P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

Toxicology Letters 232 (2015) 37–45

Contents lists available at ScienceDirect

Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions Christopher S. Mazur a, * ,1, Satori A. Marchitti a,1, Jason Zastre b a U.S. Environmental Protection Agency, National Exposure Research Laboratory, Ecosystems Research Division, 960 College Station Road, Athens, GA 30605, USA b Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA

H I G H L I G H T S







Propiconazole and CYP450 metabolites inhibited P-gp transport in membrane vesicles. Propiconazole acts as an inhibitor of P-gp rather than a substrate. Propiconazole chemosensitized NIH-3T3/MDR1 cells to the P-gp substrate paclitaxel. P-gp inhibition by propiconazole may influence cellular pesticide–drug interactions.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 August 2014 Received in revised form 24 September 2014 Accepted 26 September 2014 Available online 28 September 2014

The human efflux transporter P-glycoprotein (P-gp, MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure assessment because of their use as both pesticides and drugs. Propiconazole is an agricultural pesticide undergoing evaluation by the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program. In this study, the P-gp interaction of propiconazole and its hydroxylated metabolites were evaluated using MDR1expressing membrane vesicles and NIH-3T3/MDR1 cells. Membrane vesicle assays demonstrated propiconazole (IC50,122.9 mM) and its metabolites (IC50s, 350.8 mM, 366.4 mM, and 456.3 mM) inhibited P-gp efflux of a probe substrate, with propiconazole demonstrating the strongest interaction. P-gp mediated transport of propiconazole in MDR1-expressed vesicles was not detected indicating propiconazole interacts with P-gp as an inhibitor rather than a substrate. In NIH-3T3/MDR1 cells, propiconazole (1 and 10 mM) led to decreased cellular resistance (chemosensitization) to paclitaxel, a chemotherapeutic drug and known MDR1 substrate. Collectively, these results have pharmacokinetic and risk assessment implications as P-gp interaction may influence pesticide toxicity and the potential for pesticide–drug interactions. Published by Elsevier Ireland Ltd.

Keywords: P-gp MDR1 Conazoles Propiconazole Chemosensitization ABC transporters

1. Introduction Chemical clearance from the body is complex and involves both biotransformation (metabolism) and cellular excretion of the

Abbreviations: ABC, ATP-binding cassette; ER, endoplasmic reticulum; U.S. FDA, U.S. Food and Drug Administration; NMQ, N-methyl quinidine; P-gp, P-glycoprotein. * Corresponding author. Tel.: +1 706 355 8233; fax: +1 706 355 8302. E-mail addresses: [email protected] (C.S. Mazur), [email protected] (S.A. Marchitti), [email protected] (J. Zastre). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.09.020 0378-4274/ Published by Elsevier Ireland Ltd.

parent chemical and its metabolites via various transportermediated processes (Pang et al., 2009). Transporters are membrane-bound proteins that govern the influx and efflux for a variety of xenobiotics across cellular membranes (Giacomini et al., 2010). Highly expressed in various epithelial and endothelial tissues, such as the intestine, liver, kidney, blood–brain barrier, and placenta, transporters play critical roles in determining intracellular and systemic concentrations of xenobiotics (Chu et al., 2013). P-glycoprotein (P-gp), also known as multidrug resistance protein 1 (MDR1), is a well-characterized member of the ATP Binding Cassette (ABC) superfamily of ATP-dependent efflux

38

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

many triazole-derived pharmaceutical compounds. Considered a potential mouse liver carcinogen, propiconazole demonstrates adverse reproductive and developmental toxicities in experimental animals (Kjeldsen et al., 2013; Nesnow 2013). Propiconazole has also been shown to have endocrine disrupting capabilities in both in vitro mammalian systems and fish and is currently included in the endocrine disruptor screening program (EDSP) of the U.S. Environmental Protection Agency (U.S., EPA) (EPA, 2006; Federal Register, 2009; Kjærstad et al., 2010; Skolness et al., 2013). The metabolism of propiconazole is predominantly catalyzed by CYP3A enzymes to produce three hydroxylated (—OH) metabolites (a-OH, b-OH, and g-OH propiconazole, respectively) (Fig. 1). Thus, a complete exposure assessment should include evaluation of propiconazole and its metabolites (Mazur and Kenneke, 2008). To date, limited information exists regarding the interaction of propiconazole with the efflux transporter P-gp and no studies have evaluated its associated metabolites (Pivcevic and Zaja 2006; Bain and LeBlanc 1996). In this study, we investigate the interaction of propiconazole and its hydroxylated metabolites with P-gp using an in vitro P-gp vesicle assay system and human P-gp (MDR1) transfected NIH-3T3 cells.

