Xenobiotic Nuclear Receptors PXR and CAR Regulate...

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Xenobiotic Nuclear Receptors PXR and CAR Regulate Antiretroviral Drug Efflux

Transporters at the Blood-Testis Barrier

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, Canada (S.W., M.T.H., R.B.); Department of Biological

Sciences, Université du Québec à Montréal, Montréal, Canada, (M-A.J.); Chronic Viral

Illness Service, McGill University Health Centre, Montréal, Canada (J-P.R.)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 28, 2017 as DOI: 10.1124/jpet.117.243584

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Running Title: Regulation of ABC Transporters in the testis

Address correspondence to Reina Bendayan,

Reina Bendayan, Pharm. D.

Professor and Career Scientist, Ontario HIV Treatment Network, MHO

Department of Pharmaceutical Sciences

Leslie Dan Faculty of Pharmacy

University of Toronto

144 College Street, Toronto, ON M5S 3M2

Tel: 416-978-6979

Fax: 416-978-8511

Email: [email protected]

Number of text pages: 20

Number of figures: 8

Number of references: 45

Number of words in abstract: 242

Number of words in introduction: 734

Number of words in discussion: 1537

Recommended section assignment: Metabolism, Transport and Pharmacogenomics

Abbreviations:

ABC transporter – ATP-binding cassette membrane transporter


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ARV – Antiretroviral drug

BBB – Blood-brain barrier

BCRP – Breast cancer resistance protein

BTB – Blood-testis barrier

CAR – Constitutive androstane receptor

CsA – Cyclosporine A

GR – Glucocorticoid receptor

HIV – Human immunodeficiency virus

MRPs – Multidrug resistance-associated proteins

MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NNRTI - Non-nucleoside reverse transcriptase inhibitors

NRTI - Nucleoside reverse transcriptase inhibitors

PCN - Pregnenolone 16α carbonitrile

P-gp – P-glycoprotein

PI - Protease inhibitor

PSC833 - Valspodar

PXR – Pregnane X receptor

TCPOBOP - 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene


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Abstract

Poor antiretroviral drug (ARV) penetration in the testes could, in part, be due to the

presence of ATP-binding cassette membrane-associated drug efflux transporters such as

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug

resistance-associated proteins (MRPs) expressed at the blood-testis barrier (BTB). The

functional expression of these transporters is known to be regulated by ligand-activated

nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor

(CAR), in various tissues. The objective of this study was to investigate in vitro and ex

vivo the role of the PXR and CAR in the regulation of ABC transporters at the BTB. Both

PXR and CAR proteins were expressed in human testicular tissue and in mouse TM4

Sertoli cells, an in vitro cell line model of BTB. In addition, we demonstrated an

upregulation of P-gp, Bcrp and Mrp4 mRNA and protein expression, following exposure

to PXR or CAR ligands in TM4 cells. siRNA downregulation of PXR or CAR attenuated

the expression of these transporters, suggesting the direct involvement of these nuclear

receptors in regulating P-gp, Bcrp and Mrp4 in this system. In an ex vivo study using

freshly isolated mouse seminiferous tubules, we found that exposure to PXR or CAR

ligands, including ARVs, significantly increased P-gp expression and function. Together,

our data suggest that ABC transporters could be regulated at the BTB during chronic

treatment with ARVs that can serve as ligands for PXR and CAR, which could in turn

further limit testicular ARV concentrations.


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INTRODUCTION

Despite the use of highly active antiretroviral therapy, HIV-1 persistence in

anatomic reservoirs and sanctuary sites such as lymph nodes, brain and testes can, in

part, be attributed to the low antiretroviral drug (ARV) concentrations reached in these

tissues (Dahl et al., 2010; Fletcher et al., 2014; Huang et al., 2016; Jenabian et al., 2016).

Semen is the most common vector for HIV transmission globally and limited ARV

penetration in the testis could, in part, restrict ARV concentrations in the seminal fluid of

HIV infected men (Else et al., 2011). The testis is an immunoprivileged environment

where the blood-testis barrier (BTB), due to its physical and biochemical properties, can

limit the penetration of exogenous agents into this tissue. This barrier is primarily

composed of adjacent epithelial Sertoli cells which form tight junction complexes near the

basement membrane and physically divides the seminiferous epithelium into apical and

basolateral compartments. This allows for spermatid development in a microenvironment

within the seminiferous tubules where the entrance of anti-sperm antibodies and harmful

xenobiotics are restricted (Su et al., 2011).

ARV disposition in rodents and humans primarily involves transport by members

of the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies of drug

transport proteins across biological membranes, as well as intracellular drug metabolism.

The ABC family includes several drug efflux transporters such as P-glycoprotein (Pgp,

ABCB1), multidrug resistance-associated proteins (MRPs, ABCC), and breast cancer

resistance protein (BCRP, ABCG2), while the SLC transporters comprise organic anion-

transporting polypeptides, organic anion and organic cation transporters, as well as

equilibrative and concentrative nucleoside transporters (Kis et al., 2010). Our group and

others have demonstrated in vitro and in vivo that several efflux transporters are


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functionally expressed at the BTB, and could potentially limit the penetration of ARVs into

rodent and human testis (Choo et al., 2000; Robillard et al., 2012, 2014; Huang et al.,

2016).

Most ARVs from the protease inhibitor (PI) class are known to be substrates of P-

gp while BCRP is primarily involved in the transport of several of the nucleoside and non-

nucleoside reverse transcriptase inhibitors (NRTI/NNRTI). Both P-gp and BCRP have

also been implicated in the efflux of integrase strand transfer inhibitor drugs such as

dolutegravir and raltegravir. The MRP isoforms are also known to be involved in the

transport of ARVs, particularly, MRP4 is involved in the efflux of several NRTIs such as

lamivudine, tenofovir and abacavir. In addition to being substrates, many ARVs also

interact with these transporters as inducers and/or inhibitors (supplemental table S-1).

