Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier S

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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2017/09/28/jpet.117.243584.DC1

1521-0103/363/3/324–335$25.00 https://doi.org/10.1124/jpet.117.243584 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 363:324–335, December 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier s

Sana-Kay Whyte-Allman, Md Tozammel Hoque, Mohammad-Ali Jenabian, Jean-Pierre Routy, and Reina Bendayan Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (S.-K.W.-A., M.T.H., R.B.); Department of Biological Sciences, Université du Québec à Montréal, Montréal, Quebec, Canada (M.-A.J.); and Chronic Viral Illness Service, McGill University Health Centre, Montréal, Quebec, Canada (J.-P.R.) Downloaded from Received June 28, 2017; accepted September 14, 2017

ABSTRACT Poor antiretroviral drug (ARV) penetration in the testes could addition, we demonstrated an upregulation of P-gp, Bcrp, and be due, in part, to the presence of ATP-binding cassette Mrp4 mRNA and protein expression, after exposure to PXR or (ABC) membrane–associated drug efflux transporters such as CAR ligands in TM4 cells. Small interfering RNA downregulation jpet.aspetjournals.org P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), of PXR or CAR attenuated the expression of these transporters, and multidrug resistance–associated proteins (MRPs) expressed suggesting the direct involvement of these nuclear receptors in at the blood-testis barrier (BTB). The functional expression of regulating P-gp, Bcrp, and Mrp4 in this system. In an ex vivo these transporters is known to be regulated by ligand-activated study using freshly isolated mouse seminiferous tubules, we nuclear receptors pregnane X receptor (PXR) and constitutive found that exposure to PXR or CAR ligands, including ARVs, androstane receptor (CAR) in various tissues. This study aimed significantly increased P-gp expression and function. Together, to investigate in vitro and ex vivo the role of PXR and CAR in our data suggest that ABC transporters could be regulated at the regulation of ABC transporters at the BTB. Both PXR and the BTB during chronic treatment with ARVs that can serve at ASPET Journals on September 24, 2021 CAR proteins were expressed in human testicular tissue and in as ligands for PXR and CAR, which could in turn further mouse TM4 Sertoli cells (an in vitro cell line model of the BTB). In limit testicular ARV concentrations.

Introduction the testis could, in part, restrict ARV concentrations in the seminal fluid of HIV-infected men (Else et al., 2011). The testis Despite the use of highly active antiretroviral therapy, is an immunoprivileged environment where the blood-testis human immunodeficiency virus (HIV)-1 persistence in ana- barrier (BTB), due to its physical and biochemical properties, tomic reservoirs and sanctuary sites such as the lymph nodes, can limit the penetration of exogenous agents into this tissue. brain, and testes can be attributed, in part, to the low This barrier is primarily composed of adjacent epithelial antiretroviral drug (ARV) concentrations reached in these Sertoli cells, which form tight junction complexes near the tissues (Dahl et al., 2010; Fletcher et al., 2014; Huang et al., basement membrane and physically divide the seminiferous 2016; Jenabian et al., 2016). Semen is the most common vector epithelium into apical and basolateral compartments. This for HIV transmission globally and limited ARV penetration in allows for spermatid development in a microenvironment within the seminiferous tubules where the entrance of anti- sperm antibodies and harmful xenobiotics is restricted (Su This work was supported by the University of Toronto [Leslie Dan Faculty of et al., 2011). Pharmacy Internal Grant 923465 (to R.B.)] and by the Canadian Institutes of Health Research [Catalyst Operating Grant 296635 and Canadian HIV Cure ARV disposition in rodents and humans primarily involves Enterprise Team Grant HIG-133050 (to J.-P.R., M.-A.J., and R.B.)] in intracellular drug metabolism and transport by members of partnership with the Canadian Foundation for AIDS Research and the the ATP-binding cassette (ABC) and solute carrier (SLC) International AIDS Society. R.B. is a career scientist of the Ontario HIV Treatment Network. S.-K.W.-A. is a recipient of the Connaught International superfamilies of drug transport proteins across biologic mem- Scholarship for Doctoral Students. M.A.J. holds the Canada Research Chair branes. The ABC family includes several drug efflux trans- Tier 2 in Immuno-Virology. doi.org/10.1124/jpet.117.243584. porters such as P-glycoprotein (P-gp; ABCB1), multidrug s This article has supplemental material available at jpet.aspetjournals.org. resistance–associated proteins (MRPs; ABCC), and breast

ABBREVIATIONS: ABC, ATP-binding cassette; ARV, antiretroviral drug; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; BTB, blood- testis barrier; CAR, constitutive androstane receptor; CsA, cyclosporine A; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; GR, glucocorticoid receptor; HEK, human embryonic kidney; HIV, human immunodeficiency virus; MRP, multidrug resistance–associated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P-gp, P-glycoprotein; PCN, pregnenolone 16a carbonitrile; PI, protease inhibitor; PSC833, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-L-valine-cyclosporin A; PXR, pregnane X receptor; qPCR, quantitative real- time polymerase chain reaction; siRNA, small interfering RNA; SLC, solute carrier; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.

324 Regulation of ABC Transporters in the Testis 325 cancer resistance protein (BCRP; ABCG2), whereas the SLC purchased from Moravek Biochemicals (Brea, CA). Valspodar transporters comprise organic anion-transporting polypep- (PSC833; 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic tides, organic anion and organic cation transporters, and acid]-7-L-valine-cyclosporin A) was a generous gift from Novartis equilibrative and concentrative nucleoside transporters (Kis Pharma (Basel, Switzerland). Cyclosporine (CsA) was obtained from et al., 2010). Our group and others have demonstrated in vitro Tocris Biosciences (Ellisville, MO). 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), dexamethasone, ketoconazole, and 3-(4,5- and in vivo that several efflux transporters are functionally dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were expressed at the BTB and could potentially limit the penetra- purchased from Sigma-Aldrich (Oakville, ON, Canada). Pregnenolone tion of ARVs into rodent and human testes (Choo et al., 2000; 16a carbonitrile (PCN) was purchased from Cayman Chemicals (Ann Robillard et al., 2012, 2014; Huang et al., 2016). Arbor, MI). Type I collagen was purchased from BD Biosciences Most ARVs from the protease inhibitor (PI) class are known to (San Jose, CA). Small interfering RNA (siRNA) against mouse PXR be substrates of P-gp, whereas BCRP is primarily involved in the (sc-44058), CAR (sc-43663), and glucocorticoid receptor (GR) (sc-35506) transport of several of the nucleoside and non-nucleoside reverse and nonsilencing negative control siRNA (sc-37007) were purchased transcriptase inhibitors. Both P-gp and BCRP have also been from Santa Cruz Biotechnology (Santa Cruz, CA). Each siRNA product implicated in the efflux of integrase strand transfer inhibitor contained a pool of three target-specific 19- to 25-nt siRNAs designed to drugs such as dolutegravir and raltegravir. The MRP isoforms knock down gene expression. The mouse anti-actin, goat anti-PXR, rabbit anti-CAR, and rabbit anti-GR antibodies were also obtained are also known to be involved in the transport of ARVs. In from Santa Cruz Biotechnology Inc. Please note that the rabbit anti- particular, MRP4 is involved in the efflux of several nucleoside

