs could also affect homeostasis of endogenous substrates of zebrafish Oat1 and Oat3, like bilirubin, deoxycholic acid, αketoglutarate, pregnenolone sulfate, estrone-3-sulfate, corticosterone (Dragojević et al., 2019). Our data showed that antineoplastic methotrexate is also a substrate of both zebrafish Oat transporters (Tables 2 and 3). In comparison, human OAT3 is shown to mediate a high-affinity transport of methotrexate (Km 5–10.9 μM). Methotrexate is a chemotherapy agent used to treat specific types of cancer like choriocarcinomas, acute lymphoblastic leukemia, and some nonneoplastic diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus and dermatomyositis (Balis et al., 1998; Ohno et al., 1993; Zonneveld et al., 1996; Kipen et al., 1997; Itoh et al., 1999). As methotrexate can cause severe toxic effects, like bone marrow suppression and intestinal epithelial damage (Iqbal et al., 1993; Nakamaru et al., 1997), understanding its pharmacokinetics is of high relevance. Methotrexate is in humans eliminated by urine in its original form, both by glomerular filtration and tubular secretion. It was found that the use of methotrexate with acidic drugs, such as NSAIDs and b-lactam antibiotics, causes significant suppression of bone marrow, and it seemed to be linked to, or caused by, competitive inhibition of the transport of organic anions (Cha et al., 2001). A strong interaction with zebrafish Oat1 and Oat3 was observed for loop diuretic furosemide. Furosemide was shown to be a substrate of hOAT1 and hOAT3, while it was inhibitor of hOAT2. To reach their target transporters from the luminal space, these substances have to be transported in the proximal tubules (Burckhardt and Burckhardt, 2011). Our data show that zebrafish Oat1 and Oat3, just like human OAT1 and OAT3, could be transporters responsible for this secretion, 6
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
120
A oat1 mock
100
80
% viability
% viability
60 40 EC50
0 10 -4
oat1 0.2226
oat3 mock
100
80
20
B
120
60 40 20
mock 1.002
0 10 -4
10 2
10 0
10 -2
oat3 0.2264
EC50
mock 1.002
10 -2
10 2
10 0
log c (TPrT)
log c (TPrT)
Fig. 3. Modulation of TPrT cytotoxicity due to the overexpression of the zebrafish Oat1 (A) and Oat3 (B) in FlpIn/HEK293 cells, as determined using the CKK-8 assay. The modulation is seen as a shift of the dose-response curve to the left (lower EC50 in comparison with the mock-transfected cells). For the purpose of EC50 calculations, data were fitted to the sigmoidal four-parameter dose-response model (variable slope) in GraphPad Prism version 6. Each data point represents the mean ± SD from typical experiment out of three independent determinations.
A
120
120
oat1 mock
100 % viability
% viability
100 80 60 40 20 0 10 -2
oat1 3.334
EC50
B
60 40 20
mock 3.496
0 10 0
10 2
oat3 mock
80
10 -4
10 4
EC50
10 -2
10 0
10 2
oat3 2.690
mock 3.806
10 4
log c (TMT)
log c (TMT)
Fig. 4. TMT cytotoxicity in the zebrafish Oat1- (A) and Oat3-overexpressed (B) FlpIn/HEK293 cells, as determined using the MTT assay. No difference was observed in Oat1- or Oat3-overexpressed FlpIn/HEK293 cells in comparison to mock-transfected cells (similar EC50 values were obtained). For the purpose of EC50 calculations, data were fitted to the sigmoidal four-parameter dose-response model (variable slope) in GraphPad Prism version 6. Each data point represents the mean ± SD from typical experiment out of three independent determinations.
interests or personal relationships that could have appeared to influence the work reported in this paper.
assays resulted in higher susceptibility of overexpressing cells to toxic effect of TPrT, identified in this study as Oat1 and Oat3 substrate, in comparison to mock-transfected cells. TPrT showed EC50 value of 0.22 μM for Oat1 and 0.23 μM for Oat3 (Fig. 2). On the contrary, no significant difference in sensitivity of Oat1 and Oat3-overexpressed cells compared to mock-transfected cells was determined upon exposure to TMT, characterized as a non-competitive inhibitor, suggesting that it is not transported into the cell (Fig. 3). Therefore this approach offered a reliable verification of our determination of the type of interaction performed using Michaelis-Menten kinetics.
Acknowledgments This research described here was financially supported by Project No. 4806 granted by the Croatian National Science Foundation, the SCOPES programme joint research project granted by the Swiss National Science Foundation (SNSF) (Grant No. SCOPES IZ73ZO_152274/1), and by the project STIM – REI, Contract Number: KK.01.1.1.01.0003, a project funded by the European Union through the European Regional Development Fund – the Operational Programme Competitiveness and Cohesion 2014-2020 (KK.01.1.1.01).