transporters that is encoded by the human ABCB1 gene. Known to have broad substrate specificity, P-gp effluxes a wide variety of structurally unrelated xenobiotics with vastly different physicochemical properties (Kiki-Mvouaka et al., 2010). P-gp thus plays an important role in limiting the intestinal absorption for a variety of chemicals into the systemic circulation and contributes to the hepatic biliary excretion of xenobiotics. The similar expression and localization of P-gp and major xenobiotic metabolizing enzymes, such as the cytochromes P450 (CYPs), suggests that these proteins may work synergistically to limit entry of xenobiotics into the body and promote removal (Wacher et al., 1995). The metabolism of P-gp substrates, inhibitors or non-substrates by CYP enzymes may result in metabolites with higher or lower affinity for P-gp. This in turn may impact the systemic exposure of the parent or other coadministered xenobiotics. The inhibition or modulation of P-gp efflux activity can greatly impact the pharmacokinetics and safety of drug therapies (Akhtar et al., 2011; Härtter et al., 2013). In this regard, the U.S. FDA currently provides decision tree criteria to assess the risk of potential drug to drug (mixture) interactions resulting from P-gp modulation (Bentz et al., 2013). While understanding how drugs interact with P-gp is critical to the pharmaceutical industry for evaluating drug absorption, chemotherapeutic resistance, and potential drug–drug interactions, few studies have been conducted to understand the interaction of P-gp with environmental chemicals and their metabolites (Mazur et al., 2012). Conazoles, which include triazole and imidazole compounds, constitute an important class of systemic fungicides designed to inhibit ergosterol synthesis in fungal cell wall formation (Chen et al., 2008). Conazoles provide a unique challenge in exposure assessment as this chemical class is used for both pesticide and drug applications and their metabolites can be biologically active (Kjærstad et al., 2010). Propiconazole (1-[2-(2,4-dichlorophenyl)4-propyl-1,3-dioxolan-2-yl]methyl-1H-1,2,4-triazole) is an agricultural fungicide commonly used on fruits, vegetables, and cereal crops and shares a common 1,2,4-triazole moiety with

2. Materials and methods 2.1. Chemicals and reagents Propiconazole was obtained from the U.S. EPA National Pesticide Standard Repository (Fort Meade, MD). The a-OH (CGA 136,735), b-OH (CGA 118,244), and g-OH (CGA 118,245) hydroxylated propiconazole metabolites were a generous gift from Syngenta (Greensboro, NC). Verapamil and erythromycin were obtained from Sigma (St. Louis, MO). All organic solvents were Optima grade and purchased from Fisher Scientific (Pittsburgh, PA). The specific P-gp inhibitor (PSC833) was purchased from Solvo Biotechnology (Szeged, Hungary).

Cl

Cl O O

N

N

N

CH3

PROPICONAZOLE CYP3A

CYP3A CYP3A

O

OH

N N

CGA 136735 (α-OH)

N

OH CH3

Cl

Cl

O

O

CH3

Cl

Cl

Cl

Cl

O O

N

O

N

N

N

N

N OH

CGA 118244 (β-OH)

CGA 118245 (Y-OH)

Fig. 1. The CYP3A-mediated metabolism of propiconazole to a-, b-, and g-hydroxylated metabolites CGA 136,735, CGA 118,244, and CGA 118,245, respectively.

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

2.2. P-gp membrane vesicle transport assays Human P-gp expressed “inside–out” membrane vesicles were used to screen the ability of propiconazole and its hydroxylated metabolites to inhibit P-gp transport of the probe substrate Nmethyl quinidine (NMQ). In this system, the inverted membrane conformation allows for P-gp transport (efflux) of substrates into the vesicle lumen. After membrane solubilization, effluxed substrate concentration can be determined. Human P-gp vesicles and all chemical reagents were purchased in PREDIVEZ (Cat #

A.

39

MDR1-K-VT) assay kits from Solvo Biotechnology (Szeged, Hungary). All transporter assay kit components and reagents were stored at 80  C until use. The ATP-dependent efflux of NMQ (2 mM final concentration) into human P-gp vesicles was measured using a rapid filtration technique in the absence and presence of ATP following the manufacturer's protocol. Stock solutions of test chemicals were prepared in DMSO with final assay concentrations ranging from 0.2 mM to 1500 mM. Briefly, human P-gp vesicle suspensions (50 mL) were loaded onto 96-well flat bottom tissue culture plates (50 mg protein) followed by the subsequent addition

140

P-gp (MDR1) Transport Activity (%)

120 100 80 60 40 20 0 0.1

1

10

100

1000

Concentration (μ μM)

B.

140

P-gp (MDR1) Transport Activity (%)

120 100 80 60 40 20 0

10

100

1000

Concentration (μ μM) Fig. 2. P-gp (MDR1) mediated transport of the probe substrate NMQ in membrane vesicles in the presence of varying concentrations of (A) propiconazole ð
Þ, verapamil (D), and erythromycin ð&Þ; and (B) propiconazole ð
Þ and its hydroxylated metabolites CGA 118,245 (D), CGA 118,244 (^), and CGA 136,735 ð&Þ. Data are expressed relative to vehicle control (DMSO) treated membranes. Each experiment was performed in duplicate and the data presented represent the mean  SEM of multiple independent experiments.