CAR (sc-13065) antibody obtained from Santa Cruz Biotechnology has Downloaded from reverse transcriptase inhibitors such as lamivudine, tenofovir, been reported by the company to identify CAR at around 55–60 kDa. In and abacavir. In addition to being substrates, many ARVs also addition, several groups including ours have detected CAR at around interact with these transporters as inducers and/or inhibitors 50–60 kDa using this antibody (Hernandez et al., 2009; Wang et al., (Supplemental Table 1). Recently, we characterized the expres- 2010; Chan et al., 2011), even though the predicted molecular weight of sion and localization of drug transporters and metabolic en- CAR has been reported at around 40–45 kDa based on its amino acid zymes involved in ARV transport in the testes of HIV-infected sequence. This potentially could be due to changes in the phosphory- individuals receiving ARV therapy and we measured plasma lation of the molecule (Mutoh et al., 2009) or to other post-translational jpet.aspetjournals.org and testicular drug concentrations. In particular, darunavir, a modifications that could affect the migration of the protein in different tissues. Mouse anti–P-gp antibody, rat anti-Bcrp antibody, and rat anti- P-gp substrate that is currently recommended as a preferred PI Mrp4 antibody were purchased from Enzo Life Sciences (Farmingdale, regimen (DHHS Panel on Antiretroviral Guidelines for Adults NY), Abcam Inc. (Boston, MA), and Kamiya Biomedical Company and Adolescents, 2016, Available at http://www.aidsinfo.nih. (Seattle, WA), respectively. Horseradish peroxidase–conjugated sec- gov/ContentFiles/Adul- tandAdolescentGL.pdf.), displayed sub- ondary antibodies were ordered from Jackson ImmunoResearch Lab- therapeutic levels in the testis that reached approximately 19% oratories Inc. (West Grove, PA). of plasma concentrations (Huang et al., 2016), thus demonstrat- Human Testicular Tissue. In collaboration with Drs. Jean-Pierre at ASPET Journals on September 24, 2021 ing the role that these transporters may play in limiting ARV Routy (McGill University Health Center, Montréal, QC, Canada) and testicular tissue concentrations. Pierre Brassard and Maud Bélanger (Metropolitan Centre of Plastic The functional expression of P-gp, BCRP, and MRP isoforms Surgery, Montréal, QC, Canada), we were able to obtain testicular is known to be regulated through the activity of ligand- tissue from individuals who were undergoing orchiectomy for sex reassignment. We obtained ethics approval for this study from the activated nuclear receptors pregnane X receptor (PXR) and/or McGill University Health Centre Ethical Review Board. All study constitutive androstane receptor (CAR) in tissues such as subjects provided written informed consent for participation in the the brain, liver, and intestine as well as in peripheral blood study. Tissue samples were stored in saline solution immediately after mononuclear cells (Assem et al., 2004; Albermann et al., 2005; surgery and were processed within 1–2 hours, in which they were cut Burk et al., 2005; Chan et al., 2011). These two nuclear into small pieces, snap frozen in liquid nitrogen, and stored at 280°C receptors are known to interact with endogenous ligands and until study assessments. A small portion of each testis was sent to a wide range of pharmacological agents and hence are recog- the pathology laboratory (Metropolitan Centre of Plastic Surgery, nized as major xenobiotic sensors (Urquhart et al., 2007). Our Montreal, QC, Canada) for routine tests to exclude any infection or group previously demonstrated that ligand-activated PXR and malignancy, whereas the remaining portion was used in our laboratory CAR upregulated P-gp in vitro and in vivo at the level of the to examine nuclear receptor expression by western blot analyses. Cell Culture Systems. The continuous mouse Sertoli cell line blood-brain barrier (BBB), resulting in further impairment of (TM4) and the hepatocellular carcinoma cell line (HepG2) were drug permeation and distribution in the central nervous system obtained from American Tissue Culture Collection (Manassas, VA). (Chan et al., 2011, 2013b). Our group and others have also TM4 cells were grown on culture flasks and multiwell dishes precoated demonstrated that ARVs can directly activate PXR and CAR, with rat tail collagen type 1 (100 mg/ml) to enhance cell adherence causing an induction in the expression and activity of their and proliferation. The BCRP-overexpressing human breast cancer cell target genes (Dussault et al., 2001; Chan et al., 2013a). line (MCF7-MX100) was a generous gift from Dr. Susan Bates To our knowledge, the regulation of drug efflux transporter (Columbia University, New York, NY). The human breast cancer cell expression and function by nuclear receptors at the BTB has line DA435/LCC6 stably transfected with human MDR1 cDNA (MDA- not been addressed. This study aimed to investigate the role of MDR1) was kindly provided by Dr. Robert Clarke (Georgetown PXR and CAR in the regulation of ABC drug efflux transporters University, Washington, DC). The human embryonic kidney (HEK) cell line stably transfected with human MRP4 cDNA (HEK-MRP4) was (P-gp, Bcrp, and Mrp4) at the BTB in vitro and ex vivo in mice. kindly provided by Dr. Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands). All cell culture systems were grown – Materials and Methods and maintained at 37°C humidified 5% CO2 95% air with fresh media replaced every 2–3 days, according to our previously published Chemicals and Reagents. Unlabeled ARVs were obtained from protocols (Hoque et al., 2012; Robillard et al., 2012). the National Institutes of Health AIDS Research and Reference Re- Mouse Seminiferous Tubule Isolation. C57BL/6 male mice agent Program (Bethesda, MD). [3H]-atazanavir (0.900 Ci/mmol) was were a kind gift from Dr. Finnell (University of Texas, Austin, TX). All 326 Whyte-Allman et al. experiments, procedures, and animal care were conducted in accor- samples, mRNA was normalized by the Gapdh housekeeping gene. dance with the Canadian Council on Animal Care guidelines and Results are expressed as the fold change in mRNA levels 6 S.E.M. and approved by the University of Toronto Animal Care Committee. were calculated using the comparative threshold cycle method in which Animals were housed under a 12-hour/12-hour light/dark cycle at changes in transporter mRNA expression were calibrated to vehicle- room temperature with free access to food and water. Seminiferous treated cells. tubules were isolated as previously described (Lie et al., 2010). In Immunoblot Analysis. Western blot analysis was performed as brief, testes from wild-type adult mice were decapsulated, washed, described previously by our group with minor modifications (Robillard and incubated in collagenase (0.5 mg/ml) in a 1:1 Dulbecco’s modified et al., 2012). Briefly, lysates from human testicular tissue, mouse Eagle’s medium (DMEM)/F12 medium nutrient mixture at 35°C for seminiferous tubules, or cell lines were extracted using a modified 25 minutes. Seminiferous tubules were then washed five times by radioimmunoprecipitation lysis buffer containing 50 mM Tris-HCl, sedimentation under gravity to remove Leydig cells. The tubules were pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium ortho-vanadate, then incubated in DMEM/F12 media containing the desired ligands 0.5 mM phenylmethylsulfonyl fluoride, 1% (v/v) Nonidet-P40, and for 2 hours. Samples were then washed and stored at 280°C for future 0.1% (v/v) PI cocktail. Homogenates were subjected to brief sonication, analysis or used immediately to perform transport assays. followed by centrifugation for 15 minutes at 20,000g and 4°C, and the Ligand Treatment. Monolayers of TM4 cells grown on collagen- supernatants were isolated as whole tissue or cell lysates. The protein coated six-well plates were treated with the established PXR ligands content of each cell lysate sample was quantified using a Bio-Rad dexamethasone (25–100 mM) or PCN (10–50 mM) alone or in the protein assay kit (Bio-Rad, Hercules, CA). Proteins were separated on presence of antagonist ketoconazole (10 mM) or with the CAR ligand 7% or 10% SDS-polyacrylamide gel with 5% stacking gel and were –

