Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines

Toxicology Letters 235 (2015) 189–198

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Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines Abigail M. Dalzell a, * , Pratibha Mistry b , Jayne Wright b , Faith. M. Williams c , Colin. D.A. Brown a a b c

Epithelial Cell Research Group, Institute for Cell and Molecular Biosciences, UK Syngenta Limited, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK Medical Toxicology Centre, Institute of Cellular Medicine Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4AA, UK

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


We tested abamectin, emamectin & ivermectin in SH-SY5Y & N2a neuroblastoma cells.
All compounds inhibited H33342 dye efflux in human & mouse MDR1/ mdr1, Ki  1 mM.
All compounds inhibited GSMF dye efflux in human & mouse MRPs/ mrps, IC50  2.75 mM.
There were no significant affinity differences between human & mouse MDR1/MRPs.
MRP isoforms may offer an alternative route of efflux to MDR1 for avermectins.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 December 2014 Received in revised form 6 April 2015 Accepted 7 April 2015 Available online 9 April 2015

ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24  0.08 and 0.18  0.02 mM (ivermectin); 0.60  0.07 and 0.56  0.02 mM (emamectin) and 0.95  0.08 and 0.77  0.25 mM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin > emamectin  abamectin, P <0.05 by One-Way ANOVA), but importantly, the Ki values for

Keywords: Avermectins Multidrug resistance protein Multidrug resistance-associated protein SH-SY5Y cells N2a cells

Abbreviations: ABC, ATP-binding cassette; ATCC, American Type Culture Collection; CMFDA, 5-chloromethylfluorescein diacetate; CSA, cyclosporin A; GSMF, glutathione methylfluorescein; H33342, 20 -(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,50 -bi-1H-benzimidazole3HCl; MDR1, human multidrug resistance protein 1; Mdr1a/b, mouse isoforms of MDR1; BCRP, human breast cancer resistance protein; Bcrp, mouse isoform of BCRP; MRP, human multidrug resistance-associated protein; Mrp, mouse isoforms of multidrug resistance protein(s); P-gp, permeability associated glycoprotein; SNP, single nucleotide polymorphism. * Corresponding author at: Centre for Chemical Safety and Stewardship, Fera Science Ltd, Sand Hutton, York YO41 1LZ, UK. Tel.: +44 1904 462184\\7984 444114. E-mail addresses: [email protected], [email protected] (A.M. Dalzell). http://dx.doi.org/10.1016/j.toxlet.2015.04.005 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.

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individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58  0.51 and 1.94  0.72 mM (ivermectin); 1.87  0.57 and 2.74  1.01 mM (emamectin) and 2.25  0.01 and 1.68  0.63 mM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05–0.1 mM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of toxicity seen in the Mdr1-deficient animal models CF-1 mice, Mdr1ab (/) double knockout mice and Collie dogs. We also confirm MRP/Mrp-mediated avermectin transport in both N2a and SH-SY5Y cell lines. ã 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Avermectins are a family of macrocyclic lactone compounds widely used as agricultural insecticides and as antihelminthics in man (http://ec.europa.eu/sanco_pesticides/public/?event=activesubstance.selection&language=EN; http://www.merckmanuals. com/vet/pharmacology/anthelmintics/macrocyclic_lactones.html). Chemical structures are given as supplementary data. Although human toxicity is low and there is rapid photolysis, and exposure levels of less than 0.025 ppm on crops (Lasota and Dybas, 1990), understanding the relevance of avermectin toxicities seen in animal is important. In CF-1 mice without functional Mdr1 (Lankas et al., 1997), in some Collie dogs (Mealey et al., 2001) and in Mdr1ab(–/–) double knockout mice (Schinkel et al., 1995), with Mdr1 deficiency there is increased avermectin neurotoxicity; whereas SNPs discovered in humans to date exhibit full MDR1 expression (Macdonald and Gledhill, 2007; Macdonald and Gledhill, 2007). Much evidence has so far focussed on the effects of individual SNPs of MDR1, where transport specificity has been shown to differ for individual substrates (Gow et al., 2008; Schaefer et al., 2006). SH-SY5Y and N2a cells are both widely used in differentiated and undifferentiated states in studies of neurotoxicity (e.g., Li et al., 2010; Bates et al.,1989; Shastry et al., 2001; Kim et al., 2013; Xie et al., 2010; De Girolamo et al., 2001; Flaskos et al., 1999; Sun et al., 2010; Nicolae et al., 2013; Schulze et al., 2015) and are a relevant in-vitro tool for assessment of native avermectin transport kinetics in model neuronal cells. Transporter proteins, particularly at barriers such as the intestine, liver, kidney and the blood–brain barrier, play a key role in determining the disposition of xenobiotics, including avermectins. At the blood brain barrier, members of the ABC family of transporters are key in preventing the accumulation of xenobiotics within the CNS, acting as efficient efflux pumps to remove xenobiotics and so limiting brain exposure to potentially toxic molecules. Most in vivo experimental evidence underpinning a role for ABC transporters in protecting the CNS from toxicity has focussed on the role of Mdr1a (Abcb1a, P-glycoprotein) in rodents. Data from double knockout Mdr1a/1b (/) mice has shown that the absence of a functional Mdr1a is associated with a significant increase in brain to plasma ratios of a wide range of xenobiotics (Schinkel et al., 1995; Uhr et al., 2007; Endres et al., 2006). In normal mice, similar increases in xenobiotic penetration have been observed when Mdr1a/1b function is inhibited chemically by cyclosporin A (Hendrikse and Vaalburg, 2002; Kemper et al., 2003). Although our understanding of the importance of the equivalent human ABCB1 transporter, MDR1, at the human blood–brain barrier is less developed than for mouse, there is compelling in vivo evidence to support a key role for MDR1 in protecting the human CNS from xenobiotic penetration. PET imaging studies in man have demonstrated significant increases in brain to plasma verapamil ratios in the presence of cyclosporin A (an MDR1 inhibitor) (Sasongko et al., 2005; Muzi et al., 2009; Eyal et al., 2010).