5. Conclusions In this study we provide the first data on zebrafish Oat1 and Oat3 interaction with environmental contaminants. We confirmed broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs. Moreover, data obtained using both in vitro transport activity determinations and cytotoxicity assays imply that some of the identified interactors could be of environmental and/or human health concern, making them candidates for more specific follow-up in vivo studies, preferably using zebrafish gene knockouts. Additionally, interaction of Oat1 and Oat3 with pharmaceuticals suggests they could be potential sites of drug-drug interactions.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2020.108742. References Agarwal, S., Chinn, L., Zhang, L., 2013. An overview of transporter information in package inserts of recently approved new molecular entities. Pharm. Res. 30, 899–910. Balis, F.M., Holcenberg, J.S., Poplack, D.G., Ge, J., Sather, H.N., Murphy, R.F., Ames, M.M., Waskerwitz, M.J., Tubergen, D.G., Zimm, S., Gilchrist, G.S., Bleyer, W.A., 1998. Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children's
Declaration of competing interest The authors declare that they have no known competing financial 7
Comparative Biochemistry and Physiology, Part C 231 (2020) 108742
J. Dragojević, et al.
626–638. Mihaljević, I., Popović, M., Zaja, R., Maraković, N., Šinko, G., Smital, T., 2017. Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics. Aquat. Toxicol. 187, 18–28. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Murray, K.E., Thomas, S.M., Bodour, A., 2010. Prioritizing research for trace pollutants and emerging contaminants in the freshwater environment. Environ. Pollut. 158, 3462–3471. Nakagawa, H., Hirata, T., Terada, T., Jutabha, P., Miura, D., Harada, K.H., Inoue, K., Anzai, N., Endou, H., Inui, K-i., Kanai, Y., Koizumi, A., 2007. Roles of organic anion transporters in the renal excretion of perfluorooctanoic acid. Basic Clin. Pharmacol. Toxicol. 103, 1–8. Nakamaru, M., Masubuchi, Y., Narimatsu, S., Awazu, S., Horie, T., 1997. Evaluation of damaged small intestine of mouse following methotrexate administration. Cancer Chemother. Pharmacol. 41 (2), 98–102. Nigam, S.K., 2018. The SLC22 transporter family: a paradigm for the impact of drug transporters on metabolic pathways, signaling, and disease. Annu. Rev. Pharmacol. Toxicol. 58, 663–687. Nigam, S.K., Bush, K.T., Martovetsky, G., Ahn, S.-Y., Liu, H.C., Richard, E., Bhatnagar, V., Wu, W., 2015. The organic anion transporter (Oat) family: a systems biology perspective. Physiol. Rev. 95, 83–123. Nishimura, M., Naito, S., 2005. Tissue specific mRNA expression profiles of human ATP binding cassette and solute carrier transporter families. Drug Metab. Pharmacokinet. 20, 452–477. Ohno, Y., Yamauchi, T., Ueda, T., Aizawa, T., Kawakami, S., Tachibana, Y., Kawai, T., Nakagawa, K., Tsuchiya, S., 1993. A case of testicular choriocarcinoma achieving pathological complete response by “COMPE” chemotherapy, consisting of cisplatin, vincristine, methotrexate, peplomycin, and etoposide. Hinyokika Kiyo 39 (2), 183–187. Popovic, M., Zaja, R., Fent, K., Smital, T., 2014. Interaction of environmental contaminants with zebrafish uptake transporter Oatp1d1 (Slco1d1). Toxicol. Appl. Pharmacol. 280, 149–158. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008. Nonylphenol in the environment: A critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ. Int. 34, 1033–1049. Srimaroeng, C., Perry, J.L., Pritchard, J.B., 2008. Physiology, structure, and regulation of the cloned organic anion transporters. Xenobiotica 38 (7–8), 889–935. Tweedie, D., Polli, J.W., Berglund, E.G., Huang, S.M., Zhang, L., Poirier, A., Chu, X., Feng, B., 2013. International Transporter Consortium.Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium. Clin. Pharmacol. Ther. 94, 113–125. VanWert, A.L., Gionfriddo, M.R., Sweet, D.H., 2010. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm. Drug Dispos. 31 (1), 1–71. Westholm, D.E., Salo, D.R., Viken, K.J., Rumbley, J.N., Anderson, G.W., 2009. The blood–brain barrier thyroxine transporter organic anion-transporting polypeptide 1c1 displays atypical transport kinetics. Endocrinology 150, 5153–5162. Whalen, M.M., Loganathan, B.G., Kannan, K., 1999. Immunotoxicity of Environmentally Relevant Concentrations of Butyltins on Human Natural Killer Cells in Vitro. Environ. Res. 81, 108–116 Section A. Yamazaki, M., Li, B., Louie, S.W., Pudvah, N.T., Stocco, R., Wong, W., Abramovitz, M., Demartis, A., Laufer, R., Hochman, J.H., Prueksaritanont, T., Lin, J.H., 2005. Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport. Xenobiotica 35, 737–753. Zonneveld, I.M., Bakker, W.K., Dijkstra, P.F., Bos, J.D., van Soesbergen, R.M., Dinant, H.J., 1996. Methotrexate osteopathy in long-term, low-dose methotrexate treatment for psoriasis and rheumatoid arthritis. Arch. Dermatol. 132 (2), 184–187.