TCPOBOP (50 500 nM). At the beginning of each experiment, the then electrotransferred onto a polyvinylidene difluoride membrane. Downloaded from culture medium was aspirated and fresh medium containing ligands The membranes were blocked for 2 hours at room temperature in 5% dissolved in dimethylsulfoxide (DMSO) was added. Seminiferous skim-milk/Tris-buffered saline containing 0.1% Tween 20 and in- tubules were exposed to PCN (100 mM), TCPOBOP (5 mM), efavirenz cubated overnight at 4°C with the following primary antibodies (50 mM), or darunavir (100 mM). Control cells and seminiferous recognizing each protein: mouse anti–P-gp (C219, 1:250 dilution), tubules were exposed to 0.1% (v/v) DMSO (vehicle) in the absence of rat anti-Mrp4 (M4-I80, 1:500 dilution), rat anti-Bcrp (BXP-53, 1:500 ligands. mRNA or protein expression of ABC transporters was dilution), goat anti-PXR (A-20, 1:250 dilution), rabbit anti-CAR

examined between 6 and 72 hours in TM4 cells and between 1 and (M-127, 1:250), rabbit anti-GR (M-20, 1:2000), and mouse anti-actin jpet.aspetjournals.org 24 hours in seminiferous tubules post-treatment, with data shown for (AC40, 1:500 dilution). Primary antibody incubation was followed by time points where consistent upregulation of transporters was incubation with respective anti-mouse, anti-rat, anti-goat, or anti- observed (TM4 cells: 24 hours for mRNA, 72 hours for protein; rabbit horseradish peroxidase–conjugated secondary antibodies seminiferous tubules: 2 hours for mRNA and protein). All of the (1:10,000) for 1.5 hours. HepG2, MDA-MDR1, HEK-MRP4, and ligands were tested over a range of concentrations based on their EC50 MX-100 cell lysates were used as positive controls for nuclear receptor, values to study their effects on the transporters, and final concentra- P-gp, Mrp4, and Bcrp expression, respectively, whereas actin was tions used were based on their effectiveness in inducing transporter used as the loading control in all experiments. Protein bands were expression and cell viability. To ensure that cells would remain viable visualized by the West Pico enhanced chemiluminescence system at ASPET Journals on September 24, 2021 during ligand treatment, all ligand concentrations used were tested by (Thermo Fisher Scientific, Waltham, MA) after exposure to an X-ray applying the MTT assay as described previously by our laboratory film. Quantitative protein expression was analyzed using densitom- with minor modifications (Mosmann, 1983; Chan et al., 2011). After etry analysis with Alpha DigiDoc RT2 imaging software (Alpha treatment with ligands for up to 72 hours, cells were incubated for Innotech, San Leandro, CA). 2 hours at 37°C with 5 mg/ml MTT solution in phosphate-buffered siRNA Studies. TM4 Sertoli cells were seeded in six-well plates saline. The formazan content, dissolved in DMSO, from each well was with a density of 0.2 Â 106 cells/well. After 24 hours, cell monolayers at determined by UV analysis at 580 nm using a SpectraMax 384 micro- approximately 80% confluence were subjected to mPXR or mCAR plate reader (Molecular Devices, Sunnyvale, CA). Cell viability was targeting siRNA transfection. Transfection mix was prepared in Opti- assessed by comparing the absorbance of cellular reduced MTT in MEM GlutaMax (Invitrogen) medium with siRNA and Lipofectamine ligand-treated cells to that of vehicle-treated cells. 2000 (Invitrogen) according to the manufacturer’s protocol. The final RNA Extraction, Reverse Transcription, and Quantitative concentration of siRNA and Lipofectamine added to the cells were Real-Time Polymerase Chain Reaction. Total RNA was extracted 100 nM and 2 ml/ml, respectively, and the cells were incubated for from TM4 cells and mouse seminiferous tubules using TRIzol reagent 24 hours in the presence of transfection mixture. The following day, (Invitrogen, Carlsbad, CA) according to the manufacturer’sinstructions. the transfection mixture was replaced with fresh prewarmed TM4 cell RNA concentration (absorbance at 260 nm) and purity (absorbance ratio medium, and cell culture was pursued for an