In addition to MDR1, recent studies have implicated both BCRP (ABCG2) and a number of MRPs (ABCC 1–6) as important efflux pumps at the blood brain barrier. Evidence of a key role for BCRP in limiting the brain penetration of prototypic substrate drugs imatinib and topotecan has come mainly from experiments using Bcrp (/)/ Mdr1a/1b(–/–) mouse models, or chemical inhibition of BCRP/Bcrp function by the proton pump inhibitor pantoprazole and the MDR1/BCRP inhibitor elacridar. BCRP and MDR1 often work synergistically to clear the CNS of toxicants (Breedveld et al., 2005; de Vries et al., 2007; Zhou et al., 2009; Agarwal et al., 2012). MRP 1, 2, 4 and 5 have been identified at the blood–brain barrier of several species including human and mouse (Nies et al., 2004; Leslie et al., 2005; Löscher and Potschka, 2005; Bronger et al., 2005; Soontornmalai et al., 2006; Dallas et al., 2006; Miller, 2010). Mice deficient in Mrp1 (/) and Mdr1a/1b (/) (triple knockout) show a tenfold greater accumulation of etoposide in CSF compared to Mdr1a/1b (/) double knockout mice (Wijnholds et al., 2000); this indicates that Mrp1 and MDR1/Mdr1a mediate drug efflux from CSF in the mouse, with overlapping substrate specificity. Naturally occurring Mrp2 deficient rats (TR rat model) have been shown to accumulate higher phenytoin concentrations in brain tissue than Mrp2-expressing rats, suggesting that Mrp2 regulates the entry of phenytoin into the brain (Potschka et al., 2003). There is a clear role for the transporter multidrug resistance protein (Mdr1a, Abcb1a, P-gp) in the disposition of avermectins in animal models (Schinkel et al., 1995; Pouliot et al., 1997). In engineered double-knockout strains of mouse (Schinkel et al., 1995; Lankas et al., 1997) absence of a functional Mdr1a (Mdr1a -/-) is associated with an 87-fold increase in ivermectin accumulation within the brain compared to normal (Mdr1+/+) mice. Similarly, CF-1-Abcb1a mice, which exhibit a spontaneous mutation resulting in premature termination of Mdr1 protein translation, exhibit neurotoxicity in response to ivermectin doses of 0.2 mg/kg, whereas normal CF-1 mice expressing functional Mdr1 tolerate 2.5 mg/kg without symptoms (Lankas et al., 1997). In certain breeds of dog, notably Collie dogs (Mealey et al., 2001), a nonfunctional, 4 bp deletion within Mdr1 is associated with a significant increase (16-fold) in peak plasma concentrations and CNS penetration of ivermectin which results in neurological damage and death. In contrast to relatively well defined animal models, the role of MDR1 in the handling of avermectins in man is less well understood, although a wealth of studies have suggested that, unlike in mouse or dog, there is no data demonstrating gross mutations or SNPs of MDR1 that impact its function. In vitro studies using expression systems have demonstrated that several avermectins including ivermectin, abamectin, doramectin, eprinomectin, and selamectin are substrates for MDR1 (Lespine et al., 2007) and that ivermectin is a substrate for multiple MRPs (Lespine et al., 2006). The observation that avermectins may be substrates for more than one ABC transporter is important, in that

