Effect of GenX on P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance–Associated Protein 2 at the Blood–Brain Barrier
2020; National Institute of Environmental Health Sciences; Volume: 128; Issue: 3 Linguagem: Inglês
10.1289/ehp5884
ISSN1552-9924
AutoresRonald E. Cannon, Alicia Richards, Andrew W Trexler, Christopher T. Juberg, Birandra K. Sinha, Gabriel A. Knudsen, Linda S. Birnbaum,
Tópico(s)Prenatal Substance Exposure Effects
ResumoVol. 128, No. 3 ResearchOpen AccessEffect of GenX on P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance–Associated Protein 2 at the Blood–Brain Barrier Ronald E. Cannon, Alicia C. Richards, Andrew W. Trexler, Christopher T. Juberg, Birandra Sinha, Gabriel A. Knudsen, and Linda S. Birnbaum Ronald E. Cannon Address correspondence to R.E. Cannon, NIEHS/NTP/NTP Labs, 111 T W Alexander Drive, Building 101, Mail Drop C2-02, Research Triangle Park, NC 27709. Telephone: 984-287-3937. Email: E-mail Address: [email protected] Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , Alicia C. Richards Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , Andrew W. Trexler Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , Christopher T. Juberg Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , Birandra Sinha Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , Gabriel A. Knudsen Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA , and Linda S. Birnbaum Laboratory of Toxicology and Toxicokinetics, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA Published:26 March 2020CID: 037002https://doi.org/10.1289/EHP5884Cited by:5AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground:Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) is a replacement for perfluorooctanoic acid in the production of fluoropolymers used in a variety of consumer products. GenX alters fetal development and antibody production and elicits toxic responses in the livers and kidneys of rodents. The GenX effect on the blood–brain barrier (BBB) is unknown. The BBB protects the brain from xenobiotic neurotoxicants and harmful endogenous metabolites.Objectives:We aimed to investigate the effects of GenX on the transport activity and expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance–associated protein 2 (MRP2) at the BBB.Methods:Transporter activities were measured in isolated rat brain capillaries by a confocal microscopy–based method. ATPase (enzymatic hydrolysis of adenosine triphosphate to inorganic phosphate) levels were measured in vitro. Western blotting determined P-gp and BCRP protein levels. Cell survival after GenX exposure was determined for two human cell lines.Results:Nanomolar levels of GenX inhibited P-gp and BCRP but not MRP2 transport activities in male and female rat brain capillaries. P-gp transport activity returned to control levels after GenX removal. GenX did not reduce P-gp- or BCRP-associated ATPase activity in an in vitro transport assay system. Reductions of P-gp but not BCRP transport activity were blocked by a peroxisome proliferator–activated receptor γ (PPARγ) antagonist. GenX reduced P-gp and BCRP transport activity in human cells.Conclusion:In rats, GenX at 0.1–100 nM rapidly (in 1–2 h) inhibited P-gp and BCRP transport activities at the BBB through different mechanisms. PPARγ was required for the GenX effects on P-gp but not BCRP transport activity. https://doi.org/10.1289/EHP5884IntroductionAmmonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate (GenX) (CAS no. 62037-80-3) is a chemical precursor used in the production of polytetrafluoroethylene (Teflon) and as a replacement for perfluorooctanoic acid (Wang et al. 2013). The production volume of GenX in the European Union is estimated to be 10–100 tons per year; however, the worldwide production volume is unknown (Beekman et al. 2016). Although GenX is unlikely to pose a significant aquatic hazard, its bioactivity and persistence in environmental media are problematic (Hoke et al. 2016). GenX is a contaminant in rivers of the Netherlands, Germany, and China (Heydebreck et al. 2015). In June 2017, GenX was detected in the Cape Fear River in eastern North Carolina, United States (Sun et al. 2016). Further surveys