Recently, we characterized the expression and localization of drug transporters and

metabolic enzymes involved in ARV transport in the testes of HIV-infected individuals on

ARV therapy, and measured plasma and testicular drug concentrations. In particular,

darunavir a P-gp substrate which is currently recommended as a preferred PI regimen

(DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents, 2016), displayed

sub-therapeutic levels in the testis, reaching approximately 19% of plasma concentrations

(Huang et al., 2016), thus demonstrating the role that these transporters may play in

limiting ARV testicular tissue concentrations.

The functional expression of P-gp, BCRP and MRP isoforms is known to be

regulated through the activity of ligand-activated nuclear receptors pregnane X receptor

(PXR) and/or constitutive androstane receptor (CAR) in tissues such as the brain, liver,

intestine, as well as in PBMCs (Assem et al., 2004; Albermann et al., 2005; Burk et al.,


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2005; Chan et al., 2011). These two nuclear receptors are known to interact with

endogenous ligands and a wide range of pharmacological agents and hence are

recognized as major xenobiotic sensors (Urquhart et al., 2007). Our group previously

demonstrated that ligand-activated PXR and CAR upregulated P-gp, in vitro and in vivo

at the level of the blood-brain barrier (BBB), resulting in further impairment of drug

permeation and distribution in the central nervous system (Chan et al., 2011; Chan,

Saldivia, et al., 2013). Our group and others have also demonstrated that ARVs can

directly activate PXR and CAR causing an induction in the expression and activity of their

target genes (Dussault et al., 2001; Chan, Patel, et al., 2013).

To the best of our knowledge, the regulation of drug efflux transporter expression

and function by nuclear receptors at the BTB has not been addressed. The objective of

this study was to investigate the role of PXR and CAR in the regulation of ABC drug efflux

transporters (P-gp, Bcrp and Mrp4) at the BTB in vitro and ex vivo in mice.

MATERIALS AND METHODS

Chemicals and reagents. Unlabeled ARVs were obtained from the National Institutes of

Health AIDS Research and Reference Reagent Program (Bethesda, MD, USA). [3H]-

atazanavir (0.900 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA).

Valspodar (PSC833) was a generous gift from Novartis Pharma (Basel, Switzerland).

Cyclosporine (CsA) was obtained from Tocris Biosciences (Ellisville, MO, USA). 1,4-

Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), dexamethasone, ketoconazole, 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenly-tetrazolium bromide (MTT) were purchased from

Sigma-Aldrich (Oakville, ON, Canada). Pregnenolone 16α carbonitrile (PCN) was

purchased from Cayman Chemicals (Ann Arbor, MI, USA). Type I collagen was


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purchased from BD Biosciences (San Jose, CA, USA). Small interfering RNA (siRNA)

against mouse PXR (sc-44058), CAR (sc-43663) and GR (sc-35506), with non-silencing

negative control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology Inc

(Santa Cruz, CA, USA). Each siRNA product contained a pool of 3 target-specific 19-25

nt siRNAs designed to knock down gene expression. The mouse anti-actin, goat anti-

PXR, rabbit anti-CAR and rabbit anti-GR antibodies were also obtained from Santa Cruz

Biotechnology Inc (Santa Cruz, CA, USA). Please note that the rabbit anti-CAR (sc-

13065) antibody obtained from Santa Cruz Biotechnology has been reported to identify

CAR at around 55-60 kDa by the company. Additionally, several groups including ours

have detected CAR at around 50-60 kDa using this antibody (Hernandez et al., 2010;

Wang et al., 2010; Chan et al., 2011), even though the predicted molecular weight of CAR

has been reported at around 40-45 kDa based on its amino acid sequence. This

potentially could be due to changes in the phosphorylation of the molecule (Mutoh et al.,

2009), or other post-translational modifications that could affect the migration of the

protein in different tissues. Mouse anti-P-gp antibody, rat anti-Bcrp antibody and rat anti-

Mrp4 antibody were purchased from Enzo Life Sciences (Farmingdale, NY, USA), Abcam

Inc (Boston, MA, USA) and Kamiya Biomedical Company (Seattle, WA, USA),

respectively. Horseradish peroxidase-conjugated secondary antibodies were ordered

from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Human testicular tissue. In collaboration with Dr. Jean-Pierre Routy at the McGill

University Health Center, Dr. Pierre Brassard and Dr. Maud Bélanger from the

Metropolitan Centre of Plastic Surgery, in Montréal, Quebec, we were able to obtain

testicular tissue from individuals who were undergoing orchiectomy for gender


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reassignment. This study obtained ethics approval from the McGill University Health

Centre Ethical Review Board. All study subjects provided written informed consent for

participation in the study. Tissue samples were stored in saline solution immediately after

surgery, and processed within 1-2 h by cutting into small pieces, snap freezing in liquid

nitrogen and storing at -80°C until study assessments. A small portion of each testis was

sent to pathology for routine tests to exclude any infection, or malignancy, while the

remaining portion was used in our laboratory to examine nuclear receptor expression by

western blot analyses.