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promiscuity of avermectins for more than one transporter may provide a number of alternative routes of exit for avermectins from cells. The interpretation of data across species is complicated by species differences in the specificity and affinity of human MDR1/ MRP for a range of substrates compared with non-human homologues of these transporters (Tang-Wai et al., 1995; Yamazaki et al., 2001; Suzuyama et al., 2007). To better understand the handling of avermectins by mouse and human MDR1/Mdr1a and MRP/Mrp, this study set out to characterise the effects of avermectins, abamectin, emamectin and ivermectin, in two different established cell lines in vitro, by determining the ability of the three avermectins to inhibit either MDR1/Mdr1a mediated H33342 efflux or MRP/Mrp mediated GSMF efflux from human SHSY5Y cells and mouse N2a neuroblastoma cells. 2. Materials and methods

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quantification.net/RIN.pdf). A first strand cDNA synthesis reaction was carried out using RT2 First Strand Kit (SABiosciences). The first strand cDNA synthesis reaction was diluted with dH2O and added to x2 SABiosciences RT2 qPCR Master Mix (SABiosciences). 25 ml of this reaction mix was added to each well of a RT2 ProfilerTM PCR array plate (PAHS-0707Z (human) and PAMM070Z (mouse); SABiosciences) coated with primers for a range of either human or mouse transport proteins. Real time qPCR was carried out using a Roche Lightcycler 480 machine using the following program: 95  C, 10 min for 1 cycle followed by 45 cycles of 95  C for 15 s, 60  C for 1 min. qPCR data was analysed using software on the SABiosciences website (http://pcrdataanalysis.sabiosciences.com/ pcr/arrayanalysis.php). All data are presented as expression of the gene of interest relative to the expression of the GAPDH/Gapdh housekeeping gene detected on the same plates with the same mRNA samples and analysed using the DDCt method. Amplification efficiency was never less than 1.8 (where 2 is the maximum possible efficiency).

2.1. Materials 2.4. Fluorescent dye retention assay Cell culture flasks and well plates were from Corning Life Sciences (Amsterdam, Netherlands). Cell culture reagents (FBS, DMEM growth media, amino acids and antibiotics) were obtained from Sigma, (Poole, UK). Abamectin and emamectin benzoate (both GLP certified) were a gift from Syngenta, Jealott’s Hill International Research Center, Bracknell, UK. Ivermectin was obtained from ChemService, West Chester, PA. Cyclosporin A (CSA) was purchased from Merck UK (Nottingham, UK), CMFDA (5chloromethylfluorescein diacetate) from Invitrogen (Paisley, UK) and Hoechst 33342 (H33342) from Sigma (Poole, UK). RT2 ProfilerTM PCR Arrays for human and mouse drug transporters and all other qPCR reagents were purchased from SABiosciences (Frederick, MD, USA). All other reagents (DMSO solvent, salts for Krebs buffer) were obtained from Sigma (Poole, UK) and of the highest grade commercially available. 2.2. Cell culture systems SH-SY5Y cells were obtained from the ATCC and maintained in high glucose DMEM, supplemented with 2 mM L-glutamine (1%), 10% Foetal Calf Serum (FCS) and antibiotics; penicillin (10,000 units/ml) and streptomycin (10,000 mg/ml). They were sub cultured every 7–9 days. N2a cells obtained from the ATCC were maintained in high-glucose DMEM containing 2 mM L-glutamine (1%), 10% Foetal Calf Serum (FCS), 1 mM pyruvate, 0.1% v/v nonessential amino acids and antibiotics; penicillin (10,000 units/ml) and streptomycin (10,000 mg/ml). Cells were sub cultured every 4– 6 days. Both cell types were incubated at 37  C in 5% CO2/95% air. SH-SY5Y cells were used from passage 29–39 and N2a cells from passage 5–20 (available, characterised stocks in our laboratory). After this period the cells were replaced from frozen stocks. In order to avoid likely fluctuations in MDR1 expression levels in differentiated SH-SY5Y cells (Bates et al., 1989), both cell lines were maintained in culture in their undifferentiated phenotype. 2.3. RNA and qPCR Total RNA was isolated from 3 separate batches of SH-SY5Y or N2a cells grown to 80% confluence in T75 flasks using a SV Total RNA Isolation System (Promega). RNA integrity (purity and intactness) was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies) and all samples had RIN (RNA integrity number) greater than 8, which was intact and full length. Samples with RIN 8 performed well in rt-qPCR test sets and are intact and pure (Fliege and Pfaffl, 2006; Shroeder et al., 2006; http://gene-