40

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

of various concentrations of the test chemical (0.75 ml). Plates were preincubated for 15 min at 37  C. The reaction was started by the addition of 25 ml assay buffer (Solvo kit) with and without ATP and allowed to proceed for 3 min at 37  C. The reaction was terminated with 200 ml of ice cold ‘washing mix’ (Solvo kit). The solution was transferred to a glass fiber (Type B) filter plate (Millipore, Billerica, MA) and washed five times with ‘washing mix’ using a Millipore MultiscreenTM rapid filtration vacuum manifold. Vesicles were solubilized in methanol:water (70:30) at room temperature and vacuum collected. Quinidine (25 ng/ml in DMSO) was added to all experimental samples as the internal standard for LC/MS/MS analysis. NMQ concentrations measured in experimental wells lacking ATP were treated as negative background controls and were subtracted from measurements taken from wells with ATP (representing active transport). DMSO served as the vehicle control for P-gp mediated active transport of NMQ. Test chemical interaction with P-gp was measured by dividing NMQ concentrations for all test chemicals by those measured for DMSO to determine % P-gp transport activity. Verapamil and erythromycin, known inhibitors of P-gp, were used as controls (Eberl et al., 2007; Heredi-Szabo et al., 2013). Analyses of P-gp mediated transport of propiconazole (at 1 mM, 10 mM, and 100 mM final concentrations) in membrane vesicles was performed using the same protocol (as with NMQ) with the exception that reaction time varied (3– 30 min). NMQ (with and without ATP) served as a control. All experiments were performed in duplicate; the data presented are the mean  standard error of the mean (SEM) of multiple experiments. 2.3. LC MS/MS analysis Quantitation of NMQ was performed using an Agilent 1200 high-performance liquid chromatograph (HPLC) coupled to a 6420 triple quad mass spectrometer (Agilent, Santa Clara, CA). Injections (2 ml) at a 0.65 ml/min flow rate were made onto a Waters Atlantis C18 column (2.1 mm  100 mm, 5 mm particle diameter; Milford, MA) maintained at 35  C. Gradient elution with 0.2% formic acid in methanol (solvent A) or water (solvent B) was applied under the following conditions: 15% A for 0.1 min, followed by a linear gradient to 45% A at 2.5 min, increasing to 100% A at 2.6 min and held for a 6 min stop time. The column was then allowed to re-equilibrate under the original conditions for a 3 min post time. MS/MS detection was conducted using ESI+ in multiple reaction mode under the following conditions: NMQ quantifying transition ion m/z 339 to 172, with qualifying ion transitions m/z 339–160 and 339–96.2 (collision energies 44 V, 34 V and 40 V, respectively) with the fragmenting voltage set to 165 V. The internal standard quinidine was quantified based on the transition ion m/z 325 to 81 with the fragmenting voltage, collision energy, and cell accelerator set to 140 V, 40 V, and 4 V, respectively. ESI source parameters were applied according to the following: source gas temperature 350  C, gas flow 11 L/min, nebulizer 50 psi, capillary 4500 V. NMQ standard curves (0.5–70 ng/ml) were prepared in methanol:water (70:30) using quinidine as an internal standard. Instrument calibration based on a response factor was conducted for each vesicle transport assay and was verified during analysis by running a check standard every ten samples. A valid Table 1 IC50 values in P-gp (MDR1) vesicle transport assays. Propiconazole CGA 136,735 CGA 118,244 CGA 118,245 Erythromycin Verapamil

122.9 mM 366.4 mM 456.3 mM 350.8 mM 145.1 mM 1.8 mM

sample analysis required less than a 20% degree of uncertainty in the ratio of each qualifying ion to quantifying ion. Analysis of propiconazole was performed by injecting 2 ml at a 0.65 ml/min flow rate onto a Waters Atlantis C18 column (2.1 mm  100 mm, 5 mm particle diameter; Milford, MA) maintained at 35  C. Gradient elution with 0.2% formic acid in methanol (solvent A) or water (solvent B) was applied under the following conditions: 15% A for 0.5 min, followed by a linear gradient to 100% A at 5.0 min. The column was then allowed to re-equilibrate under the original conditions for a 3 min post time. MS/MS detection was conducted using ESI+ mode using multiple reaction mode under the following conditions: propiconazole quantifying transition ion m/z 342 to 158, with qualifying ion transitions m/z 342–69 and 339 with the fragmenting voltage, collision energy, and cell accelerator set to 135 V, 20 V, and 4 V, respectively. ESI source parameters were applied according to the following: source gas temperature 325  C, gas flow 11 l/min, nebulizer 50 psi, capillary 3500 V. Propiconazole standard curves (0.5–70 ng/ml) were prepared in methanol:water (70:30). 2.4. Cell culture The parental NIH-3T3 murine fibroblast cell line and its human MDR1 transfected counterpart, the NIH-3T3-G185 cell line (NIH3T3/MDR1), were kindly provided by Dr. M.M. Gottesman (the National Cancer Institute at the National Institutes of Health, Bethesda, MD, USA) (Cardarelli et al., 1995). The overexpression of P-gp in NIH-3T3/MDR1 cells was verified by Western blot analysis in crude cell lysates probed with the C219 monoclonal antibody (Covance, Dedham, MA), which recognizes all P-gp isoforms (data not shown). Characterization of the multidrug resistance phenotype of NIH-3T3/MDR1 cells to the known P-gp substrates colchicine and paclitaxel was performed, as described (Bruggemann et al., 1992). Cell culturing was performed under standard conditions in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), both from American Type Culture Collection (ATCC, Rockville, MD). The drug resistant, NIH-3T3/MDR1 cell line was maintained in medium supplemented with 60 ng/ml of colchicine (Sigma, St. Louis, MO). All cells were maintained in a humidified atmosphere with 5% CO2 at 37  C. 2.5. Cytotoxicity assays The cytotoxicities of propiconazole (0.01–100 mM) and paclitaxel (0.01–10 mM) to parental NIH-3T3 cells and NIH3T3/MDR1 cells were determined using a colorimetric MTS (3(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) cell viability assay following the manufacturer's guidelines (Promega, Madison, WI). This assay is based on the reduction of the MTS tetrazolium compound by metabolically viable cells to a colored formazan product soluble in tissue culture medium. Absorbance of the formazan product at 490 nm was measured with a BioTek Instruments Synergy HT microplate reader (Winooski, VT) and cell viability was expressed relative to vehicle-treated (DMSO) control cells. Propiconazole was further tested for its potential to decrease the resistance of (chemosensitize) NIH 3T3/MDR1 cells to paclitaxel, a chemotherapeutic agent and known substrate of MDR1 (Oosterhuis et al., 2008). Parental NIH-3T3 and NIH-3T3/ MDR1 cells were exposed to paclitaxel (0.1 mM) in the presence of propiconazole (0.01–100 mM) and cell viability was determined by the MTS assay. The specific P-gp inhibitor PSC833 (10 mM) was used as a positive control. In all experiments, cells were seeded at a density of 3  103 cells/well in 96-well tissue culture treated plates (Corning Costar, Tewksbury, MA) and were