nearby and downstream from a PFAS manufacturing facility detected GenX in air, private well water, and some local food products including oysters and honey (Pritchett et al. 2019; Clabby 2018). Most importantly, GenX was detected in finished drinking water. Since GenX has the potential to disrupt biological signaling pathways known to regulate ABC [adenosine triphosphate (ATP) binding cassette] transporters (Conley et al. 2019; Miller and Cannon 2014; More et al. 2017; Zhang et al. 2014), we investigated its effects on three transporters at the BBB: ABCB1, [P-glycoprotein (P-gp)], ABCG2 [breast cancer resistance protein (BCRP)], and ABCC2 [multidrug resistance–associated protein 2 (MRP2)]. We hypothesized that disruption of signaling pathways by GenX would change basal levels of transporter expression and/or activity. ABC transporters are primary active transporters that derive their energy from the hydrolysis of ATP to adenosine diphosphate (ADP) + inorganic phosphate (Pi). Within biological barriers, they function to restrict the access of toxic drugs, endobiotics, and xenobiotics to sensitive cells, tissues, and organs (Locher 2016). In tumor cells, their overexpression leads to multidrug resistance and presents major obstacles in cancer therapies (Bockor et al. 2017; Robey et al. 2018). In most human tissues, P-gp, BCRP, and ABCC2/MRP2 are ubiquitously expressed at low levels; however, in the biological barriers, they are highly expressed and function to protect the brain, retina, testes, and the developing fetus (Nagy et al. 2016). Their localization and expression levels in liver and kidney also serve to eliminate harmful agents from the body (Leslie et al. 2005). Specifically, in the canalicular membrane of liver, they transport conjugates and harmful metabolites into bile. In the proximal tubules of the kidney, they aid in excretory transport of substrates into urine. They are also expressed in the enterocytes of the intestine, where they function to limit the absorption of harmful substrates into the body (Murakami and Takano 2008). The substrates of P-gp, BCRP, and MRP2 are chemically diverse and include chemotherapeutics, lipids, steroids, bilirubin, bile acids, platelet-activating factor, dietary flavonoids, and conjugated endogenous and xenobiotic metabolites (Kim 2002).Signaling pathways dynamically regulate the activity and expression of P-gp, BCRP, and MRP2 at the BBB (Miller and Cannon 2014). To examine the effects of GenX on these transporters, we exposed rat brain capillaries ex vivo to low nanomolar concentrations of GenX. We also dosed rats in vivo by oral gavage with 30, 300, or 3,000 pmol/kg GenX and measured transport activity ex vivo using a steady-state confocal microscopy–based assay. Lastly, we expanded our study to humans by measuring the effect of GenX on the survival of two human cell lines exposed to increasing concentrations of cytotoxic substrates of P-gp and BCRP.Materials and MethodsMaterialsCrystalline GenX (molecular weight of 347.084 g/mol, >97% purity] was purchased from SynQuest Labs. P-gp fluorescent substrate [N-ε-(4-Nitrobenzofurazan-7-yl)-D-Lys8] cyclosporine A (NBD-CSA) was custom synthesized by R. Wenger (Sandoz) (Schramm et al. 1995). MRP2 fluorescent substrate Texas Red®, Sigma-Aldrich (sulforhodamine MRP2 inhibitor MK-571, corn oil, and ß-actin mouse monoclonal antibody A1978 were purchased from Sigma-Aldrich. P-gp inhibitor PSC-833 and BCRP inhibitor KO-134 were purchased from Tocris Bioscience. Adriamycin was the gift of the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program of the National Cancer Institute (NCI), National Institutes of Health (NIH). Mitoxantrone hydrochloride was purchased from Sigma-Aldrich. Both Adriamycin and mitoxantrone were dissolved in double-distilled water (10mg/mL) and stored at −80°C. P-gp rabbit monoclonal antibody ab170904 was purchased from Abcam. Secondary antibodies, Alexa Fluor® 647 Goat anti-Mouse IgG and Alexa Fluor® 647 Goat anti-Rabbit IgG, were purchased from Thermo Fisher Scientific. Western blotting lysis buffer CelLytic™ MT Mammalian Tissue Lysis/Extraction Reagent with complete mini protease inhibitor was purchased