The functional activity of MDR1/Mdr1a was determined by the level of intracellular retention of H33342 (1 mM) after blockade of MDR1/Mdr1a-mediated efflux of H33342. The functional activity of MRPs was determined by the retention of GSMF (a fluorescent metabolite of CMFDA, used at 1 mM) after blockade of MRPmediated efflux of GSMF (Forster et al., 2008). For all experiments, cells were seeded onto 96-well plates (Costar) at 20,000 N2a cells/ well and 15,000 SH-SY5Y cells/well to achieve 90 to 95% confluence. Three to four days after seeding, medium was aspirated from the wells and the cells were washed twice with warmed Krebs buffer (mM: 137 NaCl, 5.4 KCl, 0.99 MgSO4 7H2O, 0.34 KH2 PO4, 0.30 NaH2PO4 2H2O, 10 Glucose, 10Hepes, 2.8CaCl2 2H2O made up to pH 7.4 with Tris base, at 37  C). Cells were then incubated with the appropriate inhibitor for 40 min at 37  C. CSA and Ko143 were used to inhibit efflux of H33342 by MDR1/Mdr1a and BCRP/Bcrp respectively and MK571 was used to inhibit GSMF efflux by MRPs. Inhibitor substrates were removed and cells were incubated with the inhibitor plus the appropriate dye for a further 40 min at 37  C. The composition of each test solution is detailed in the appropriate figure. All solutions within each experiment, including the vehicle control, contained the same concentration of DMSO solvent (0.3%). Cells were washed twice with Krebs and fresh Krebs buffer was added to the wells. Cellular fluorescence levels were determined using either a Dynex MFX Microplate Fluorimeter (Dynex Technologies, Chantilly, VA) or a FLUOstar Omega plate reader (BMG Labtech, Aylesbury, UK). Cell monolayer integrity was confirmed using a wellscan read method (5 5 points) over the whole growth surface (Fluostar) or by visual check (Dynex). Results were not accepted if the cell monolayer had washed off.

2.5. Statistical analysis Data are expressed as mean  S.E.M., fitted to a non-linear least-squares regression curve (one-site binding, hyperbola) to generate IC50 values. IC50 values were converted to Ki values according to the Cheng and Prusoff method (Cheng and Prusoff, 1973) using the mean H33342 EC50 values for MDR1 and Mdr1a (Table 1). Statistical differences were tested using Student’s unpaired t-test or a One-Way ANOVA with Dunnett’s post-hoc test as appropriate. Statistical significance was assessed as P < 0.05 or lower. Analysis was performed using Prism 4 software (Graphpad, San Diego, CA).

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3. Results 3.1. Comparison of mRNA expression of ABC transporters in human SHSY5Y and mouse N2a neuroblastoma cells using qPCR The pattern of expression of efflux transporters in human SHSY5Y and mouse N2a cells is summarised in Fig. 1. Both cell lines showed a broadly similar pattern of expression of ABC transporters at the mRNA level., MRP1 in human SH-SY5Y cells and the mouse homologue Mrp1 in N2a cells had the highest expression levels of the ABC-transporters assayed, relative to the expression of the housekeeping genes GAPDH/Gapdh. In human SH-SY5Y cells, the rank order for ABC transporter expression was MRP1 > MRP6 > > MRP4 > MRP2 = MRP5 > MRP3. In mouse, the relative expression pattern was Mrp1 > Mrp6 > Mrp4 > Mrp2 > Mrp3 = Mrp5. mRNA for human MDR1 and mouse Mdr1a were expressed at levels approximately 30% of those for MRP1. There was no evidence for the expression of either human BCRP or mouse Bcrp mRNA in either SH-SY5Y or N2a cells respectively. 3.2. Demonstration of expression of MDR1/Mdr1a but not BRCP/Bcrp in human SH-SY5Y and mouse N2a cells To demonstrate the functional expression of MDR1/Mdr1a and BCRP/Bcrp in SH-SY5Y and N2a cells a H33342 dye retention assay was used, similar to that used previously (Shapiro et al., 1997; Müller et al., 2007). H33342 is a substrate for MDR1/Mdr1 and BCRP/Bcrp (Nefakh, 1998Nefakh, 1998 Goodell et al., 1996; Shapiro et al., 1997; Kim et al., 2013; Scharenberg et al., 2002). The magnitude of the fluorescent signal represents the amount of dye retained within the cells which is a balance between passive dye uptake, binding to DNA and dye efflux. Initial experiments were designed to investigate the kinetics of H33342 dye uptake and efflux in both cell lines. Fig. 2a shows curves for the retention of H33342 within SH-SY5Y or N2a cells. Exposure to a range of concentrations of H33342 (0–15 mM) resulted in a concentrationdependent and saturable increase in intracellular fluorescence with apparent EC50 values of 7.61  2.27 (n = 3) in SH-SY5Y cells and 5.33  1.17 mM (n = 4) in N2a cells. To demonstrate functional expression of MDR1/Mdr1a, the impact of inhibiting MDR1/Mdr1a-mediated H33342 dye efflux was investigated at a H33342 dye concentration of 1 mM in the presence of a range of CSA concentrations (0–10 mM, chosen based on a reported IC50 for substrate probe inhibition for CSA of 2.2 mM with calcein-AM (Rautio et al., 2006), and CSA transport by MDR1 (Tsuji et al., 1993). Initial experiments established greatest sensitivity to competitive inhibition at 1 uM H33342. The results summarised in Fig. 2b show a saturable concentration-dependent increase in intracellular fluorescence as MDR1/Mdr1a-mediated dye efflux is inhibited by CSA. Non-linear regression analysis of the data generated IC50 and Ki values for the competition with MDR1/ Table 1 Comparison of apparent Ki values in mM for inhibition of MDR1/Mdr1a-mediated Hoechst 33342 efflux in human and mouse neuroblastoma cells by avermectins. SH-SY5Y Ki