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

41

Fig. 3. P-gp (MDR1) mediated transport (pmol/mg/min) of propiconazole in membrane vesicles in the presence or absence of ATP. The probe substrate (NMQ) with (+ATP) and without (ATP) served as positive and negative controls, respectively. Each experiment was performed in duplicate and the data presented represent the mean  SEM of multiple independent experiments.

exposed to propiconazole and/or paclitaxel (in DMSO) for 48 hours. Final DMSO concentrations did not exceed 1%. All experiments were performed in triplicate; the data presented are the mean  SEM of multiple experiments.

propiconazole (at 1 mM, 10 mM, and 100 mM final concentrations) occurred (Fig. 3). Reactions allowed to proceed for longer times demonstrated similar results (data not shown). 3.2. Chemosensitization of NIH-3T3/MDR1 cells by propiconazole

2.6. Statistical and data analysis MDR1-expressed membrane vesicle IC50 values were determined for each test chemical by curve fitting the data using nonlinear regression with a four parameter logistic fit (SigmaPlot, version 11.0). For analysis of cytotoxicity results in parental NIH3T3 and NIH-3T3/MDR1 cells, groups were compared by Student's t-test (SigmaPlot). P < 0.05 was considered to be significant. 3. Results 3.1. Interaction of propiconazole and metabolites with P-gp in membrane vesicle assays Propiconazole inhibited the transport of the probe substrate NMQ in P-gp expressed vesicles in a dose-response manner with an estimated IC50 value of 122.9 mM (Fig. 2A). Positive controls with the known P-gp modulators, verapamil and erythromycin, displayed IC50 values of 1.8 mM and 145.1 mM (Fig. 2A), respectively, which were consistent with the manufactures quality assurance reports and assay kit documentation (Solvo) (HerediSzabo et al., 2013). These results indicated propiconazole inhibited the transport activity of P-gp more similarly to erythromycin than verapamil. Propiconazole (IC50, 122.9 mM) inhibited P-gp mediated transport of NMQ significantly greater than its three CYP3A hydroxylated metabolites CGA 118,245 (IC50, 350.8 mM), CGA 136,735 (IC50, 366.4 mM), and CGA 118,244 (IC50, 456.3 mM) (Table 1) (Fig. 2B). Control vesicle membranes lacking P-gp expression displayed no appreciable transport of NMQ (data not shown). To further delineate whether propiconazole interacts with P-gp as a competitive substrate or inhibitor, P-gp mediated transport of propiconazole was evaluated in MDR1-expressed membrane vesicles. Results indicate that no appreciable P-gp transport of