additional 48 hours. After 260/280 nm) was assessed using a DU Series 700 Scanning UV-visible 72 hours of siRNA transfection, cells were harvested to analyze P-gp, spectrophotometer (Beckman Coulter, Mississauga, ON, Canada). Mrp4, Bcrp, PXR, and CAR protein expression by western blot. Isolated RNA was digested in DNase I (0.1 U/ml) to remove genomic Ex Vivo Transport Assay with Seminiferous Tubules. The DNA. Then 2.0 mg DNase-treated total RNA was reverse transcribed tubular accumulation of [3H]-atazanavir, a known ARV substrate of using the high-capacity cDNA reverse transcriptase kit (Applied P-gp, was determined by applying a radioactive transport assay as Biosystems, Waltham, MA). Reverse transcription was performed at described previously (Robillard et al., 2012; Klein et al., 2013). 25°C for 10 minutes, followed by 37°C for 120 minutes and then 85°C Seminiferous tubules were dissected from the mouse testis and cut for 5 minutes using a Mastercycler ep Realplex 2S thermal cycler into 10- to 15-mm pieces for data normalization before transfer to a (Eppendorf, Mississauga, ON, Canada). mRNA expression of genes 96-well cell culture plate for incubation with ligands and/or transport encoding ABC transporters (Abcb1a, Abcb1b, Abcc4, and Abcg2) and assay. Tubules were either isolated and incubated at 35°C in the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase DMEM/F12 medium containing ligands prior to washing and trans- (Gapdh) or cyclophilin B was analyzed by quantitative real-time port experiments using transport buffer (Ringer’s solution containing polymerase chain reaction (qPCR) on a Mastercycler ep Realplex 2S 103 mM NaCl, 25 mM NaHCO3, 19 mM sodium gluconate, 1 mM thermal cycler using TaqMan qPCR chemistry. Primers and probes sodium acetate, 1.2 mM NaH2PO4, 5 mM KCl, 1 mM CaCl2,1mM (Supplemental Table 2) were designed and validated by Life Technol- MgCl2, and 5.5 mM glucose, at pH 7.4), or they were isolated and ogies (Burlington, ON, Canada). All reactions were performed in washed directly in transport buffer without ligand treatment. For triplicate with each 20-ml reaction containing 100 ng cDNA, 1 ml20Â transport experiments, seminiferous tubules were preincubated at primer mix, and 10 ml TaqMan qPCR master mix. To analyze the 35°C for 15 minutes in transport buffer alone or in transport buffer regulation of Abcb1b, Abcg2, and Abcc4 genes in ligand-treated containing standard P-gp inhibitors CsA (25 mM) or PSC833 (5 mM), Regulation of ABC Transporters in the Testis 327 a cyclosporine derivative and specific inhibitor of P-gp (Atadja et al., 1998). To initiate transport, the preincubation buffer was replaced with transport buffer containing 1 mM[3H]-atazanavir with or without P-gp inhibitors. At the desired time points, the reaction was stopped by washing the tubules with ice-cold transport buffer. Seminiferous tubules were then dissolved in 1 N NaOH for extraction of accumu- lated radioactivity. The content of each well was collected and mixed with 3 ml Pico-Fluor 40 scintillation fluid (PerkinElmer Life and Analytical Sciences, Waltham, MA), and the total radioactivity was measured using an LS5600 liquid scintillation counter (Beckman Coulter). Background accumulation was estimated by determining the retention of radiolabeled compound in the tubules after a minimum (zero) time of exposure by removing the radiolabeled solution immediately after its addition into each well. Each data point within an individual experiment represents the average of triplicate Fig. 1. PXR and CAR protein expression in human testicular tissue and TM4 values. For the accumulation experiments, data are reported as Sertoli cells. PXR and CAR proteins were examined in testicular tissue accumulation of the substrate at steady state, as determined by the obtained from three healthy human subjects (S1–S3), as well as in different – time-dependent uptake assays, in the absence or presence of inhibitor. passages of TM4 Sertoli cells (P1 P3). Whole tissue or cell lysates were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene

Statistical Analyses. All experiments were repeated at least difluoride membrane. HepG2 cells served as the positive control. The goat Downloaded from three times in cells pertaining to different passages, or in seminiferous polyclonal A-20 antibody was used to detect PXR, whereas CAR was detected tubules isolated from two to three animals per experiment. Results are using rabbit polyclonal M-127 antibody. Actin was used as an internal control expressed as the mean 6 S.E.M. Statistical analyses were performed and was detected using anti–b-actin mouse monoclonal antibody. using GraphPad Prism 7 software (GraphPad Software Inc., San Diego, CA). Comparison between groups was performed as appropri- ate, applying either the two-tailed t test for unpaired experimental performed 72 hours post-treatment with both PXR ligands values or one-way analysis of variance with the Bonferroni multiple- (Fig. 2, C–F). Densitometric analysis revealed approximately jpet.aspetjournals.org , comparisons post hoc test or Dunnett post hoc t test. P 0.05 was 50% induction in P-gp expression when cells were exposed to considered statistically significant. dexamethasone or PCN, whereas Bcrp demonstrated close to 30% and 40% increases in cells exposed to dexamethasone or Results PCN, respectively. Effect of PXR Inhibitor on Abcb1b and Abcg2 In- PXR and CAR Protein Expression in Human Testic- duction. Ketoconazole is an established antagonist of PXR ular Tissue and TM4 Sertoli Cells. To investigate the (Mani et al., 2013); therefore, we tested whether the observed at ASPET Journals on September 24, 2021 expression of PXR and CAR in human testicular tissue obtained induction of P-gp or Bcrp was primarily mediated by PXR in from three noninfected individuals, as well as in the TM4 Sertoli the TM4 Sertoli cells using this compound. Cells were exposed cell line derived from mouse testis, western blot analyses were to ketoconazole (10 mM) in the presence of dexamethasone or performed using whole tissue or cell lysates, respectively. PCN for 24 hours. As expected, the addition of ketoconazole HepG2 cells known to express PXR and CAR served as a with dexamethasone or PCN prevented Abcb1b and Abcg2 positive control in these experiments (Fig. 1). CAR was robustly upregulation (Fig. 3), further suggesting that PXR is involved expressed in both the human testicular tissue samples and TM4 in the regulation of these transporters. Sertoli cells, with bands visible at a molecular weight of 60 kDa. CAR-Mediated Upregulation of Abcb1b (P-gp) and PXR was also detected with protein bands at the expected Abcc4 (Mrp4) mRNA and Protein Expression. Treat- molecular weight of approximately 52 kDa in all samples ment of TM4 cells with the CAR ligand TCPOBOP (50–500 nM) evaluated. Considering the exceptional rarity of human testic- resulted in significant increases in both Abcb1b and Abcc4 ular tissue availability, the rest of the experimental work was (which encodes Mrp4) mRNA about 1.5-fold (Fig. 4, A and B). performed in vitro in TM4 Sertoli cells and ex vivo in seminif- Densitometry analysis also revealed a moderate but significant erous tubules freshly isolated from mice testes. increase in the protein expression of P-gp and Mrp4 after PXR-Mediated Upregulation of Abcb1b (P-gp) and 72-hour treatment with TCPOBOP (Fig. 4, B and C). Together, Abcg2 (Bcrp) mRNA and Protein Expression. Applying these results suggest a pathway involving CAR in the regula- qPCR analysis, we examined the expression of Abcb1b and tion of these ABC transporters. Abcg2 mRNA, which encode P-gp and Bcrp, respectively, in Downregulation of P-gp, Bcrp, and Mrp4 Expression TM4 Sertoli cells exposed to PXR ligands PCN or dexameth- by PXR- and CAR-Targeting siRNA. PXR- and CAR- asone. We also investigated the expression of Abcb1a and found targeting siRNA were used to further examine the direct that of the two murine Abcb1 isoforms encoding P-gp, Abcb1b involvement of each nuclear receptor in the regulation of the was most abundant at the BTB (Supplemental Fig. 1). This was transporters in TM4 Sertoli cells. In PXR siRNA-transfected cells also demonstrated previously by our group (Robillard et al., compared with cells treated with control nonsilencing siRNA, 2012); therefore, we only investigated the regulation of Abcb1b PXR protein expression was downregulated by approximately in our studies. TM4 cells treated with increased concentrations 50%, whereas P-gp, Bcrp, and Mrp4 expression was significantly of PCN (10–50 mM) or dexamethasone (50–100 mM) had reduced by 32%, 27%, and 33%, respectively (Fig. 5A). Experi- significantly higher levels of Abcb1b (up to 2-fold) and Abcg2 ments using CAR siRNA demonstrated an approximately 35% (up to 3-fold) mRNA expression compared with vehicle-treated decrease in CAR expression as well as P-gp and Bcrp and a 25% control cells at 24 hours (Fig. 2, A and B). To determine whether decrease in Mrp4 expression, compared with cells treated with the increases in mRNA expression would result in changes in control siRNA (Fig. 5B). The PXR ligand dexamethasone is also P-gp and Bcrp protein expression, western blot analysis was knowntoactivateGR(Bledsoeetal.,2002),andtheinductionof 328 Whyte-Allman et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 2. PXR-mediated upregulation of Abcb1b (A) and Abcg2 (B) mRNA expression as well as P-gp (C and E) and Bcrp (D and F) protein expression in TM4 Sertoli cells. Cells were exposed to PXR ligands dexamethasone or PCN at various concentrations for 24 hours. mRNA expression was measured by qPCR. All ligand- treated and vehicle (DMSO-treated) samples were examined in triplicates in each experiment. Threshold cycle (CT) values of each gene of interest were normalized with the housekeeping gene Gapdh mRNA and compared with the vehicle control using the comparative CT method (ΔΔCT). Results are expressed as the mean fold change 6 S.E.M. of three separate experiments. (C–F) Induction of P-gp and BCRP protein expression by PXR in ligand-treated cells compared with the vehicle control for 72 hours. The MDA-MDR1 and MCF7-MX100 cell systems were used as positive controls for P-gp and Bcrp expression, respectively. The relativeprotein expression was determined by densitometry analysis. Representative immunoblots (top) are shown with their corresponding densitometry analyses (bottom). For each loaded sample, the protein of interest was first normalized against b-actin. Results are expressed as the percent change of ligand-treated samples compared with vehicle-treated control samples and reported as the mean 6 S.E.M. for three separate experiments. *P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001 (statistically significant differences between treatment and vehicle as determined by Student’s t test). Regulation of ABC Transporters in the Testis 329 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 3. Effects of PXR antagonist ketoconazole on Abcb1b and Abcg2 mRNA expression in TM4 Sertoli cells. Cells were exposed to ketoconazole with or without PXR ligands dexamethasone (A and B) or PCN (C and D) for 24 hours. mRNA expression was measured by qPCR. All treatment and vehicle samples were examined in triplicates in each experiment. Threshold cycle values of each gene of interest were normalized with the housekeeping gene Gapdh mRNA and compared with the vehicle (DMSO-treated) control. Results are expressed as the mean fold change 6 S.E.M. for three separate experiments. *P , 0.05; **P , 0.01; ***P , 0.001 (statistically significant differences between treatment groups and vehicle as determined by one-way analysis of variance with Bonferroni’s correction for multiple comparisons).