Cell culture systems. The continuous mouse Sertoli cell line (TM4) and the

hepatocellular carcinoma cell line (HepG2) were obtained from the American Tissue

Culture Collection (Manassas, VA, USA). TM4 cells were grown on culture flasks and

multi-well dishes pre-coated with rat tail collagen type 1 (100 µg/ml) to enhance cell

adherence and proliferation. The BCRP overexpressing human breast cancer cell line

(MCF7-MX100) was a generous gift from Dr. Susan Bates (Bethesda, MD, USA). The

human breast cancer cell line DA435/LCC6 stably transfected with human MDR1 cDNA

(MDA-MDR1) was kindly provided by Dr. Robert Clarke from Georgetown University

(Washington, DC, USA). The human embryonic kidney cell line stably transfected with

human MRP4 cDNA (HEK-MRP4) was kindly provided by Dr. Piet Borst (Netherlands

Cancer Institute, Amsterdam, The Netherlands). All cell culture systems were grown and

maintained at 37°C humidified 5% CO2-95% air with fresh media replaced every 2 to 3

days, according to our previously published protocols (Hoque et al., 2012; Robillard et al.,

2012).


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Mouse seminiferous tubule isolation C57BL/6 male mice were a kind gift from

Dr. Finnell at the University of Texas (Austin, TX, USA). All experiments, procedures and

animal care were conducted in accordance with the Canadian Council on Animal Care

guide lines and approved by the University of Toronto Animal Care Committee. Animals

were housed under a 12 h light/ 12 h dark cycle at room temperature with free access to

food and water. Seminiferous tubules were isolated as previously described (Lie et al.,

2010). In brief, testes from wildtype adult mice were decapsulated, washed and incubated

in collagenase (0.5 mg/ml) in DMEM: F12 [1:1] medium nutrient mixture at 35 oC for 25

min. Seminiferous tubules were then washed five times by sedimentation under gravity

to remove Leydig cells. The tubules were then incubated in DMEM: F12 media containing

desired ligands for 2 h. Samples were then washed, and stored at -80 oC for future

analysis, or used immediately to perform transport assays.

Ligand treatment. Monolayers of TM4 cells grown on collagen-coated 6-well

plates were treated with the established PXR ligands, dexamethasone (25-100 µM) or

PCN (10-50 µM) alone or in the presence of antagonist ketoconazole (10 µM), or the CAR

ligand TCPOBOP (50-500 nM). At the beginning of each experiment, culture medium was

aspirated and fresh medium containing ligands dissolved in DMSO was added.

Seminiferous tubules were exposed to PCN (100 µM), TCPOBOP (5 µM), efavirenz (50

µM) or darunavir (100 µM). Control cells and seminiferous tubules were exposed to 0.1%

(v/v) DMSO (vehicle) in the absence of ligands. mRNA or protein expression of ABC

transporters was examined between 6 and 72 h in TM4 cells and 1 and 24 h in

seminiferous tubules post-treatment, with data shown for timepoints where consistent

upregulation of transporters was observed (TM4 cells: 24 h mRNA, 72 h protein;


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seminiferous tubules: 2 h mRNA and protein). Each ligand was tested over a range of

concentrations based on their EC50 values to study their effects on the transporters, final

concentrations used were based on their effectiveness in inducing transporter expression,

as well as cell viability. To ensure cells would remain viable during ligand treatment, all

ligand concentrations used were tested by applying the MTT assay as described

previously by our laboratory with minor modifications (Mosmann, 1983; Chan et al., 2011).

Following treatment with ligands for up to 72 h, cells were incubated for 2 h at 37°C with

5 mg/ml MTT solution in phosphate buffered saline (PBS). The formazan content,

dissolved in DMSO, from each well was determined by UV analysis at 580 nm using a

SpectraMax 384 microplate reader (Molecular Devices, Sunnyvale, CA). Cell viability was

assessed by comparing the absorbance of cellular reduced MTT in ligand-treated cells to

that of vehicle-treated cells.

RNA extraction, reverse transcription and quantitative real-time PCR (qPCR).

Total RNA was extracted from TM4 cells and mouse seminiferous tubules using TRIzol

reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

RNA concentration (absorbance at 260nm) and purity (absorbance ratio 260/280nm) was

assessed using a DU Series 700 Scanning UV/VIS Spectrophotometer (Beckman

Coulter, Mississauga, ON, Canada). Isolated RNA was digested in DNase I (0.1 U/ml) to

remove genomic DNA. 2.0 µg of DNase-treated total RNA was then reverse transcribed

using the high capacity cDNA reverse transcriptase kit (Applied Biosystems, Waltham,

MA, USA). Reverse transcription was performed at 25 oC for 10 min, followed by 37 oC

for 120 min and then 85 oC for 5 min using a Mastercycler EP Realplex 2S thermal cycler

(Eppendorf, Mississauga, ON, Canada). The mRNA expression of genes encoding ABC


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transporters (Abcb1a, Abcb1b, Abcc4 and Abcg2) and the housekeeping genes

glyceraldehyde 3-phosphate dehydrogenase Gapdh or cyclophilin B was analyzed by

qPCR on a Mastercycler ep Realplex 2S thermal cycler using TaqMan qPCR chemistry.

Primers and probes (supplemental table S-2) were designed and validated by Life

Technologies (Burlington, ON, Canada). All reactions were performed in triplicate with

each 20uL reaction containing 100ng of cDNA, 1 µL of 20X primer mix and 10 µL of

TaqMan qPCR mastermix. To analyze the regulation of Abcb1b, Abcg2, Abcc4 genes in

ligand-treated samples, mRNA was normalized by Gapdh housekeeping gene. Results

are expressed as the fold change in mRNA levels ± S.E.M., and were calculated using

the comparative CT (∆∆CT) method where changes in transporter mRNA expression were

calibrated to vehicle-treated cells.

Immunoblot analysis. Western blot analysis was performed as described

previously by our group with minor modifications (Robillard et al., 2012). Briefly, lysates

from human testicular tissue, mouse seminiferous tubules, or cell lines were extracted

using a modified radioimmunoprecipitation lysis buffer containing 50 mM Tris-HCl, pH 7.4,

150 mM NaCl, 1 mM EGTA, 1 mM sodium ortho-vanadate, 0.5 mM phenylmethylsulfonyl

fluoride, 1% (vol/vol) Nonidet-P40, and 0.1% (vol/vol) protease inhibitor cocktail.