Abamectin Emamectin Ivermectin

N2a Ki Mean

SEM

N

Mean

SEM

N

P value

0.95 0.60* 0.24**

0.08 0.07 0.08

4 4 4

0.77 0.56* 0.18**

0.25 0.02 0.02

4 4 4

P > 0.05 P > 0.05 P > 0.05

Student’s unpaired t-test was performed for all data between cell lines; for all comparisons P > 0.05. * P < 0.05 compared to Abamectin in the same cell line by One-way ANOVA with Dunnett’s post-hoc test. ** P < 0.01 compared to Abamectin in the same cell line by One-way ANOVA with Dunnett’s post-hoc test.

Fig. 1. qPCR analysis of ABC transporter gene expression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. Relative transporter gene expression levels of ABC efflux transporters in cDNA isolated from human SH-SY5Y or mouse N2a cell lines. Data were analysed using online software http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php. Results shown are the mean  S.E.M. of relative gene expression from 3 separate mRNA isolations using either a human or a mouse drug transporter qPCR array RT2 ProfilerTM PCR array plate (PAHS-0707Z (human) and PAMM070Z (mouse); SABiosciences) All data are presented as expression of gene of interest relative to expression of GAPDH/Gapdh housekeeping gene (DDCt) included on the same plate. Amplification efficiency was never less than 1.8 out of 2.

Mdr1a-mediated H33342 dye efflux were 1.66  0.33 mM, n = 5 (Ki: 1.42  0.29 mM) in SH-SY5Y cells and 0.44  0.13 mM, n = 4 (Ki: 0.37  0.10 mM) in N2a cells. The affinity of CSA competition with H33342 dye efflux for human MDR1 was significantly higher than for mouse Mdr1a (P < 0.05). To investigate functional expression of BCRP/Bcrp, the impact of inhibiting BCRP/Bcrp-mediated H33342 dye efflux was assayed at a H33342 dye concentration of 1 mM in the presence of a range of Ko143 concentrations (0–5 mM, Fig. 3a). The reported EC90 for reversal of mitoxantrone efflux by BCRP was 25 nM Ko143 (Allen et al., 2002), indicating high specificity. In contrast to the competition for MDR1/Mdr1a by cyclosporin A, no similar concentration dependent increase in intracellular fluorescence was found with Ko143. Only at concentrations of 2.5 mM and 5 mM Ko143 (200–500 fold greater than reportedIC50 of 10 nM for BCRP (Weiss et al., 2007) was there a small but significant increase in intracellular fluorescence, likely to be due to nonspecific effects at high concentrations. The use of the specific BCRP/Bcrp inhibitor Ko-143 confirmed the functional absence of human BCRP and mouse Bcrp, showing that effects on H33342 dye retention were MDR1/mdr1-dependent.

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Fig. 2. Hoechst 33342 dye retention in SH-SY5Y and N2a cells with and without Cyclosporin A. The retention of H33432 in cells exposed to a range of external H33342 concentrations (0–15 mM) in SH-SY5Y and N2a neuroblastoma cells (Fig. 2a) and the impact of cyclosporin A (0–10mM) upon H33342 (1 mM) retention (Fig. 2b) were measured. Data are expressed as mean  S.E.M. n = 6 wells per concentration from a single experiment representative of 3 (SHSY-5Y) or 4 (N2a) experiments, curves were fitted using nonlinear least squares regression analysis. Curve fitting of data from 4 experiments gave estimated EC50 values of 7.61  2.27 (n = 3) and 5.33  1.17 mM (n = 4) for the retention of H33342 dye in SH-SY5Y cells and N2a mouse neuroblastoma cells respectively (P > 0.05 with Student’s t-test). Curve fitting of data gave estimated mean IC50 values for competition by cyclosporin A with MDR1/Mdr1a-mediated H33342 dye efflux of 1.66  0.33 mM, n = 5 (Ki: 1.42  0.29 mM) in SH-SY5Y cells and 0.44  0.13 mM, n = 4 (Ki: 0.37  0.10 mM) in N2a cells respectively (P < 0.05 with Student’s t-test).