from Sigma-Aldrich. The BCRP substrate BODIPY™ FL prazosin and the Western blotting materials, including 10-well Invitrogen NuPAGE 4-12% Bis-Tris Gels NP0321 and polyvinylidene difluoride (PVDF) Western blot membranes, were obtained from Invitrogen (Thermo Fisher Scientific).AnimalsMale and female Hsd:Sprague-Dawley® (SD®) rats (age 12–15 wk) were purchased from Envigo. Animals were housed in the NIEHS Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)–approved animal care facility [∼49% humidity, ∼72°F room temperature, 12-h light/dark cycle, polycarbonate shoebox cages (Tecniplast), and Sani-Chip® bedding (PJ Murphy Forest Products)] for at least 1 wk prior to use and allowed access to food (NIH #31) and tap water ad libitum consumption. Animals were euthanized by CO2 inhalation followed by decapitation. All animal protocols were approved by the Animal Care and Use Committee at the NIEHS according to the guidelines from the NIH. All data are reported in compliance with the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines.Capillary Isolation and ex Vivo ABC Transport AssayTo measure ex vivo transport activity of P-gp and BCRP at the blood–brain barrier (BBB), capillaries from male and female rats were isolated as previously reported (Chan and Cannon 2017). Briefly, the brains from four to six male or female rats were harvested following euthanasia and placed in assay buffer [1×phosphate-buffered saline (PBS), pH 7.4, supplemented with 900mg/mL of glucose and 110mg/mL of sodium pyruvate] on ice. Cortical gray matter was isolated by discarding white matter, meninges, midbrain, choroid plexus, and olfactory lobes using dissecting forceps and a stereomicroscope. The remaining cortical gray matter enriched for capillaries was minced with a razor blade, suspended in 15mL isolation buffer, and homogenized by 40 up-and-down strokes using a Thomas (size C) mechanical tissue grinder (Thomas Scientific; catalog no. 3431E55) matched with a size C serrated pestle (Thomas Scientific; catalog no. 3431F25; clearance: 150 to 230μM) rotating at 50 rpm. A final homogenization was performed by 10 up-and-down strokes using a 15-mL KONTES® Dounce tissue grinder (pestle, size B; catalog no. 885300-0015; clearance: 165 to 889μm; VWR). The resulting homogenate was suspended in an equal volume of ice-cold 30% Ficoll PM400 dissolved in assay buffer to achieve a final Ficoll PM 400 content of 15% wt/vol and centrifuged for 20 min at 5,800×g at 4°C using an RC-5B Centrifuge (Sorvall) with a SS-34 rotor. Following centrifugation, supernatants were removed, and pelleted capillaries were resuspended in assay buffer containing 1% bovine serum albumin (BSA) (Sigma-Aldrich) and captured by passage through a 30-μM filter [pluriStrainer (Pluriselect), 43-50030-03]. BSA from Sigma was removed from isolated capillaries by three sequential centrifugations at 900×g following resuspension in 15mL isolation buffer (no BSA). After the third centrifugation, the capillaries were resuspended in 0.5mL assay buffer, and equal volumes were loaded into sterile borosilicate chamber slides purchased from Thermo Scientific (catalog no. 155380). The chamber slides were incubated at room temperature for 15 min to allow capillary settling and binding. Next, the chamber slides were rinsed with 1mL assay buffer. To measure transporter activity, chamber slide bound capillaries were incubated at 0–4 h in 2mL of assay buffer containing a fluorescent substrate (2μM) specific for each transporter (NBD-CSA for P-gp, BODIPY™ FL prazosin for BCRP, and Texas Red® for MRP2). Transporter activity was determined as the mean measurement of steady-state luminal fluorescence for 15–20 individual capillaries per chamber. Luminal fluorescence reaches a steady state in 30 min at room temperature (Chan and Cannon 2017). Nonspecific background fluorescence was determined by measuring the luminal fluorescence of fully inhibited capillaries. All inhibitors were administered as a 30-min pretreatment before fluorescent substrates were added. The inhibitors used were PSC-833 (10μM, P-gp), KO134 (20μM, BCRP), and MK-571 (20μM, MRP2). Specific transport for each transporter was calculated as the