Homogenates were subjected to brief sonication, followed by centrifugation for 15 min at

20,000 X g and 4oC, and the supernatants were isolated as whole tissue or cell lysates.

The protein content of each cell lysate sample was quantified using a Bio-rad protein

assay kit. Proteins were separated on 7% or 10% SDS-polyacrylamide gel with 5%

stacking gel, then electro-transferred onto a polyvinylidene difluoride membrane. The

membranes were blocked for 2 h at room temperature in 5% skim milk–Tris-buffered


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saline containing 0.1% Tween 20 and incubated overnight at 4 oC with primary antibodies

recognizing each protein: mouse anti-Pgp (C219, 1:250 dilution), rat anti-Mrp4 (M4-I80,

1:500 dilution), rat anti-Bcrp (BXP-53, 1:500 dilution), goat anti-PXR (A-20, 1:250

dilution), rabbit anti-CAR (M-127, 1:250), rabbit anti-GR (M-20, 1:2000) and mouse anti-

actin (AC40, 1:500 dilution). Primary antibody incubation was followed by incubation with

respective anti-mouse, anti-rat, anti-goat or anti-rabbit horseradish peroxidase-

conjugated secondary antibodies (1:10,000) for 1.5 h. HepG2, MDA-MDR1, HEK-MRP4

and MX-100 cell lysates were used as positive controls for nuclear receptors, P-gp, Mrp4

and Bcrp expression respectively, while actin was used as the loading control in all

experiments. Protein bands were visualized by West Pico enhanced chemiluminescence

system (Thermo-Fisher Scientific, Waltham, MA, USA) after exposure to an X-ray film.

Quantitative protein expression was analyzed using densitometry analysis with the Alpha

DigiDoc RT2 imaging software (Alpha Innotech, San Leandro, CA, USA).

siRNA studies. TM4 Sertoli cells were seeded in six-well plates with a density of

0.2 X 106 cells/well. After 24 h, cell monolayers at approximately 80% confluence were

subjected to mPXR or mCAR targeting siRNA transfection. Transfection mix was

prepared in Opti-MEM GlutaMax (Invitrogen, Carlsbad, CA, USA) medium with siRNA

and lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The final

concentration of siRNA and lipofectamine added to the cells were 100 nM and 2 µl/ml,

respectively, the cells were incubated for 24 h in the presence of transfection mixture.

The following day, transfection mixture was replaced with fresh prewarmed TM4 cell

medium, and cell culture was pursued for an additional 48 h. After 72 h of siRNA


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transfection, cells were harvested to analyze P-gp, Mrp4, Bcrp, PXR and CAR protein

expression by western blot.

Ex vivo transport assay with seminiferous tubules. The tubular accumulation

of [3H]-atazanavir a known ARV substrate of P-gp was determined by applying a

radioactive transport assay as described previously (Robillard et al., 2012; Klein et al.,

2013). Seminiferous tubules were dissected from mouse testis and cut into 10-15 mm

pieces for data normalization before transferring to a 96-well cell culture plate for

incubation with ligands and/or transport assay. Tubules were either isolated and

incubated at 35 oC in DMEM:F12 medium containing ligands prior to washing and

transport experiments using transport buffer (Ringer’s solution containing (mM): 103

NaCl, 25 NaHCO3, 19 sodium gluconate, 1 sodium acetate, 1.2 NaH2PO4, 5 KCl, 1 CaCl2,

1 MgCl2, and 5.5 glucose, at pH 7.4), or they were isolated and washed directly in

transport buffer without ligand treatment. For transport experiments, seminiferous tubules

were preincubated at 35°C for 15 min in transport buffer alone or transport buffer

containing standard P-gp inhibitors CsA (25 µM) or PSC833 (5 µM), a cyclosporine

derivative and specific inhibitor of P-gp (Atadja et al., 1998). To initiate transport,

preincubation buffer was replaced with transport buffer containing 1 µM [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 accumulated radioactivity. The content of each

well was collected and mixed with 3 ml of Pico-Fluor 40 scintillation fluid (PerkinElmer

Life and Analytical Sciences, Waltham, MA, USA), and the total radioactivity was

measured using an LS5600 liquid scintillation counter (Beckman Coulter). Background


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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 values. For the accumulation experiments,

data are reported as accumulation of the substrate at steady state, as determined by the

time-dependent uptake assays, in the absence or presence of inhibitor.

Statistical analyses. All experiments were repeated at least three times in cells

pertaining to different passages, or in seminiferous tubules isolated from two to three

animals per experiment. Results are expressed as means standard error of the mean

(S.E.M). Statistical analyses were performed using GraphPad Prism 7 software

(GraphPad Software Inc., San Diego, CA). Comparison between groups was performed

as appropriate, applying the two-tailed Student’s t test for unpaired experimental values,

or one-way analysis of variance (ANOVA) with Bonferroni multiple comparison post hoc

test or Dunnett’s post hoc t test. P < 0.05 was considered statistically significant.

RESULTS

PXR and CAR protein expression in human testicular tissue and TM4 Sertoli cells.

To investigate the expression of PXR and CAR in human testicular tissue obtained from

three non-infected individuals, as well as in the TM4 Sertoli cell line derived from mouse

testis, western blot analyses were performed using whole tissue or cell lysates,

respectively. HepG2 cells known to express PXR and CAR served as a positive control

in these experiments (Fig. 1). CAR was robustly expressed in both the human testicular

tissue samples and TM4 Sertoli cells, with bands visible at a molecular weight of 60 kDa.