The cytotoxicities of paclitaxel (0.01–10 mM) and propiconazole (0–100 mM) were assessed in parental NIH-3T3 and NIH-3T3/ MDR1 cells (Fig. 4A,B). A significant protective effect was observed in NIH-3T3/MDR1 cells to the substrate paclitaxel, as compared to parental NIH-3T3 cells, resulting from enhanced P-gp transport (efflux). At 0.1 mM concentrations, paclitaxel decreased cell viability in parental NIH-3T3 cells to approximately 40%, whereas this concentration of paclitaxel demonstrated no cytotoxic effect in NIH-3T3/MDR1 cells (Fig. 4A). No significant cytotoxicity from propiconazole was observed in either parental NIH-3T3 or NIH3T3/MDR1 cells at concentrations of 10 mM and below (Fig. 4B). To determine if propiconazole decreases the cellular resistance of (chemosensitizes) MDR1 expressing cells to the chemotherapeutic agent paclitaxel, parental NIH-3T3 and NIH-3T3/MDR1 cells were exposed to paclitaxel (0.1 mM) in combination with propiconazole at varying concentrations (0–100 mM) (Fig. 5A and B). Results indicate propiconazole decreased the cellular resistance of NIH-3T3/MDR1 cells to paclitaxel in a concentration dependent manner (Fig. 5A). Cell viability of NIH-3T3/MDR1 cells was significantly decreased by approximately 20% and 35% in response to 1 mM and 10 mM propiconazole, respectively, as compared to paclitaxel alone. In the presence of the specific P-gp inhibitor PSC833 (10 mM), cell viability of NIH-3T3/ MDR1 decreased by approximately 70%. In contrast to NIH-3T3/ MDR1 cells, treatment of parental NIH-3T3 cells with propiconazole or PSC833 did not further reduce cell viability compared to cells treated with paclitaxel alone (Fig. 5B). 4. Discussion Humans are exposed to a variety of environmental pesticides on a daily basis via dietary and environmental exposures (water, soil, air) (Kjeldsen et al., 2013). Transporter proteins are present in

42

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

A.

120 Parental NIH-3T3 NIH-3T3-MDR1

* 100

Cell Viability (%)

* 80

60

40

20

0 0.01

0.1

1

10

Paclitaxel Concentration (μ μM)

B.

120

Parental NIH-3T3 NIH-3T3-MDR1

Cell Viability (%)

100

80

*

60

40

20

0 0.01

0.1

1

10

100

Propiconazole Concentration (μM) Fig. 4. Cell viability of parental NIH-3T3 and NIH-3T3/MDR1 cells in response to varying concentrations of paclitaxel (A) or propiconazole (B). Data are expressed relative to vehicle control (DMSO) treated cells. Each experiment was performed in triplicate and the data presented represent the mean  SEM of multiple experiments. *Statistically significant difference in the cytotoxic dose response between parental NIH-3T3 and NIH-3T3/MDR1 cells (P < 0.05).

virtually all tissues throughout the body and play critical roles in xenobiotic absorption, distribution, and elimination. These processes influence systemic and intracellular xenobiotic concentrations subject to metabolizing and detoxifying enzymes (Hillgren et al., 2013). In particular, P-gp is an ABC transporter known to efflux a wide range of structurally and functionally diverse xenobiotics out of cells (Kiki-Mvouaka et al., 2010). Previous screening studies evaluating the interaction of propiconazole with P-gp have provided conflicting results (Pivcevic and Zaja, 2006; Bain and LeBlanc, 1996). In one study the intracellular uptake of the P-gp substrate calcein acetoxymethyl ester in NIH-3T3/ MDR1 cells was measured and both propiconazole and verapamil exhibited similar P-gp inhibition (Pivcevic and Zaja, 2006). However,

a second study measuring the cellular efflux of the P-gp substrate doxorubicin in MDR1 expressing B16/10 murine melanoma cells reported that propiconazole inhibited P-gp transport significantly less than verapamil (Bain and LeBlanc,1996). Such differences are not unexpected as P-gp and other endogenous transporters may be expressed at varying levels between cell line and passage number (Lumen et al., 2010). In addition, differing physicochemical properties of probe substrates can influence the potency of a xenobiotic to inhibit P-gp transport in a given assay (Zastre et al., 2008). Recently, a comprehensive study across multiple pharmaceutical and research laboratories found high variability among IC50 values determined for known P-gp inhibitors across various in vitro and cell based experimental systems (Bentz et al., 2013). For verapamil, differences

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

model substrate NMQ with an IC50 value similar to erythromycin but substantially less then verapamil. Although several cellular studies have screened propiconazole for P-gp interactions using inhibition assays, none have provided assessment of P-gp substrate transport. To our knowledge, these

in IC50 values varied by greater than a 100-fold across the different assay systems. Utilizing a high throughput P-gp vesicle transport system, which does not have multiple transporter expression, require cell passaging, and is independent of cellular ATP-synthesis, our results demonstrated propiconazole inhibited P-gp transport of a

A.

43

120

Cell Viability (%)

100

NIH-3T3-MDR1

*

80

* 60

40

*

*

20

0

C PA

C PA

C PA

C PA

C PA

C PA

C PA

+

+

+

+

+

+

3 83 C PS

00 i1

μ μM

μ μM

μ μM

μ μM

μ μM

0

i1

.1 i0

1 .0 i0

i1

op Pr

op Pr

op Pr

op Pr

op Pr

e cl hi Ve

B.

120

100

Cell Viability (%)

Parental NIH-3T3 80

60

40

20

0

C PA

C PA

C PA

C PA

C PA

C PA

+

+

+

+

+

+

0

μ μM

μ μM

μ μM

μ μM

μ μM

00 i1

i1

i1

.1 i0

1 .0 i0

3 83 C PS

op Pr

op Pr

op Pr

op Pr

op Pr

e cl hi

C PA

Ve

Fig. 5. Cell viability of NIH-3T3/MDR1 (A) and parental NIH-3T3 (B) cells to paclitaxel (PAC; 0.1 mM) in the presence of propiconazole (Propi; 0.01–100 mM). The specific P-gp (MDR1) inhibitor PSC833 (10 mM) served as a positive control. Data are expressed relative to vehicle control (DMSO) treated cells. Each experiment was performed in triplicate and the data presented represent the mean  SEM of multiple experiments. *Statistically significant difference in the cytotoxic dose response of NIH-3T3/MDR1 cells exposed to paclitaxel and propiconazole (or PSC833) as compared to paclitaxel alone (P < 0.05).