3.3. Demonstration of the expression of MRP/Mrp isoforms in human SH-SY5Y and mouse N2a cells To determine whether MRP/Mrp isoforms were expressed in either SH-SY5Y or N2a neuroblastoma cells a dye retention assay using CMFDA was employed (Fig. 3b). CMFDA is a non-fluorescent lipophilic compound which is cleaved by intracellular esterases then modified to generate a hydrophilic membrane impermeant fluorescent product, GSMF, within the cells. GSMF is a specific substrate for MRP/Mrp isoforms (Forster et al., 2008; Lebedeva et al., 2011 Lebedeva et al., 2011). SH-SY5Y or N2a cells were exposed to a range of concentrations of CMFDA (0–1.5 mM) and intracellular fluorescence was measured in the absence and presence of MK571 (10 mM), a specific inhibitor of MRP/Mrp isoforms (Luders et al., 2009). In contrast to the saturable dependence of intracellular fluorescence upon the concentration of H33342, exposure of cells to CMFDA resulted in a linear increase in intracellular GSMF fluorescence in response to increasing external CMFDA concentration over the range 0–1.5 mM. External CMFDA concentrations above 1.5 mM were associated with the onset of cell toxicity. Linear regression analysis of the data gave a slope of the relationship between external CMFDA concentration and intracellular fluorescence of 29,970  685.3 and 6803  236.4 Fluorescence Units/mM for SH-SY5Y and N2a (Fig. 3b) respectively. The presence of 10 mM MK571 resulted in a significant increase in

the slope of the relationship between external CMFDA and intracellular fluorescence in both SH-SY5Y (106,000  1670 versus 29,970  685.3 Fluorescence Units/mM, n = 4, P < 0.001) and N2a cells (37,050  1475 versus 6803  236.4 Fluorescence Units/mM, n = 4 P < 0.001), consistent with MK571 competition with MRP/Mrp-mediated GSMF dye efflux. 3.4. Effect of avermectins on H33324 dye retention in SH-SY5Y and N2a cells To investigate the interactions of selected avermectins with MDR1/Mdr1a, SH-SY5Y and N2a cells were loaded with H33342 (1 mM) in the presence and absence of abamectin, emamectin or ivermectin (all at 5 mM, Fig. 4). Cytotoxicity was minimal at 5 mM in preliminary experiments. The results summarised in Fig. 4a show a significant increase in intracellular H3342 fluorescence in the presence of each avermectin in either SH-SY5Y cells or N2a cells. The increase in fluorescence was of the same magnitude as found after exposure of cells to 5 mM cyclosporin A. Fig. 5a shows concentration–dependence curves for the ability of ivermectin (0– 7 mM) to increase intracellular H33342 dye retention in SH-SY5Y and N2a cells. Non-linear regression analysis generated estimated IC50 and Ki values for the competition for MDR1/Mdr1a-mediated H33342 dye efflux by ivermectin of 0.27  0.08 mM (Ki: 0.24  0.08 mM) in SH-SY5Y cells and 0.21  0.03 mM (Ki:

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Fig. 3. Dose–response curves for the ability of Ko143 and MK-571 to inhibit BCRP/Bcrp or MRP/Mrp-mediated dye efflux in SHSY-5Y and N2a cells. Fig. 3a. The impact of a range of inhibitor concentrations upon the intracellular retention of H33342 (1 mM) or the MRP-specific GSMF was examined in SH-SY5Y and N2a cells. Ko143 (0–2.5 mM) did not inhibit BCRP or Bcrp. Extracellular CMFDA was at 0–1.5 mM. As positive controls competition by cyclosporin A (5 mM) for MDR1/Mdr1-mediated H33342 efflux or MK-571 (10 mM), for MRP/Mrp-mediated GSMF efflux was assessed. Data in Fig. 3a are expressed as the mean  S.E.M. n = 6 wells per concentration from a single experiment representative of 4 independent experiments, analysed by One-Way ANOVA and Dunnett’s post-hoc test; *P < 0.05; **P < 0.01 versus control. Fig. 3b: Data are expressed as the mean  S.E.M. from 4 independent experiments. Linear regression analysis fitted a line with a slope of 29,970  685.3 Fluorescence Units/mM in the absence of MK571 and 106,000  1670 Fluorescence Units/mM in the presence of MK571 (P < 0.001 by Student’s t-test) for SH-SY5Y cells and a slope of 6803  236.4 Fluorescence Units/mM in the absence of MK571 and 37,050  1475 Fluorescence Units/mM (P < 0.001 by Student’s t-test) in the presence of MK571 in N2a cells.

0.18  0.02 mM) in N2a cells. Similar concentration curves were produced for abamectin and emamectin and the estimated Ki values are summarised in Table 1. In both cell lines, a rank order of affinity of ivermectin > emamectin > abamectin was found. Both ivermectin (P < 0.01) and emamectin (P < 0.05) had a significantly higher affinity to inhibit MDR1/Mdr1a-mediated dye efflux than abamectin. Importantly, the rank order of affinities to inhibit MDR1/Mdr1a-mediated dye efflux were identical for human MDR1 and mouse Mdr1a and there was no significant difference in Ki values for individual avermectins for either human or mouse isoforms of MDR1. 3.5. Effect of avermectins on GSMF dye retention in SH-SY5Y and N2a cells SH-SY5Y and N2a cells were loaded with CMFDA (1 mM) in the presence and absence of avermectins. The CMFDA intracellular metabolite GSMF is a substrate for MRP transporters (Förster et al., 2008; Lebedeva et al., 2011). Exposure to either abamectin, emamectin or ivermectin (all at 5 mM, Fig. 4b) resulted in significant increases in intracellular GSMF fluorescence in both SH-SY5Y and N2a cells, of a similar magnitude as achieved with