difference between total noninhibited transport minus nonspecific inhibited transport. To measure luminal fluorescence, confocal images of capillaries were captured using a Zeiss 710 confocal microscope. Luminal fluorescence was quantified using FIJI/ImageJ (File version, ImageJ 1.52a; Java 1.8.0_112) (NIH) analysis software. The solvent [0.1% vol/vol dimethylsulfoxide (DMSO)] was used as the vehicle control (VC) to match the concentration of the treatment solvents. GenX in 1×PBS compared to GenX in DMSO 0.1% produced no significant effects on P-gp transport (Figure S1). For all relevant experiments, GenX was dissolved in DMSO immediately prior to use. Negative controls employed inhibitors specific for each transporter. Where relevant, GenX and the peroxisome proliferator–activated receptor γ (PPARγ) antagonist, GW9662 (50 nm) were freshly dissolved in DMSO and used alone or in cotreatments (inhibitor studies) in volumes (0.1% vol/vol) to match VC volumes. When needed, staggered time courses were used to accommodate confocal image acquisitions at 10 min/dose group.Measuring Transport Activity after GenX RemovalThe reversibility assays are a modified design of the ex vivo transport assay. Briefly, isolated capillaries from four to six rats were pooled and equally distributed into chamber slides and incubated at room temperature in 2mL assay media with a VC (DMSO vol/vol 0.1%) and the P-gp- or BCRP-specific fluorescent substrate (2μM). In our VC group, steady-state transport activity was established by measuring luminal fluorescence after 1–5 h incubation at room temperature. In a second group, GenX was added to achieve a 100-nM final concentration in media containing the fluorescence substrates and incubated for 1 h for P-gp and 2 h in the BCRP transport assays. At 1–2 h, respectively, P-gp and BCRP transport activities were measured. Following transport measurements, GenX was removed by two washes (equal volumes) with assay media containing fluorescence substrates specific for each transporter. After GenX removal, the samples were incubated in assay media with appropriate substrates. Next, P-gp and BCRP transport activities were measured at 0.5, 1, 2, and 3 h after GenX removal. All transporter activities were determined as the mean measurement of steady-state luminal fluorescence minus background fluorescence for 15–20 individual capillaries per chamber. Capillaries were imaged using a Zeiss 710 confocal microscope. Luminal fluorescence was quantified using FIJI/ImageJ analysis software. All transport experiments, including the reversibility assays, were performed two times to ensure reproducibility.ATPase Activation AssayThe ATPase assay was used to determine if GenX at concentrations of 0.001, 0.01, 0.1, and 1.0μM influenced the ATPase activity associated with P-gp and BCRP transport activity in a purified system. The assay is based on the spectrophotometric quantitation of Pi produced from P-gp- or BCRP ATPase-mediated conversion of ATP to ADP + Pi when the substrate was stimulated with 10μM paclitaxel for P-gp and 10μM sulfasalazine for BCRP. To accomplish this, 10μL of membrane vesicles, provided by the assay kit, containing either 2μg of P-gp or BCRP proteins, were diluted 10-fold in assay buffer and transferred to a well of a 96-well plate in triplicate. The wells contained either a) P-gp or BCRP plus substrates (stimulated positive control), b) P-gp or BCRP plus substrates with the ATPase inhibitor vanadate (1.25 mM) (ATPase negative control), c) P-gp or BCRP minus substrates (negative control), d) P-gp or BCRP minus substrates with 1.0μM GenX, or e) P-gp or BCRP plus substrates and GenX in DMSO 0.1% at 0.001, 0.01, 0.1, or 1.0μM final concentration. The mixtures were preincubated at 37°C for 10 min, and the reaction was started by the addition of 10μL Mg-ATP (200 mM) and transporter-specific substrates or DMSO 0.1% where appropriate. The plate was incubated at 37°C for 20 min. All reactions were terminated by the addition of 40μL of 5% sodium dodecyl sulfate. Pi levels are a function of ABC transporter activity, which requires the hydrolysis of ATP to ADP + Pi. To measure Pi, a colorimetric detection solution was