cancer group and pediatric oncology branch study. Blood 92, 3569–3577. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burckhardt, G., 2012. Drug transport by organic anion transporters (OATs). Pharmacol. Ther. 136, 106–130. Burckhardt, B.C., Burckhardt, G., 2003. Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev. Physiol. Biochem. Pharmacol. 146, 95–158. Burckhardt, G., Burckhardt, B.C., 2011. In vitro and in vivo evidence of the importance of organic 504 anion transporters (OATs) in drug therapy. In: Drug Transporters, Edited by Fromm, M. F. and Kim, 505 R. B. vol. 2011. Springer, Berlin, pp. 29–104. Cha, S.H., Sekine, T., Fukushima, J.I., Kanai, Y., Kobayashi, Y., Goya, T., Endou, H., 2001. Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol. 59, 1277–1286. Colas, C., Ung, P.M., Schlessinger, A., 2016. SLC transporters: structure, function, and drug discovery. Med. Chem. Commun. 7, 1069–1081. Cole, R.F., Mills, G.A., Parker, R., Bolam, T., Birchenough, A., Kröger, S., Fones, G.R., 2015. Trends in the analysis and monitoring of organotins in the aquatic environment. Trends Environ. Analyt. Chem. 8, 1–11. Dragojević, J., Mihaljević, I., Popović, M., Smital, T., 2019. Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds. Comp. Biochem. Physiol. B 236 (1–11). Fent, K., Weston, A.A., Caminada, D., 2006. Ecotoxicology of human pharmaceuticals. Aquat. Toxicol. 76, 122–159. Giacomini, K.M., Huang, S.M., Tweedie, D.J., Benet, L.Z., Brouwer, K.L., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., et al., 2010. International transporter consortium. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215–236. Giesy, J.P., Kannan, K., 2002. Perfluorochemical Surfactants in the Environment. Environ. Sci. Technol. 36 (7), 146A–152A. Goodman & Gilman's The Pharmacological Basis of Therapeutics by. Joel Griffith Hardman, Lee E. Limbird, Alfred G. Gilman, 1994. 10th Ed. Gui, C., Wahlgren, B., Lushington, G.H., Hagenbuch, B., 2009. Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17beta-glucuronide transport. Pharmacol. Res. 60, 50–56. Hjelmborg, P.S., Andreassen, T.K., Bonefeld-Jørgensen, E.C., 2008. Cellular uptake of lipoproteins and persistent organic compounds - an update and new data. Environ. Res. 108, 192–198. Hoch, M., 2001. Organotin compounds in the environment — an overview. Appl. Geochem. 16 (7–8), 719–743. Iqbal, M.P., Ali, A.A., Alvi, A.A., 1993. Severe bone marrow suppression in a patient with rheumatoid arthritis on methotrexate. J. Pak. Med. Assoc. 43 (12), 262–263. Itoh, T., Mitsuoka, S., Uji, M., Matsushita, H., 1999. Successful combination chemotherapy with low-dose methotrexate and steroids for dermatomyositis complicated by interstitial pneumonitis. J. Jpn Respir. Soc. 37 (8), 636–640. Kindla, J., Mu, F., Mieth, M., Fromm, M.F., 2011. Influence of non-steroidal antiinflammatory drugs on organic anion transporting polypeptide (OATP) 1B1- and OATP1B3-mediated drug transport. Drug Metab. Dispos. 39, 1047–1053. Kipen, Y., Littlejohn, G.O., Morand, E.F., 1997. Methotrexate use in systemic lupus erythematosus. Lupus 6 985–389. Lee, S.C., Arya, V., Yang, X., Volpe, D.A., Zhang, L., 2017. Evaluation of transporters in drug development: current status and contemporary issues. Adv. Drug Deliv. Rev. 116, 100–118. Lončar, J., Smital, T., 2018. Interaction of environmental contaminants with zebrafish (Danio rerio) multidrug and toxin extrusion protein 7 (Mate7/Slc47a7). Aquat. Toxicol. 205, 193–203. Mihaljevic, I., Popovic, M., Zaja, R., Smital, T., 2016. Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio). BMC Genomics 17,
8