44

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

Intestinal Epithelia

Hepatocytes

Intestinal Lumen Oral Absorption

Systemic Circulation

Portal Blood Supply (Liver)

CYP3A Metabolism

CYP3A Metabolism

P-gp efflux P-gp efflux Apical

Apical

Basolateral

P-gp Inhibitor (Propiconazole) P-gp Inhibitor Metabolite P-gp Substrate (Conazole drug) P-gp (MDRI)

Bile Duct Basolateral

Fig. 6. The interplay of P-gp (MDR1) mediated efflux and intracellular CYP3A metabolism of xenobiotics in intestinal epithelial cells and liver hepatocytes. Inhibition of P-gp transport (efflux) by propiconazole and/or its hydroxylated metabolites may affect the cellular efflux of pharmaceutical P-gp substrates (e.g., conazole drugs) leading to potential pesticide–drug (mixture) interactions.

results are the first evidence that the fungicide propiconazole is not significantly transported by P-gp but rather functions as a P-gp inhibitor. Because P-gp influences the absorption and excretion of xenobiotics (both drugs and pesticides), modulation or inhibition of P-gp can significantly affect the pharmacokinetics and efficacy/ safety of drugs used for therapeutic applications. For instance, inhibition of P-gp activity by erythromycin enhances the intestinal absorption and alters systemic exposure of many P-gp substrates with potentially serious implications (Kiso et al., 2000; Schwarz et al., 2000; Takano et al., 1998). The similar potency observed for P-gp inhibition with propiconazole and erythromycin in this study suggests that potential environmental chemical-drug interactions may also occur with significant ingestion of propiconazole. Our results indicate that at low micromolar concentrations (1 mM and 10 mM), propiconazole decreased cellular resistance in (chemosensitized) NIH-3T3/MDR1 cells to paclitaxel, a known P-gp substrate. At these concentrations, no significant cytotoxic effect was observed by propiconazole alone (in either parental or MDR1 expressing cells). These findings support the conclusion that P-gp inhibition is the specific mechanism by which propiconazole decreases cellular resistance. As a chemical class, conazoles provide a unique challenge in exposure and safety assessment as these chemicals are used for both pesticide and drug applications (Kjaerstad et al., 2010). The aromatase inhibitors anastrozole and letrozole are conazole drugs used in breast cancer treatment, while ketoconazole is a commonly used drug for various fungal infections (Geisler 2011; Ying et al., 2013). Ketoconazole is a well-established P-gp inhibitor and is not appreciably transported by P-gp, similar to our results for propiconazole in the present study (Takano et al., 1998; Wang et al., 2002). Significant drug-drug interactions have been demonstrated for P-gp transport with co-administered ketoconazole and other pharmaceuticals that are P-gp substrates (Floren et al., 1997). In addition, both anastrozole and letrozole have been suggested to be susceptible to P-gp mediated efflux (Miyajima et al., 2013). Thus, the 1,2,4-triazole moiety common to all conazole based pharmaceutical and pesticide compounds may predispose these chemicals towards P-gp interactions. Currently, the U.S. EPA and the U.S. FDA are evaluating the risks posed by concomitant exposure to triazole-containing pharmaceutical and pesticide products (EPA, 2006).

Importantly, xenobiotic metabolites may contribute to pesticide– drug interactions that may result in potentially toxic effects. Because P-gp activity greatly influences the intestinal absorption and hepatic biliary excretion of xenobiotics (both drugs and pesticides) entering the systemic circulation, modulation or inhibition of P-gp by xenobiotics and/or their biologically active metabolites can affect the pharmacokinetics, tissue levels and safety of therapeutic applications (Fig. 6). CYP3A enzymes share many common substrates with P-gp and the interplay of P-gp mediated efflux and CYP3A-mediated metabolism is an important component in the pharmacokinetic assessment of xenobiotics. Little information exists on whether pesticides or their metabolites interact with P-gp, and to date no studies have investigated the interaction of CYP3A hydroxylated metabolites of propiconazole with P-gp. Our results suggest that propiconazole metabolites also inhibit P-gp transport, albeit to a lesser extent than the parent compound. These results contrast with studies of the pharmaceutical compounds tamoxifen and indinavir where hydroxylation of the parent chemical results in enhanced P-gp interaction (Hochman et al., 2000; Teft et al., 2010). Additional studies are needed to assess how the addition of a polar hydroxyl group through CYP-mediated metabolism may influence the intermolecular interaction with P-gp. 5. Conclusion Our results demonstrate that the systemic fungicide propiconazole and its three hydroxylated metabolites inhibit P-gp efflux activity. Greater inhibition of P-gp activity was seen for the parent compound propiconazole, which was also shown to decrease the resistance of NIH-3T3/MDR1 cells to the chemotherapeutic agent paclitaxel, a substrate of MDR1. Analysis of propiconazole transport in membrane vesicles demonstrated that propiconazole acts as a Pgp inhibitor rather than a substrate. Results from membrane vesicle and cell based systems demonstrating P-gp inhibition correlated well, suggesting in vitro membrane vesicle assays may be used to rapidly screen chemicals for transporter interactions in a highthroughput manner. P-gp mediated transport processes are determinants of systemic exposure concentrations and play an important role in human health risk assessment. Further investigation into potential environmental chemical-drug interactions via modulation of P-gp activity is warranted.