prepared by mixing one part of 35 mM ammonium molybdate in 15 mM zinc acetate (pH 5.0) with three parts of 10% ascorbic acid. The final detection solution was mixed by inversion, and 200μL was added to each sample and incubated for 20 min at 37°C. Assays were read at 650 nm on a Gemini™ spectrometer (Molecular Devices) and graphed as relative units of ATPase activity. The means of triplicate samples were calculated using Prism software (version 7.05; GraphPad). Data are expressed as mean±standard error (SE).GenX in Vivo Dosing StudiesMale and female rats (five/dose group) received a single oral gavage dose of high-performance liquid chromatography (HPLC)–grade water as a VC or GenX in HPLC-grade water at 30, 300, or 3,000 pmol/kg (10, 100, or 1,000 ng/kg, respectively). All doses were given in a volume of 4mL/kg. Animals were euthanized at 5 h postdosing. Brains from each dose group were pooled, and capillaries were isolated as previously described above. P-gp and BCRP transport activities were measured ex vivo as described above.GenX Cytotoxicity in Human CellsThe P-gp overexpressing the human ovarian NCI/ADR-RES cell line was obtained from NCI at Frederick Cancer Center (Scudiero et al. 1998). It was selected for its reliable high expression of P-gp. A second line, MX-MCF-7, was selected for its high expression levels of BCRP. This mitoxantrone-resistant MX-MCF-7 BCRP-expressing cell line (Nakagawa et al. 1992) was a generous gift of Dr. E. Schneider, Wadsworth Center, New York State Department of Health. Both low passage lines (<10 passages) were grown in Phenol Red-free RPMI media supplemented with 10% fetal bovine serum and antibiotics and used up to 15–20 passages, after which the cells were discarded and a new cell culture started from fresh frozen stock. Both cell lines were used in cytotoxicity studies that measured cell growth inhibition. First, we determined the cytotoxic effect of GenX (alone) for each line. Cell growth inhibition assays were performed for each line by plating 100,000–125,000 (NCI/ADR-RES for P-gp and MX-MCF-7 for BCRP) cells/well in triplicate. Each was grown for 18 h at 37°C and 5% CO2 to allow attachment. Next, they were grown in fresh media containing increasing concentrations of GenX (10−9 to 10−4 M). After 72 h, the cultures were washed three times with 50mL1×PBS to remove nonadherent cells. The remaining adherent cells were harvested by trypsinization and counted in a Beckman Coulter Counter (Beckman).To determine if GenX (100 nM) affected the toxicity of known cytotoxic substrates for P-gp or BCRP, for each line, we plated 100,000–125,000 (NCI/ADR-RES for P-gp and MX-MCF-7 for BCRP) cells/well in triplicate. Each was grown for 18 h at 37°C and 5% CO2 to allow attachment. Next, they were grown in fresh media containing 100 nM GenX and the toxic P-gp substrate, Adriamycin (10−8 to 10−5 M). MX-MCF-7 cells were grown in media for 72 h with or without 100 nM GenX plus the toxic BCRP substrate mitoxantrone (10−9 to 10−4 M). After 72 h, the cultures were washed three times with 50mL1×PBS to remove nonadherent cells. The remaining adherent cells were harvested by trypsinization and counted in a Beckman Coulter Counter (Beckman).Gel Electrophoresis and Western BlottingIsolated capillaries pooled from 6 rats were treated with VC (DMSO 0.1%) or 100 nM GenX at room temperature in 15mL Falcon tubes (Fisher Scientific; catalog no. 14-959-53A) containing 5mL assay buffer (1×PBS, pH 7.4, supplemented with 900mg/L of glucose and 110mg/L of sodium pyruvate). After treatment, capillaries were pelleted by centrifugation for 15 min at a centrifugal force of 1,860×g at 4°C and stored at −80°C until use. Membrane-containing protein lysates were isolated by adding 200μL of lysis buffer (CelLytic™ MT Cell Lysis Reagent; Sigma-Aldrich; catalog no. C3228-500; with Roche Complete Mini protease inhibitor cocktail; catalog no. 4693159001) to each pellet. Capillary pellets were kept on ice and vortexed for 30 s every 10 min for 90 min. In addition, the samples were sonicated at 4°C for 60 s at the 20-, 40-, and 60-min time points. To separate the nuclei from cytoplasm and cellular membranes, the samples were centrifuged