P-gp and Bcrp in the presence of this ligand could possibly be the expression of Abcb1b mRNA by approximately 1.5-fold, mediated via pathways involving GR. We investigated whether 1.4-fold, 1.3-fold, and 1.5-fold, respectively (Fig. 6A). We also knocking down GR with targeting siRNA would result in the observed significant upregulation in P-gp protein expression by downregulation of P-gp and Bcrp in TM4 cells. Our data 55%, 57%, 93%, and 61% 2 hours post-treatment with PCN, demonstrated that compared with control siRNA, exposure to TCPOBOP, darunavir, and efavirenz, respectively. GR siRNA significantly decreased the levels of GR significantly; P-gp Function in Mouse Seminiferous Tubules. To however there were no changes in the expression of the investigate whether P-gp was functional in the seminiferous transporters. Furthermore, dexamethasone significantly upre- tubules, substrate accumulation assays were performed using gulated the expression of P-gp and Bcrp in the presence of GR [3H]-atazanavir. Atazanavir is a known P-gp substrate and is siRNA, suggesting a PXR-mediated effect (Supplemental Fig. 2). a commonly used ARV in the clinic (atazanavir remains on the PXR- and CAR-Mediated Induction of P-gp Ex Vivo World Health’s Organization list of essential medicines, http:// in Mouse Seminiferous Tubules. Seminiferous tubules www.who.int/medicines/publications/essentialmedicines/en/. isolated from mice testes were exposed to PXR ligands (PCN, The time course of [3H]-atazanavir at 35°C showed increasing efavirenz, or darunavir) or CAR ligands (TCPOBOP or efavir- substrate uptake until a plateau was reached at approxi- enz) to determine whether these compounds, particularly the mately 1 hour under control conditions in the seminiferous clinically relevant ARV ligands, could induce the expression of tubules (Fig. 7A). The accumulation of [3H]-atazanavir was efflux pumps in a more physiologically representative BTB significantly increased by 30% and 76% in the presence of P-gp system. Our group has previously demonstrated that ARVs inhibitors PSC833 or CsA, respectively, compared with the such as darunavir, efavirenz, ritonavir, abacavir, lopinavir, and control (Fig. 7B), suggesting that [3H]-atazanavir accumula- nevirapine serve as ligands for PXR or CAR by performing tion is mediated by P-gp at the BTB. luciferase reporter gene assays (Chan et al., 2013a). After Ligand-Activated PXR and CAR Increase P-gp Func- 2-hour exposure of seminiferous tubules to PCN, darunavir, tion in Mouse Seminiferous Tubules. We examined efavirenz, or TCPOBOP, we observed a significant increase in whether an upregulation of P-gp expression at the BTB would 330 Whyte-Allman et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 4. CAR-mediated upregulation of Abcb1b (A) and Abcc4 (B) mRNA expression as well as P-gp (C) and Mrp4 (D) protein expression in TM4 Sertoli cells. Cells were exposed to different concentrations of the CAR agonist TCPOBOP for 24 hours. mRNA expression was measured by qPCR. All ligand treatment and vehicle (DMSO-treated) samples were examined in triplicates in each experiment. Threshold cycle (CT) values of each gene of interest were normalized with the housekeeping gene Gapdh mRNA and compared with the vehicle control using the comparative CT method (ΔΔCT). Results are expressed as the mean fold change 6 S.E.M. for three separate experiments. Induction of P-gp and Mrp4 protein expression by CAR in TM4 Sertoli cells treated with TCPOBOP or vehicle for 72 hours. The MDA-MDR1 and HEK-MRP4 overexpressing cell systems were used as positive controls for P-gp and Mrp4 expression, respectively. The relative protein expression was determined by densitometry analysis. Representative immunoblots (top) are shown with their corresponding densitometry analyses (bottom). For each loaded sample, the protein of interest was first normalized against b-actin. Results are expressed as the percent change of ligand- treated samples compared with vehicle-treated control samples and reported as the mean 6 S.E.M.ofthreeseparateexperiments.*P , 0.05; **P , 0.01; ***P , 0.001 (statistically significant differences between treatment and vehicle as determined by the t test). also result in increased function. Seminiferous tubules treated that P-gp induction regulated by ligand-activated PXR and with ARVs and other established ligands of PXR and CAR CAR including commonly prescribed ARVs can result in an resulted in a moderate but significant reduction in [3H]-atazanavir increase in its function at the BTB. accumulation (approximately 30% in the presence of PCN and 20% in the presence of TCPOBOP, darunavir, or efavir- Discussion enz) compared with the vehicle control (Fig. 8). Moreover, PSC833 (a specific P-gp inhibitor) abolished the differences in ARV distribution into tissues is primarily regulated by the [3H]-atazanavir accumulation between vehicle control and extent of serum protein binding, drug physicochemical proper- treatment groups, further confirming the involvement of P-gp ties, and expression and activity of drug transporters and drug in the efflux of this drug. Together, these data demonstrate metabolic enzymes (Walubo, 2007; Kis et al., 2010; Chillistone Regulation of ABC Transporters in the Testis 331 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 5. Effect of PXR (A) and CAR (B) siRNA on the protein expression of P-gp, Bcrp, and Mrp4 in TM4 Sertoli cells. Cells were treated with control siRNA (Cont siRNA) or siRNA directed against PXR and CAR. The MDA-MDR1, MCF7-MX100, and HEK-MRP4 cell systems were used as positive controls for P-gp, Bcrp, and Mrp4 expression, respectively. Representative immunoblots (left) with corresponding densitometry analyses (right) were used to determine relative protein expression. For each loaded sample, the protein of interest was first normalized against b-actin. Results are expressed as the percent change of samples exposed to PXR- or CAR-targeting siRNA compared with control siRNA and reported as the mean 6 S.E.M. for at least three experiments. *P , 0.05; **P , 0.01; ***P , 0.001 (statistically significant differences between treatment and vehicle as determined by the t test). and Hardman, 2014). Most ARVs (especially PIs) display known to be substrates of drug efflux transporters including substantial protein binding .85% (Bazzoli et al., 2010), result- P-gp, BCRP, and the MRPs (Kis et al., 2010; Alam et al., 2016). ing in lower amounts of unbound drug to distribute into tissues. We previously demonstrated the potential role of ABC trans- Numerous ARVs that are currently recommended as preferred porters in restricting ARV concentrations in the testis of HIV- and alternative regimens during HIV pharmacotherapy are infected men (Huang et al., 2016). Furthermore, low seminal 332 Whyte-Allman et al.