MK571 (10 mM), consistent with the competition between avermectins and GSMF for MRP/Mrp – mediated dye efflux. Fig. 5b summarises the concentration dependence of intracellular fluorescence upon ivermectin concentration at 1 mM external CMFDA. Non-linear regression analysis of the data gave estimated IC50 values for the competition for MRP/Mrp-mediated GSMF dye efflux by ivermectin of 1.58  0.51 mM, n = 4 in SH-SY5Y cells and 1.94  0.72 mM, n = 4 in N2a cells respectively. IC50 values for the three avermectins are summarised in Table 2. In contrast to the differences in IC50 values between the three avermectins for competition with MDR1/Mdr1a, there were no significant species differences in the affinities of abamectin, emamectin and ivermectin for either human MRP isoforms in human SH-SY5Y cells or mouse Mrp isoforms in N2a cells. 4. Discussion To understand the possible species differences in the handling of avermectins by ABC transporters, the effects of abamectin, emamectin and ivermectin upon H33342 dye efflux mediated by ABCB1/Abbc1 (MDR1/Mdr1) and GSMF dye efflux mediated by members of the ABCC/Abcc (MRP/Mrp) family of multidrug

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Fig. 4. Ability of avermectins to inhibit MDR1/Mdr1a-mediated efflux of H33342 in SHSY-5Y and N2a cells. The impact of abamectin, emamectin, ivermectin or inhibitors (all 5 mM) upon (Fig. 4a) the intracellular retention of H33342 (1 mM); Cyclosporin A 5 mM or (Fig. 4b) GSMF retention at 1 mM extracellular CMFDA; MK-571 10 mM in SH-SY5Y and N2a cells. Data are expressed as the mean  S.E.M of 4 independent experiments, each n = 6 per concentration analysed by One-Way ANOVA with Dunnett’s post-test; **P < 0.05; *P < 0.05.

resistance proteins were assessed in human SH-SY5Y and mouse N2a neuroblastoma cell lines. At the quantitative mRNA level, both human SH-SY5Y and mouse N2a neuroblastoma cell lines were shown to express MDR1/Mdr1a and MRP1-6/Mrp1-6 at roughly comparable levels. mRNA for BCRP/Bcrp (ABCG2/Abcg2) was absent from both cell lines. Recently, it has been reported that ivermectin is a substrate for human BCRP (Jani et al., 2011) and that both ivermectin and selamectin were inhibitors of BCRP-mediated mitoxantrone accumulation in MDCKII cells transfected with either human, mouse or bovine homologues of BCRP (Merino et al., 2009). In our hands, both SH-SY5Y and N2a cells showed no evidence of native, endogenous BCRP/Bcrp expression either at the mRNA or functional levels, so the interaction of avermectin, emamectin or ivermectin with BCRP/Bcrp could not be assessed. Both cell lines exhibited CSA-sensitive retention of the fluorescent dye H33342, consistent with the expression of a functional MDR1 in SH-SY5Y and Mdr1a in N2a cells respectively. Importantly, we could demonstrate a species dependent difference in the Ki value for CSA competition with MDR1-mediated dye efflux compared to Mdr1a-mediated dye efflux. A similar 2–3 fold higher affinity for quinidine competition for cyclosporin A efflux in mouse Mdr1a versus human MDR1 was reported in cells transfected with either human or mouse homologues of MDR1 (Suzuyama et al., 2007). Similar species differences in handling of a range of drug molecules (Yamazaki et al., 2001) including antiepileptics (Baltes et al., 2007) by human and mouse homologues of MDR1 have also been reported. The apparent substrate affinity differences for CSA