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PXR was also detected with protein bands at the expected molecular weight of

approximately 52 kDa in all samples evaluated. Considering the exceptional rarity of

human testicular tissue availability, the rest of the experimental work was performed in

vitro in TM4 Sertoli cells and ex vivo in seminiferous tubules freshly isolated from mice

testes.

PXR-mediated up-regulation of Abcb1b (P-gp) and Abcg2 (Bcrp) mRNA and

protein expression. Applying qPCR analysis, we examined the expression of Abcb1b

and Abcg2 mRNA which encode P-gp and Bcrp, respectively, in TM4 Sertoli cells

exposed to PXR ligands PCN or dexamethasone. We also investigated the expression of

Abcb1a and found that of the two murine Abcb1 isoforms encoding P-gp, Abcb1b was

most abundant at the BTB (supplemental fig. S-1), this was also demonstrated previously

by our group (Robillard et al., 2012), therefore we only investigated the regulation of

Abcb1b in our studies. TM4 cells treated with increased concentrations of PCN (10-50

μM) or dexamethasone (50-100 μM) had significantly higher levels of Abcb1b (up to 2-

fold) and Abcg2 (up to 3-fold) mRNA expression compared to vehicle treated control cells

at 24 h (Fig. 2A-B). To determine whether the increases in mRNA expression would result

in changes in P-gp and Bcrp protein expression, western blot analysis was performed 72

h post-treatment with both PXR ligands (Fig. 2C-F). Densitometric analysis revealed

approximately 50% induction in P-gp expression when cells were exposed to

dexamethasone or PCN, while Bcrp demonstrated close to 30% and 40% increases in

cells exposed to dexamethasone or PCN, respectively.

Effect of PXR inhibitor on Abcb1b and Abcg2 induction. Ketoconazole is an

established antagonist of PXR (Mani et al., 2013), therefore we tested whether the


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observed induction of P-gp or Bcrp was primarily mediated by PXR in the TM4 Sertoli

cells using this compound. Cells were exposed to ketoconazole (10 µM) in the presence

of dexamethasone or PCN for 24 h. As expected, addition of ketoconazole with

dexamethasone or PCN prevented Abcb1b and Abcg2 upregulation (Fig. 3), further

suggesting that PXR is involved in the regulation of these transporters.

CAR-mediated up-regulation of Abcb1b (P-gp) and Abcc4 (Mrp4) mRNA and

protein expression. Treatment of TM4 cells with the CAR ligand TCPOBOP (50 – 500

nM) resulted in significant increases in both Abcb1b and Abcc4 (which encodes Mrp4)

mRNA about 1.5-fold (Fig. 4A-B). Densitometry analysis also revealed moderate but

significant increase in the protein expression of P-gp and Mrp4 following 72 h treatment

with TCPOBOP (Fig. 4B-C). Together, these results suggest a pathway involving CAR in

the regulation of these ABC transporters.

Down-regulation of Pgp, Bcrp and Mrp4 expression by PXR- and CAR-

targeting siRNA. PXR and CAR targeting siRNA were used to further examine the direct

involvement of each nuclear receptor in the regulation of the transporters in TM4 Sertoli

cells. In PXR siRNA-transfected cells, compared to cells treated with control non-silencing

siRNA, PXR protein expression was downregulated by approximately 50%, whereas Pgp,

Bcrp and Mrp4 expression was significantly reduced by 32%, 27% and 33%, respectively

(Fig. 5A). Experiments using CAR siRNA demonstrated about 35% decrease in the

expression of CAR, as well as P-gp and Bcrp, and 25% decrease in Mrp4 expression,

compared to cells treated with control siRNA (fig. 5B). The PXR ligand dexamethasone

is also known to activate the glucocorticoid receptor (GR) (Bledsoe et al., 2002), and the

induction of P-gp and Bcrp in the presence of this ligand could possibly be mediated via


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pathways involving GR. We investigated if knocking down GR with targeting siRNA would

result in the downregulation of P-gp and Bcrp in TM4 cells. Our data demonstrated that

compared to control siRNA, exposure to GR siRNA significantly decreased the levels of

GR significantly, however there were no changes in the expression of the transporters.

Furthermore, dexamethasone significantly upregulated the expression of P-gp and Bcrp

in the presence of GR siRNA suggesting a PXR-mediated effect (supplemental Fig. S-2).

PXR- and CAR-mediated induction of P-gp ex vivo in mouse seminiferous

tubules. Seminiferous tubules isolated from mice testes were exposed to PXR ligands

(PCN, efavirenz or darunavir) or CAR ligands (TCPOBOP or efavirenz) in order to

determine if these compounds, particularly the clinically relevant ARV ligands, could

induce the expression of efflux pumps in a more physiologically representative BTB

system. Our group has previously demonstrated that ARVs such as darunavir, efavirenz,

ritonavir, abacavir, lopinavir and nevirapine serve as ligands for PXR or CAR by

performing luciferase reporter gene assays (Chan, Patel, et al., 2013). Following 2 h

exposure of seminiferous tubules to PCN, darunavir, efavirenz, or TCPOBOP, we

observed a significant increase in the expression of Abcb1b mRNA by approximately 1.5-

fold, 1.4-fold, 1.3-fold, and 1.5-fold respectively (Fig. 6A). We also observed significant

upregulation in P-gp protein expression by 55%, 57%, 93%, and 61% 2 h post-treatment

with PCN, TCPOBOP, darunavir and efavirenz, respectively.