Fig. 6. PXR- and CAR-mediated upregulation of Abcb1b mRNA (A) and protein (B) expression in mouse seminiferous tubules. Seminiferous tubules were exposed to ligands for 2 hours. (A) mRNA expression was measured by qPCR. All treatment and vehicle (DMSO-treated) control samples were examined in tripli- Downloaded from cates in each experiment. Threshold cycle (CT) values of each gene of interest were normalized with the housekeeping gene Gapdh mRNA and compared with the vehicle control using the comparative CT method (ΔΔCT). Results are expressed as the mean fold change 6 S.E.M. of three separate experiments (n = 9 animals). (B) Induction of P-gp protein expression in ligand-treated seminiferous tubules. The MDA-MDR1 overexpressing cell system served as positive control for P-gp expression. The relative protein expression was determined by densitometry analysis. Representa- jpet.aspetjournals.org tive immunoblots (top) is shown with the corresponding densitometry analysis (bottom). For each loaded sample, the protein of interest was first normalized against b-actin. Results are expressed as the percent change of ligand-treated samples compared with vehicle-treated control samples and reported as mean 6 S.E.M. (n =3–5 animals per treatment). *P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001 (statistically significant differences between treatment groups and vehicle as determined by the t test). at ASPET Journals on September 24, 2021

fluid concentrations compared with plasma levels of ARVs in regulation of ABC transporters in the human testis due to the HIV-infected men have also been observed in clinical studies limitations of obtaining this tissue. However, their expression (Else et al., 2011), suggesting a potential contribution of efflux in the human testis implicates the potential for drug-receptor transporters in restricting ARV permeability along the male interactions and regulation of drug transporters at the BTB. genital tract. The nuclear receptor–mediated induction of these Further experiments were performed in vitro using TM4 transport systems during chronic therapy could further limit Sertoli cells, a nontumorigenic cell line and BTB model that ARV testis concentrations by modulating blood to tissue was first derived from the testis of BALB/c mice (Mather, disposition across the BTB. P-gp and BCRP are known to be 1980). We previously characterized this model by performing regulated by the nuclear receptors PXR and CAR (Bauer et al., morphology studies and examining the expression of the 2004; Narang et al., 2008; Qiao and Yang, 2014), whereas the Sertoli cell marker and transcription factor GATA-binding regulation of MRP4 is primarily modulated by CAR (Assem factor 4 (GATA-4), and we also demonstrated the expression et al., 2004). In this study, we examined the role of PXR in and function of ABC transporters in the efflux of ARVs in these regulating the expression of P-gp and Bcrp, whereas we cells (Robillard et al., 2012; Hoque et al., 2015). examined CAR for its role in regulating both P-gp and Mrp4. The PXR-mediated induction of P-gp and Bcrp that we Herein, we demonstrated the protein expression of PXR and observed in this study agrees with previous work from our CAR in human testicular tissue and TM4 Sertoli cells. We group and others. In particular, our group previously demon- were not able to investigate the nuclear receptor–mediated strated PXR-mediated induction of P-gp in vivo at the mouse Regulation of ABC Transporters in the Testis 333

Fig. 7. P-gp function in mouse seminiferous tubules. (A) The time profile (up to 2 hours) of atazanavir (1 mM) was measured at 35°C. Atazanavir uptake was calculated as picomoles of intracellular radioactivity normalized to the length of the seminiferous tubules in Downloaded from millimeters. Results are expressed as the mean 6 S.E.M. of three separate experiments (n = 6 animals). (B) Accumulation of atazanavir (2 hours) in the absence (control) or presence of P-gp inhibitors PSC833 and CsA at 35°C. Results are expressed as the mean 6 S.E.M. of three separate experiments with each data point in an individual experiment representing triplicate measurements. **P , 0.01; ***P , 0.001 (statistically significant differences between inhibitor groups and control as determined by the t test). jpet.aspetjournals.org at ASPET Journals on September 24, 2021

BBB in animals administered dexamethasone (Chan et al., the presence of PXR ligands, without any downregulatory effects 2013b) and in vitro in a human BBB cell line model (Chan et al., when cells were exposed to ketoconazole only. Activation of CAR 2011). Bauer et al. (2004) also showed the involvement of PXR by the established ligand TCPOBOP, a phenobarbital-like in upregulating P-gp at the rat BBB after exposure to PCN or compound, in mice (Tzameli et al., 2000) also demonstrated that dexamethasone. The involvement of PXR in the regulation of this nuclear receptor can upregulate P-gp and Mrp4 in TM4 BCRP was also demonstrated by Narang et al. (2008) at the rat Sertoli cells. These results complement previous studies by our BBB and by Qiao and Yang (2014), in vitro, in human breast group, which showed that ligand-activated CAR was able to cancer cell lines. To further assess the role of this nuclear induce P-gp expression at the human BBB (Chan et al., 2011). receptor in regulating P-gp and Bcrp at the BTB, TM4 Sertoli To examine whether PXR or CAR could directly regulate the cells were exposed to the PXR antagonist ketoconazole, an expression of ABC transporters at the BTB, we performed antifungal drug and well established inhibitor of the phase I studies using siRNA to knockdown the nuclear receptors and metabolic enzyme CYP3A4 (Thummel and Wilkinson, 1998). measured the corresponding effects on the transporters (Fig. 5). This treatment resulted in the downregulation of these trans- We observed that both PXR and CAR gene silencing resulted in porters in the presence of dexamethasone or PCN (Fig. 3). significant downregulation of the ABC transporters, suggesting Downregulation of P-gp and Bcrp by ketoconazole could possibly that these nuclear receptors are directly involved in regulating involve other nuclear receptor pathways, since this drug the transporters at the BTB. We also demonstrated that the has been demonstrated as a pan-antagonist that could inhibit upregulation of the transporters in the presence of dexameth- the ligand-induced activation of the liver X receptor and the asone (also a GR ligand) was mediated directly through PXR farnesoid X receptor (Huang et al., 2007). However, since inhibi- (Supplemental Fig. 2), which is in agreement with the work of tion of these nuclear receptors as well as PXR, is dependent on Pascussi et al. (2000), who proposed that dexamethasone activation by their respective ligands, the inhibitory effect of concentrations above 10 mM directly activated PXR and in- ketoconazole demonstrated in our experiments was most likely duced the regulation of target genes such as CYP3A4 in human mediated through PXR as we only observed downregulation in hepatocytes. 334 Whyte-Allman et al.