at 10,000×g for 30 min. The pellets containing nuclei were discarded, and the supernatants containing cytoplasm and membranes were centrifuged at 100,000×g for 90 min. The liquid cytosolic fraction was removed, and the remaining membrane pellet was dissolved in 50μL MT cell (Sigma-Aldrich) lysis buffer and stored at −80°C until use. Membrane protein concentration was determined as described by the Coomassie Protein Assay Kit 23200 (Thermo Scientific), a modified version of the Bradford assay (Bradford 1976). BSA protein standards were provided by the kit. Electrophoresis and Western blotting were performed according to the manufacturer's instructions. Briefly, 1μg of capillary membrane lysates were mixed with 1×reducing agent and 1×loading buffer (Invitrogen), loaded into NuPAGE Bis-Tris Gels (4–12%) using the XCell SureLock Mini-Cell Electrophoresis System (Invitrogen), and electrophoresed in MOPS Running Buffer (Invitrogen) for 50 min at a constant 200V. Following electrophoresis, the resolved proteins were transferred from the gel to a PVDF membrane (Invitrogen) using the XCell II Blot Module using Invitrogen™ Bolt™ Transfer Buffer plus 10% (vol/vol) methanol. Electrotransfer was performed at constant current (0.1 up to approximately ∼0.4A) or voltage (10 to 25V) for 60 min. To visualize the P-gp-, BCRP-, and ß-actin-specific bands, the membrane was incubated at room temperature for 30 min in 50mL blocking buffer (Intercept® Blocking Buffer, Licor). The membrane was washed in 1×PBS and hybridized overnight at 4°C in 1×PBS with 0.1% Tween with 1:200 vol/vol P-gp (Abcam; catalog no. 170904) and BCRP (Abcam; catalog no. 207732) primary antibodies and 1:5,000 vol/vol ß-actin primary antibody (Abcam; catalog no. 8224). To remove excess antibody, the membrane was washed three times with 1×PBS with 0.1% Tween and incubated at room temperature for 1 h in 1×PBS with 1:10,000 vol/vol secondary antibodies [Odyssey Goat anti-Mouse IR Dye 800CW or Goat anti-Rat IR dye 680CW (Licor)] for an additional 60 min and washed three times with 1×PBS with 0.1% Tween to remove excess unbound secondary antibody. Specific protein bands were imaged and quantified for protein fluorescence using FluorChem M (ProteinSimple). For each sample, target band intensities were normalized to β-actin. Western blotting was performed in triplicate from three independent experiments. Means and standard errors of the mean (SEM) were calculated using Prism software (version 7.05; GraphPad) software. Data are expressed as a mean±SE.StatisticsLuminal fluorescence and cell survival data were analyzed and graphed using Prism software (version 7.05; GraphPad). Data are expressed as mean±SE, and significant differences between the control and treated means were determined by one-way analysis of variance (ANOVA) and Tukey multiple comparison. For the ex vivo transport assay, significance for each data point was determined by comparing treated to control: *p<0.05; **p<0.01; ***p<0.001.ResultsTransporter Activity in Rat Brain Capillaries Treated with GenXWe determined the rapid effects of GenX exposure on the BBB by measuring GenX-mediated changes in ex vivo transport activity of three well-characterized ABC transporters (P-gp, BCRP, and MRP2). To accomplish this, we used an established steady state–based confocal microscopy assay (Chan and Cannon 2017). To determine the effect of increasing concentrations of GenX on the ABC transporters at the BBB, we exposed isolated capillaries from male and female rat brains to 0.01–100 nM GenX for 3 h and measured transport activity. In males, P-gp transport activity was lowered but not significantly (p=0.13) by 0.01 nM GenX exposure. P-gp transport activity was significantly lower in male capillaries exposed to 0.01–100 nM GenX (Figure 1A). In females, P-gp transport activity was unchanged by 0.01–0.1 nM GenX exposures but significantly lowered by 1.0–100 nM GenX (Figure 1B). Next, we treated capillaries with 100 nM GenX for 1–4 h and examined the hourly changes in P-gp transport. We chose 100 nM GenX because it produced the greatest reduction in P-gp transport activity for both sexes. Results shown in Figure 1C,D show that 100 nM