C.S. Mazur et al. / Toxicology Letters 232 (2015) 37–45

Conflict of interest The views expressed in this article are those of the author(s) and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. Transparency document The Transparency document associated with this article can be found in the online version. References Akhtar, N., Ahad, A., Khar, R.K., Jaggi, M., Aqil, M., Iqbal, Z., Ahmad, F.J., Talengaonkar, S., 2011. The emerging role of P-glycoprotein inhibitors in drug delivery: a patent review. Expert Opin. Ther. Pat. 21, 561–576. Bain, L.J., LeBlanc, G.A., 1996. Interaction of structurally diverse pesticides with the humanMDR1geneproductP-glycoprotein.Toxicol.Appl.Pharmacol.141,288–298. Bentz, J., O’Connor, M.P., Bednarczyk, D., Coleman, J., Lee, C., Palm, J., et al., 2013. Variability in P-glycoprotein inhibitory potency (IC50) using various in-vitro experimental systems: implications for universal digoxin drug–drug interaction risk assessment decision criteria. Drug. Metab. Disp. 41, 1347–1366. Bruggemann, E.P., Currier, S.J., Gottesman, M.M., Pastan, I., 1992. Characterization of the azidopine and vinblastine binding site of P-glycoprotein. J. Biol. Chem. 267, 21020–21026. Cardarelli, C.O., Aksentijevich, I., Pastan, I., Gottesman, M.M., 1995. Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters. Cancer Res. 55, 1086–1091. Chen, P.J., Moore, T., Nesnow, S., 2008. Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes. Toxicol. In-Vitro. 22, 1476–1483. Chu, X., Korzekwa, K., Elsby, R., Fenner, K., Galetin, A., Lai, Y., et al., 2013. Intracellular drug concentrations and transporters: measurement modeling and implications for the liver. Nature 94, 126–141. U.S. EPA., 2006. Reregistration Eligibility Decision (RED) for Propiconazole. Available at: http://www.epa.gov/oppsrrd1/REDs/propiconazole_red.pdf (accessed 16.06.14.). Eberl, S., Renner, B., Neubert, A., Reisig, M., Bachmakov, I., Konig, J., et al., 2007. Role of P-glycoprotein inhibition for drug interactions. Clin. Pharmacokinet. 46, 1039–1049. Federal Register, 2009. Endocrine Disruptor Screening Program: Tier 1 Screening Order. Available at: http://www.regulations.gov/contentStreamer? contentType=html&disposition=attachment&objectId=0900006480a472c5 (accessed 06.13.14). Floren, L.C., Bekersky, I., Benet, L.Z., Mekki, Q., Dressler, D., lee, J.W., Roberts, J.P., Hebert, M.F., 1997. Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clin. Pharmacol. Ther. 62, 41–49. Geisler, J., 2011. Differences between the non-steroidal aromatase inhibitors anastrozole and letrozole – of clinical importance? Brit. J. Cancer 104, 1059–1066. Giacomini, K.M., Huang, S.M., Tweedi, D.J., Benet, L.Z., Brouwer, K.L., Chu, X., et al., 2010. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215–236. Härtter, S., Sennewald, R., Nehmiz, G., Reilly, P., 2013. Oral bioavailability of dabigatran etexilate (Pradaxa(1)) after co-medication with verapamil in healthy subjects. Br. J. Clin. Pharmacol. 75, 1053–1062. Heredi-Szabo, K., Palm, J.E., Andersson, T.B., Pal, A., Mehn, D., Fekete, Z., et al., 2013. A P-gp vesicular transport inhibition assay – optimization and validation for drug–drug interaction testing. Eur. J. Pharm. Sci. 49, 773–781. Hillgren, K.M., Keppler, D., Zur, A.A., Giacomini, K.M., Stieger, B., Cass, C.E., Zhang, L., 2013. Emerging transporters of clinical importance: an update from the international transporter consortium. Nature 94, 52–63. Hochman, J.H., Chiba, M., Nishime, J., Yamazaki, M., Lin, J.H., 2000. Influence of Pglycoprotein on the transport and metabolism of Indinavir in Caco-2 cells expressing cytochrome P-450 3A4. J. Pharmacol. Exp. Ther. 292, 310–318.