increased P-gp expression, we explored the effects of these ligands on the function of P-gp in the seminiferous tubules. We conducted transport assays with atazanavir, a commonly used PI in the clinic and P-gp substrate as previously demonstrated in brain, testis, and intestine of humans or rodents (Kis et al., 2013; Robillard et al., 2014). The accumulation of atazanavir was significantly increased in the presence of standard P-gp inhibitors PSC833 or CsA, suggesting that P-gp–mediated efflux is involved in the overall transport of this drug at the BTB ex vivo (Fig. 7B). Treatment of seminiferous tubules with these ligands increased P-gp function, as was evident by the decrease in atazanavir accumulation (Fig. 8). Together, these data suggest that ARVs are capable of inducing changes in the expression and function of ABC drug transporters through their interactions with nuclear receptors, potentially leading to alterations in their pharmacokinetic properties as well as drug-drug interactions at the BTB. PXR and CAR exhibit species differences in their ligand specificity. Our studies were Downloaded from Fig. 8. Atazanavir accumulation in PXR and CAR ligand-treated performed in mice and may not fully predict activation of the seminiferous tubules. Atazanavir (1 mM) accumulation was measured nuclear receptors in humans; however, our group previously after 2-hour treatment with the PXR ligands PCN or darunavir, the CAR demonstrated that efavirenz and darunavir activated human ligand TCPOBOP, or the dual PXR and CAR ligand efavirenz. For the PXR or CAR (Chan et al., 2013a). group without PSC833, results are expressed as the mean percent change in atazanavir accumulation versus the vehicle control (DMSO-treated, In summary, to our knowledge, this study is the first to without PSC833) 6 S.E.M. obtained from three independent experiments reveal that the testis is capable of altering drug pharmacoki- jpet.aspetjournals.org (n = 3 animals per experiment). ***P , 0.001 (statistically significant netic properties through nuclear receptor–mediated path- differences between groups were determined by one-way analysis of variance with the Dunnett post hoc t test). For the group with PSC833, ways. We report novel findings demonstrating upregulation results are expressed as the mean percent change in atazanavir of P-gp, Bcrp, and Mrp4 expression at the BTB in vitro in mice, accumulation normalized to the corresponding treatment without and we observed upregulation of P-gp expression and function 6 , PSC833 S.E.M. ****P 0.0001 (statistically significant differences in mouse seminiferous tubules after exposure to nuclear between groups was determined by one-way analysis of variance with the Bonferroni multiple-comparisons post hoc test). Each data point in an receptor ligands including ARVs. Our data indicate the high individual experiment represents triplicate measurements. complexities of ARV pharmacokinetics and the great potential at ASPET Journals on September 24, 2021 for drug-drug interactions during therapy. In addition to ARVs, nuclear receptors are modulated by other drugs used Our group previously demonstrated the transport of ARVs, to treat HIV coinfections. For example, patients coinfected including atazanavir and raltegravir, by ABC transporters in with tuberculosis may receive concurrent treatment including TM4 Sertoli cells (Robillard et al., 2012; Hoque et al., 2015); the rifamycin class of drugs, which are potent activators of however, these studies have not been performed in a more PXR (Dooley et al., 2008). Administration of these drugs could physiologically relevant system such as freshly isolated seminif- activate the nuclear receptor–mediated regulation of drug erous tubules. We investigated the role of the nuclear receptors transporters and metabolic enzymes, potentially leading to PXR and CAR in regulating the expression and function of P-gp reduced drug efficacy when multidrug regimens are taken ex vivo in mouse seminiferous tubules. As we have observed chronically by patients. Further studies are needed, especially in vitro at the BTB, exposure to PXR and CAR ligands, including in the human testis, to fully elucidate ARV interactions with darunavir and efavirenz (part of the current preferred and drug transporters and nuclear receptors, and their potential alternative ARV regimens; DHHS Panel on Antiretroviral Guide- contribution to the restriction of drug accumulation in this lines for Adults and Adolescents, 2016, Available at http://www. tissue and formation of a HIV sanctuary. aidsinfo.nih.gov/ContentFiles/Adul- tandAdolescentGL.pdf.), also induced the expression of P-gp at the mRNA and protein level Acknowledgments at the BTB ex vivo. It is noteworthy that the concentrations of The authors thank Drs. Pierre Brassard and Maud Bélanger ARVs used in our ex vivo experiments were higher than the (Metropolitan Centre of Plastic Surgery) and the Orchid study group clinically reported concentrations achieved in plasma. Pre- participants for the tissue donation. The authors also thank Dr. C. Yan vious work by our group demonstrated the activation of PXR Cheng and his research group (Population Council, Rockefeller or CAR, as well as upregulation in the expression and function University, New York, NY) and Drs. Nathan Cherrington and David of P-gp, using clinical plasma concentrations of darunavir Klein (University of Arizona, Tucson, AZ) for providing guidance with the methods on isolating and performing transport assays with and efavirenz, in vitro at the BBB (Chan et al., 2013a). We seminiferous tubules ex vivo. anticipate that plasma concentrations of these drugs could be effective at the human BTB but it was not feasible to perform Authorship Contributions these experiments in human samples. Based on experiments Participated in research design: Whyte-Allman, Bendayan. conducted in mouse seminiferous tubules ex vivo, lower Conducted experiments: Whyte-Allman, Hoque. concentrations of the drugs were less effective in inducing Contributed reagents or analytic tools: Jenabian, Routy, Bendayan. P-gp expression, suggesting species differences. Performed data analysis: Whyte-Allman, Hoque, Bendayan. Since we have determined that activation of PXR and Wrote or contributed to the writing of the manuscript: Whyte- CAR by PCN, TCPOBOP, darunavir, or efavirenz resulted in Allman, Bendayan. Regulation of ABC Transporters in the Testis 335

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