GenX significantly lowered P-gp transport activity in 15 and 30 min in male and female rat brain capillaries, respectively.Figure 1. Changes in P-glycoprotein (P-gp) transport activity in brain capillaries from six rats treated with ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX). (A) Male and (B) female are graphs of P-glycoprotein (P-gp) transport activity at increasing doses of GenX in brain capillaries of Hsd:Sprague-Dawley® (SD®) rats. (C) Male and (D) female are graphs of the GenX-dependent reductions in P-gp transport activity over time. Dotted horizontal line denotes vehicle control (no GenX) levels. Mean±standard error (SE) is shown. SE and significance were determined by one-way analysis of variance (ANOVA) and Tukey multiple comparison. Significance is as compared to control unless otherwise specified: **p<0.01; ***p<0.001.Next, we examined the effect of GenX on BCRP transport activity. We measured BCRP transport activity after treating male and female brain capillaries with GenX (0.1 nM–1μM) for 3 h (Figure 2A,B). BCRP transport activity in males was significantly lower in samples treated with 1.0 nM–1μM GenX. In contrast, female BCRP transport activity was significantly lower in capillaries treated with 0.1 nM–1μM GenX. We also examined the hourly changes in BCRP transport in capillaries following exposure to 100 nM GenX for 1–4 h (Figure 2C,D). Both male and female BCRP transport activity was significantly lower at 1 h. Of note, the reduction in BCRP transport activity in females was more significant than males at the 1-h time point; furthermore, GenX-dependent transport inhibition persisted throughout the 3-h assay time for both sexes.Figure 2. Changes in breast cancer resistance protein (BCRP) transport activity in brain capillaries from six rats treated with ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX). (A) Male and (B) female are graphs of BCRP transport activity at increasing doses of GenX in brain capillaries of Hsd:Sprague-Dawley® (SD®) rats. (C) Male and (D) female are graphs of the GenX-dependent reductions in BCRP transport activity over time. Dotted horizontal line denotes vehicle control (no GenX) levels. Mean±standard error (SE) is shown. SE and significance were determined by one-way analysis of variance (ANOVA) and Tukey multiple comparison. Significance is as compared to control unless otherwise specified: *p<0.05; **p<0.01; ***p<0.01.Lastly, we determined the GenX effect on MRP2 transport in identically designed experiments. We observed no GenX effect with regard to dose and time on MRP2 transport activity (Figure S2A–D).Reversibility Assays for P-gp and BCRP TransportPrevious transport studies from our laboratory have shown that chemical perturbation of signal transduction pathways can lead to rapid changes in transport activity independently of protein degradation or expression (Banks et al. 2018; Cannon et al. 2012). In general, these chemical-induced changes in transport activity revert to control levels upon chemical removal. Knowing this, we performed a reversibility assay by exposing male and female rat brain capillaries to GenX (100 nM). Shown in Figure 3, P-gp transport activity in males (Figure 3A) and females (Figure 3B) rapidly reverted to control levels within 1 h after GenX removal. In contrast to P-gp, GenX-mediated decreases in BCRP transport did not revert for either sex (Figure 3C,D).Figure 3. Transport reversibility following ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) removal. (A) Male and (B) female are graphs denoting P-glycoprotein (P-gp) transport activities, and (C) male and (D) female are graphs denoting breast cancer resistance protein (BCRP) transport activities in brain capillaries from six rats before and after GenX (100 nM) removal. Mean±standard error (SE) is shown. SE and significance were determined by one-way analysis of variance (ANOVA) and Tukey multiple comparison. Significance is as compared to control unless otherwise specified: ***p<0.001.The Effect of GenX on P-gp and BCRP ATPase Activity in VitroTo determine if GenX directly inhibited ATP hydrolysis or was a P-gp or BCRP
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