45

Kiki-Mvouaka, S., Menez, C., Borin, C., Lyazrhi, F., Foucaud-Vignault, M., Dupuy, J., et al., 2010. Role of P-glycoprotein in the disposition of macrocyclic lactones: a comparison between ivermectin, eprinomectin, and moxidectin in mice. Drug. Metab. Disp. 38, 573–580. Kiso, S., Cai, S.H., Kitaichi, K., Furui, N., Takagi, K., Takagi, K., Nabeshima, T., Hasegawa, T., 2000. Inhibitory effect of erythromycin on P-glycoproteinmediated biliary excretion of doxorubicin in rats. Anticancer Res. 20, 2827– 2834. Kjærstad, M.B., Taxvig, C., Nellemann, C., Vinggaard, A.M., Anderson, H.R., 2010. Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Repro. Tox. 30, 573–582. Kjeldsen, L.S., Ghisari, M., Bonefeld-Jørgensen, E.C., 2013. Currently used pesticides and their mixtures affect the function of sex hormone receptors and aromatase enzyme activity. Toxicol. Appl. Pharmacol. 272, 453–464. Lumen, A.A., Acharya, P., Polli, J.W., Ayrton, A., Ellens, H., Bentz, J., 2010. If the K1 is defined by the free energy of binding to P-glycoprotein, which kinetic parameters define the IC50 for the Madin–Darby canine kidney II cell line overexpressing human multidrug resistance 1 confluent cell monolayer? Drug Metab. Disp. 38, 260–269. Mazur, C.S., Kenneke, J.F., 2008. Cross-species comparison of conazole fungicide metabolites using rat and rainbow trout (Onchorhynchus mykiss) hepatic microsomes and purified human CYP 3A4. Environ. Sci. Technol. 42, 947–954. Mazur, C.S., Marchitti, S.A., Dimova, M., Kenneke, J.F., Lumen, A., Fisher, J., 2012. Human and rat ABC transporter efflux of bisphenol A and bisphenol A glucuronide: interspecies comparison and implications for pharmacokinetic assessment. Toxicol. Sci. 128, 317–325. Miyajima, M., Kusuhara, H., Takahashi, K., Takashima, T., Hosoya, T., Watanabe, Y., Sugiyama, Y., 2013. Investigation of the effect of active efflux at the blood–brain barrier on the distribution of nonsteroidal aromatase inhibitors in the central nervous system. J. Pharm. Sci. 102, 3309–3319. Nesnow, S., 2013. Integration of toxicological approaches with omic and related technologies to elucidate mechanisms of carcinogenic action: propiconazole, an example. Cancer Lett. 334, 20–27. Oosterhuis, B., Vukman, K., Vági, E., Glavinas, H., Jablonkai, I., Krajcsi, P., 2008. Specific interactions of chlororacetanilide herbicides with human ABC transporter proteins. Toxicology 248, 45–51. Pang, K.S., Maeng, H.J., Fan, J., 2009. Interplay of transporters and enzymes in drug and metabolite processing. Mol. Pharm. 6, 1734–1755. Pivcevic, B., Zaja, R., 2006. Pesticides and their binary combinations as Pglycoprotein inhibitors in NIH 3T3/MDR1 cells. Environ. Toxicol. Pharmacol. 22, 268–276. Schwarz, U.I., Grmatte, T., Krappweis, J., Oertel, R., Kirch, W., 2000. P-glycoprotein inhibitor erythromycin increases oral bioavailability of talinolol in humans. Int. J. Clin. Pharmacol. Ther. 38, 161–167. Skolness, S.Y., Blanksma, C.A., Cavallin, J.E., Churchill, J.J., Durhan, E.J., Jensen, K.M., Johnson, R.D., Kahl, M.D., Makynen, E.A., Villeneuve, D.L., Ankley, G.T., 2013. Propiconazole inhibits steroidogenesis and reproduction in the fathead minnow (Pimephales promelas). Toxicol. Sci. 132, 284–297. Takano, M., Hasegawa, R., Fukuda, T., Yumoto, R., Nagai, J., Murakami, T., 1998. Interaction with P-glycoprotein and transport of erythromycin: midazolam and ketoconazole in Caco-2 cells. Eur. J. Pharmacol. 358, 289–294. Teft, W.A., Mansell, S.E., Kim, R.B., 2010. Endoxifen, the active metabolite of tamoxifen, is a substrate of the efflux transporter P-glycoprotein (multidrug resistance 1). Drug. Metab. Disp. 39, 558–562. Wacher, V.J., Wu, C.Y., Benet, L.Z., 1995. Overlapping substrate specificities and tissue distribution of cytochrome P450 3 A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. Mol. Carcinog. 13, 129– 134. Wang, E.J., Lew, K., Casciano, C.N., Clement, R.P., Johnson, W.W., 2002. Interaction of common azole antifungals with P glycoprotein. Antimicrob. Agents Chemother. 46, 160–165. Ying, Y.J., Lu, N.X., Mei, T.Q., Yan, Z.S., De, Z.Y., 2013. Ketoconazole associated hepatotoxicity: a systemic review and metaanalysis. Biomed. Environ. Sci. 26, 605–610. Zastre, J.A., Jackson, J.K., Wong, W., Burt, H.M., 2008. P-glycoprotein efflux inhibition by amphiphilic diblock copolymers: relationship between copolymer concentration and substrate hydrophobicity. Mol. Pharm. 5, 643–653.