P-gp function in mouse seminiferous tubules. To investigate whether P-gp was

functional in the seminiferous tubules, substrate accumulation assays were performed

using [3H]-atazanavir. Atazanavir is a known P-gp substrate and a commonly used ARV

in the clinic that remains on the World Health’s Organization list of essential medicines


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(World Health Organization, 2015). The time course of [3H]-atazanavir at 35 oC showed

increasing substrate uptake until a plateau was reached at approximately 1 h under

control conditions in the seminiferous tubules (Fig. 7A). The accumulation of [3H]-

atazanavir was significantly increased by 30% and 76% in the presence of P-gp inhibitors

PSC833 or CsA, respectively, compared to control (Fig. 7B), suggesting that [3H]-

atazanavir accumulation is mediated by P-gp at the BTB.

Ligand-activated PXR and CAR increase P-gp function in mouse

seminiferous tubules. We examined whether an upregulation of P-gp expression at the

BTB would also result in increased function. Seminiferous tubules treated with ARVs and

other established ligands of PXR and CAR resulted in a moderate but significant reduction

in [3H]-atazanavir accumulation, approximately 30% in the presence of PCN, and 20% in

the presence of TCPOBOP, darunavir or efavirenz, compared to vehicle control (Fig. 8).

Moreover, PSC833 (a specific P-gp inhibitor) abolished the differences in [3H]-atazanavir

accumulation between vehicle control and treatment groups, further confirming the

involvement of P-gp in the efflux of this drug. Together, these data demonstrate that P-

gp induction regulated by ligand-activated PXR and CAR including commonly prescribed

ARVs can result in an increase in its function at the BTB.

DISCUSSION

ARV distribution into tissues is primarily regulated by the extent of serum protein

binding, drug physicochemical properties, as well as the expression and activity of drug

transporters and drug metabolic enzymes (Walubo, 2007; Kis et al., 2010; Chillistone and

Hardman, 2014). Most ARVs, especially PIs, display substantial protein binding >85%


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(Bazzoli et al., 2010) resulting in lower amounts of unbound drug to distribute into tissues.

Numerous ARVs that are currently recommended as preferred and alternative regimen

during HIV pharmacotherapy are known to be substrates of drug efflux transporters

including P-gp, BCRP and the MRPs (Kis et al., 2010; Alam et al., 2016). We previously

demonstrated the potential role of ABC transporters in restricting ARV concentrations in

the testis of HIV infected men (Huang et al., 2016). Furthermore, low seminal fluid

concentrations compared to plasma levels of ARVs in HIV-infected men have also been

observed in clinical studies (Else et al., 2011), suggesting a potential contribution of efflux

transporters in restricting ARV permeability along the male genital tract. The nuclear

receptor-mediated induction of these transport systems during chronic therapy could

further limit ARV testis concentrations by modulating blood to tissue disposition across

the BTB. P-gp and BCRP are known to be regulated by the nuclear receptors PXR and

CAR (Bauer et al., 2004; Narang et al., 2008; Qiao and Yang, 2014), while the regulation

of MRP4 is primarily modulated by CAR (Assem et al., 2004). In the present study, we

examined the role of PXR in regulating the expression of P-gp and Bcrp, while CAR was

examined for its role in regulating both P-gp and Mrp4.

Herein, we demonstrated the protein expression of PXR and CAR in human

testicular tissue and TM4 Sertoli cells. We were not able to investigate the nuclear

receptor-mediated regulation of ABC transporters in human testis due to the limitations

of obtaining this tissue. However, their expression in the human testis implicates the

potential for drug-receptor interactions and regulation of drug transporters at the BTB.

Further experiments were performed in vitro using TM4 Sertoli cells, a non-tumorigenic

cell line and BTB model which was first derived from the testis of BALB/c mice (Mather,


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1980). We previously characterized this model by performing morphology studies and

examining the expression of the Sertoli cell marker and transcription factor GATA-4, as

well as demonstrated the expression and function of ABC transporters in the efflux of

ARVs in these cells (Robillard et al., 2012; Hoque et al., 2015).

The PXR-mediated induction of P-gp and Bcrp observed in the present study

agrees with previous work from our group and others. In particular, our group previously

demonstrated PXR-mediated induction of P-gp in vivo at the mouse BBB in animals

administered dexamethasone (Chan, Saldivia, et al., 2013), and in vitro in a human BBB

cell line model (Chan et al., 2011). Bauer et al. also showed the involvement of PXR in

upregulating P-gp at the rat BBB following exposure to PCN or dexamethasone (Bauer

et al., 2004). The involvement of PXR in the regulation of BCRP was also demonstrated

by Narang et al. at the rat BBB (Narang et al., 2008) and by Qiao and Yang, in vitro, in

human breast cancer cell lines (Qiao and Yang, 2014). To further assess the role of this

nuclear receptor in regulating P-gp and Bcrp at the BTB, TM4 Sertoli cells were exposed

to the PXR antagonist ketoconazole, an antifungal drug and well-established inhibitor of

the phase I metabolic enzyme CYP3A4 (Thummel and Wilkinson, 1998). This treatment

resulted in the downregulation of these transporters in the presence of dexamethasone

or PCN (Fig. 3). Downregulation of P-gp and Bcrp by ketoconazole could possibly involve

other nuclear receptor pathways since this drug has been demonstrated as a pan-

antagonist that could inhibit the ligand-induced activation of the liver X receptor and the

farnesoid X receptor (Huang et al., 2007). However, since inhibition of these nuclear

receptors, including PXR, are dependent on activation by their respective ligands, the

inhibitory effect of ketoconazole demonstrated in our experiments was most likely


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mediated through this nuclear receptor as we only observed downregulation in the

presence of PXR ligands, without any downregulatory effects when cells were exposed

to ketoconazole only. Activation of CAR by the established ligand TCPOBOP, a

phenobarbital-like compound, in mice (Tzameli et al., 2000), also demonstrated that this

nuclear receptor can upregulate P-gp and Mrp4 in TM4 Sertoli cells. These results

complement previous studies by our group which showed that ligand-activated CAR was

able to induce P-gp expression at the human BBB (Chan et al., 2011).

To examine whether PXR or CAR could directly regulate the expression of ABC

transporters at the BTB, we performed studies using siRNA to knockdown the nuclear

receptors and measured the corresponding effects on the transporters (Fig. 5). We

observed that both PXR or CAR gene silencing resulted in significant downregulation of

the ABC transporters, suggesting that these nuclear receptors are directly involved in

regulating the transporters at the BTB. We also demonstrated that the upregulation of the

transporters in the presence of dexamethasone (also a GR ligand) was mediated directly

through PXR (supplemental fig. S-2), and in agreement with the work of Pascussi et al.

who proposed that dexamethasone concentrations above 10 µM directly activated PXR

and induced the regulation of target genes such as CYP3A4 in human hepatocytes

(Pascussi et al., 2000).

Our group previously demonstrated in TM4 Sertoli cells, the transport of ARVs

including atazanavir and raltegravir by ABC transporters (Robillard et al., 2012; Hoque et

al., 2015), however these studies have not been performed in a more physiologically

relevant system such as freshly isolated seminiferous tubules. We investigated the role

of the nuclear receptors PXR and CAR in regulating the expression and function of P-gp


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ex vivo in mouse seminiferous tubules. As we have observed in vitro at the BTB, exposure

to PXR and CAR ligands, including darunavir and efavirenz, part of the current preferred

and alternative ARV regimens (DHHS Panel on Antiretroviral Guidelines for Adults and

Adolescents, 2016), also induced the expression of Pgp at the mRNA and protein level

at the BTB ex vivo. It is noteworthy that the concentrations of ARVs used in our ex vivo

experiments were higher than the clinically reported concentrations achieved in plasma.

Previous work by our group demonstrated the activation of PXR or CAR, as well as

upregulation in the expression and function of P-gp, using clinical plasma concentrations

of darunavir and efavirenz, in vitro at the BBB (Chan, Patel, et al., 2013). We anticipate

that plasma concentrations of these drugs could be effective at the human BTB but it was

not feasible to perform these experiments in human samples. Based on experiments

conducted in mouse seminiferous tubules ex vivo, lower concentrations of the drugs were

less effective in inducing P-gp expression, suggesting species differences.

Since we have determined that activation of PXR and CAR by PCN, TCPOBOP,

darunavir or efavirenz resulted in increased P-gp expression, we explored the effects of

these ligands on the function of Pgp 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 human or rodent (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. 7-B). 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


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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 performed in mice

and may not fully predict activation of the nuclear receptors in human, however our group

previously demonstrated that efavirenz and darunavir activated human PXR or CAR

(Chan, Patel, et al., 2013).

In summary, we reveal for the first time that the testis is capable of altering drug

pharmacokinetic properties through nuclear-receptor mediated pathways. We report

novel findings demonstrating upregulation of P-gp, Bcrp and Mrp4 expression at the BTB

in vitro in mice, and observed upregulation of P-gp expression and function in mouse

seminiferous tubules following exposure to nuclear receptor ligands including ARVs. Our

data indicate the high complexities of ARV pharmacokinetics and the great potential for

drug-drug interactions during therapy. In addition to ARVs, nuclear receptors are

modulated by other drugs used to treat HIV co-infections. For example, patients co-

infected with tuberculosis may receive concurrent treatment including the rifamycin class

of drugs which are potent activators of PXR (Dooley et al., 2008). Administration of these

drugs could activate the nuclear receptor-mediated regulation of drug transporters and

metabolic enzymes, potentially leading to reduced drug efficacy when multidrug regimens

are taken chronically by patients. Further studies are needed, especially in human testis,

to fully elucidate ARV interactions with drug transporters and nuclear receptors, and their

potential contribution to restrict drug accumulation in this tissue and formation of a HIV

sanctuary.


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Acknowledgements

We would like to thank Dr. Pierre Brassard and Dr. Maud Bélanger from the Metropolitan

Centre of Plastic Surgery in Montréal, Canada, as well as the individuals from the Orchid

study group who donated their tissue. We also thank Dr. C. Yan Cheng and his research

group (Population Council, Rockefeller University), as well as Dr. Nathan Cherrington and

Dr. David Klein (University of Arizona) for providing guidance with the methods on

isolating and performing transport assays with seminiferous tubules ex vivo.

Authorship Contributions

Participated in research design: Whyte-Allman and Bendayan.

Conducted experiments: Whyte-Allman and Hoque.

Contributed reagents or analytic tools: Routy, Jenabian and Bendayan.

Performed data analysis: Whyte-Allman, Hoque, and Bendayan.

Wrote or contributed to the writing of the manuscript: Whyte-Allman and Bendayan.


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Footnotes

This work was supported by the Leslie Dan Faculty of Pharmacy Internal Grant [grant

923465] to R.B.; the Canadian Institutes of Health Research Catalyst Operating Grant

[grant 296635]; and the Canadian HIV Cure Enterprise Team Grant [grant HIG-133050]

from the Canadian Institutes of Health Research in partnership with the Canadian

Foundation for AIDS Research and the International AIDS Society to J.-P.R., M.-A.J and

R.B. Dr. Reina Bendayan is a Career Scientist of the Ontario HIV Treatment Network.

Ms. Sana-Kay Whyte-Allman is a recipient of the Connaught International Scholarship for

Doctoral Students. Dr. MA Jenabian is the holder of the Canada Research Chair Tier 2 in

Immuno-Virology.


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Figure Legends

Fig. 1. PXR and CAR protein expression in human testicular tissue and TM4 Sertoli cells.

PXR and CAR proteins were examined in testicular tissue obtained from three healthy

human subjects (S1-S3), as well as in different passages of TM4 Sertoli cells (P1-P3).

Whole tissue or cell lysates were resolved on a 10% SDS-PAGE gel and transferred to a

PVDF membrane. HepG2 cells served as positive control. The goat polyclonal A-20

antibody was used to detect PXR, while CAR was detected using rabbit polyclonal M-127

antibody. Actin was used as an internal control and detected using anti-beta Actin mouse

monoclonal antibody.

Fig. 2. PXR-mediated upregulation of Abcb1b (A) and Abcg2 (B) mRNA expression as

well as Pgp (C,E) and Bcrp (D,F) protein expression in TM4 Sertoli cells. Cells were

exposed to PXR ligands dexamethasone or PCN at various concentrations for 24 h.

mRNA expression was measured by qPCR. All ligand-treated and vehicle (DMSO-

treated) groups were performed in triplicates in each experiment. CT values of each gene

of interest were normalized with the housekeeping gene Gapdh mRNA and compared to

the vehicle control using the comparative CT method (∆∆CT). Results are expressed as

mean fold change ± S.E.M of three separate experiments. (C-F) Induction of P-gp and

BCRP protein expression by PXR in ligand-treated cells compared to vehicle control for

72 h. The MDA-MDR1 and MCF7-MX100 cell systems were used as positive controls for

P-gp and Bcrp expression respectively. The relative protein expression was determined

by densitometry analysis. Representative immunoblots (top panels) are shown with their

corresponding densitometry analyses (bottom panels). For each loaded sample, the

protein of interest was first normalised against β-actin. Results are then expressed as the


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percent change of ligand-treated samples compared to vehicle-treated control samples

and reported as mean ± 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.

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-B) or PCN (C-D) for 24 h. mRNA expression was measured by qPCR.

All treatment and vehicle groups were performed in triplicates in each experiment. CT

values of each gene of interest were normalized with the housekeeping gene Gapdh

mRNA and compared to the vehicle (DMSO-treated) control. Results are expressed as

mean fold change ± 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 ANOVA with Bonferroni’s correction for multiple comparisons.

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 h. mRNA expression was

measured by qPCR. All ligand treatment and vehicle (DMSO-treated) groups were

performed in triplicates in each experiment. CT values of each gene of interest were

normalized with the housekeeping gene Gapdh mRNA and compared to the vehicle

control using the comparative CT method (∆∆CT). Results are expressed as mean fold

change ± 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 h. The

MDA-MDR1 and HEK-MRP4 over-expressing cell systems were used as positive controls


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for P-gp and Mrp4 expression respectively. The relative protein expression was

determined by densitometry analysis. Representative immunoblots (top panels) are

shown with their corresponding densitometry analyses (bottom panels). For each loaded

sample, the protein of interest was first normalised against β-actin. Results are then

expressed as the percent change of ligand-treated samples compared to vehicle-treated

control samples and reported as mean ± S.E.M of three separate experiments. *, p <

0.05; **, p < 0.01; ***, p < 0.001; statistically significant differences between treatment

and vehicle as determined by Student’s t test.

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) was

used to determine relative protein expression. For each loaded sample, the protein of

interest was first normalised against β-actin. Results are then expressed as the percent

change of samples exposed to PXR or CAR targeting siRNA compared to control siRNA

and reported as mean ± 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 Student’s t test.

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 h. (A) mRNA expression was measured by qPCR. All treatment and vehicle (DMSO-

treated) control groups were performed in triplicates in each experiment. CT values of


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each gene of interest were normalized with the housekeeping gene Gapdh mRNA and

compared to the vehicle control using comparative CT method (∆∆CT). Results are

expressed as mean fold change ± 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 over-expressing cell system served as positive control for P-gp expression. The

relative protein expression was determined by densitometry analysis. Representative

immunoblots (top panel) is shown with the corresponding densitometry analysis (bottom

panel). For each loaded sample, the protein of interest was first normalised against β-

actin. Results are then expressed as the percent change of ligand-treated samples

compared to vehicle-treated control samples and reported as mean ± 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 Student’s

t test.

Fig. 7. P-gp function in mouse seminiferous tubules. (A) Time profile (up to 2 h) of

atazanavir (1 µM) was measured at 35 °C. Atazanavir uptake was calculated as pmol of

intracellular radioactivity normalised to the length of the seminiferous tubules in mm.

Results are expressed as the mean ± S.E.M of three separate experiments (n = 6

animals). (B) Accumulation of atazanavir (2 h) in the absence (control) or presence of P-

gp inhibitors PSC833 and CsA at 35 °C. Results are expressed as mean percentage

control ± 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 Student’s t

test.


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Fig. 8. Atazanavir accumulation in PXR and CAR ligand-treated seminiferous tubules.

Atazanavir (1 µM) accumulation was measured after 2 h treatment with the PXR ligands

PCN or darunavir, the CAR ligand TCPOBOP, or the dual PXR and CAR ligand efavirenz.

For the group without PSC833, results are expressed as the mean percent change in

atazanavir accumulation versus the vehicle control (DMSO-treated, without PSC833) ±

S.E.M obtained from three independent experiments (n = 3 animals per experiment). ***,

p < 0.001; statistically significant differences between groups were determined by One-

way ANOVA with Dunnett’s post hoc t test. For the group with PSC833, results are

expressed as the mean percent change in atazanavir accumulation normalized to the

corresponding treatment without PSC833 ± S.E.M, ****, p< 0.0001 statistically significant

differences between groups was determined by One-way ANOVA with Bonferroni multiple

comparison post hoc test. Each data point in an individual experiment represents triplicate

measurements.


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Xenobiotic Nuclear Receptors PXR and CAR Regulate...

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