between human MDR1 and mouse Mdr1 isoforms is similar to that found by others (Suzuyama et al., 2007), but not for the avermectins tested, validates this kinetic assay, showing that it is capable of detecting affinity differences in these model cell lines. There were differences in avermectin affinities within cell lines, with ivermectin having the highest affinity for MDR1 and mdr1a. The rank order of affinities for human MDR1 was ivermectin (Ki 0.24  0.08 mM) > emamectin (Ki 0.60  0.07 mM) > abamectin (Ki 0.95  0.08 mM). For mouse Mdr1a the rank order of affinities of avermectins to inhibit dye efflux was; ivermectin (Ki 0.18  0.02 mM) > emamectin (Ki 0.56  0.02 mM)  abamectin (Ki 0.77  0.25 mM). The IC50 values reported for ivermectin and abamectin inhibition of human MDR1 and mouse Mdr1a transfected into LLC-PK1 cells (Lespine et al., 2007) are very similar to those we report for the native, endogenous expression of these transporters in either human SH-SY5Y or mouse N2a neuroblastoma. Initial evidence that ivermectin was a substrate for MDR1 came from in vivo studies in which Mdr1a deficient Mdr1a(/) mice exposed to ivermectin were found to have an 87% increase in CNS ivermectin level, which was associated with significantly increased toxicity compared with normal Mdr1a(+/+) litter mates (Schinkel et al., 1994; Lankas et al., 1997; Kwei et al., 1999). In vitro studies employing LLC-PK1 cells transfected with either Mdr1a or MDR1 provided evidence for MDR1 and Mdr1a-mediated transport of ivermectin and cyclosporin A (Schinkel et al., 1995). Similarly, Pouliot and co-workers showed that the steady-state

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Fig. 5. Dose-response curves for the ability of ivermectin to inhibit transporter-mediated dye efflux in SH-SY5Y and N2a cells. The impact of a range of concentrations of ivermectin (0–7 uM) upon the intracellular retention of H33342 (1 mM) (Fig. 5a or b) ivermectin (0–4 mM) for GSMF (1 mM CMFDA) was examined in SH-SY5Y and N2a cells, Curves were fitted using nonlinear least squares regression analysis. Data in Fig. 5a are expressed as the mean  S.E.M. n = 6 wells per concentration from a single experiment representative of either 4 (SHSY-5Y) or 6 (N2a) independent experiments. Estimated mean IC50 values for competition with MDR1/ Mdr1a-mediated H33342 dye efflux by ivermectin of 0.27  0.08 mM, n = 4 (Ki: 0.24  0.08 mM) in SH-SY5Y cells and 0.21  0.03 mM, n = 6 (Ki: 0.18  0.02 mM) in N2a cells respectively. For GSMF (Fig. 5b), data are expressed as the mean  S.E.M. n = 6 wells per concentration from a single experiment representative of 4 independent experiments. Estimated mean IC50 values for the competition with MRP/Mrp-mediated GSMF dye efflux by ivermectin of 1.58  0.51 mM, n = 4 in SH-SY5Y cells and 1.94  0.72 mM, n = 4 in N2a cells respectively.

accumulation of [3H]-ivermectin was lower in drug-resistant – than drug-sensitive human lymphoma cells, an observation that replicated the effects of the known MDR1/substrate [3H]-vinblastine in these cells. They demonstrated that the efflux of both drugs was ATP-dependent, and that the efflux of doxorubicin and vinblastine was reversed in the presence of ivermectin or cyclosporin A, indicating competitive efflux, with similar IC50: 13  0.9 mM (CSA); 3.4  0.6 mM (ivermectin) for vinblastine, and Table 2 IC50 values in mM for interaction of the avermectin insecticides abamectin, emamectin and ivermectin with human MRP transporters and mouse mrp transporters in the CMFDA assay. SH-SY5Y IC50

Abamectin Emamectin Ivermectin

N2a IC50 Mean

SEM

N

Mean

SEM

N

P value

2.25 1.87 1.58

0.01 0.57 0.51

6 6 4

1.68 2.74 1.94

0.63 1.01 0.72

4 4 4

0.32 0.42 0.69

One-way ANOVA comparison to Abamectin data was performed in each cell line with Dunnett’s post-hoc test; for all comparisons P > 0.05. Student’s unpaired t-test was performed for all data between cell lines; for all comparisons P > 0.05.

35  1.5 (CSA); 24  2.0 mM (ivermectin) for doxorubicin (Pouliot et al., 1997). These observations were extended when Lespine et al., (2007) showed that ivermectin, abamectin, doramectin, eprinomectin, and selamectin were potent inhibitors of rhodamine123 dye efflux from porcine kidney epithelial cells transfected with either mouse Mdr1a or human MDR1. Equally, an increase in basolateral-to-apical flux of [3H]-ivermectin and [3H]-selamectin was found in monolayers of MDCKII-MDR1 cells compared to wildtype MDCKII; this was significantly reduced by the bilateral addition of the MDR1 inhibitor verapamil (100 mM), (Brayden and Griffin, 2008). The data presented in this paper show abamectin, emamectin and ivermectin competition for MDR1 and Mdr1amediated H33342 dye efflux comparable to CSA in both SH-SY5Y and N2a cell lines respectively, consistent with these avermectins being potential substrates for MDR1/Mdr1 in the CNS/blood brain barrier of both species. In contrast to the extensive data supporting the interactions of avermectins with MDR1/Mdr1a, there have been few studies of the interactions of avermectins with other ABC transport proteins. In addition to MDR1/Mdr1a activity, both cell lines exhibited an MK571 sensitive retention of GSMF, consistent with the functional

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expression of MRP/Mrp although in the absence of isoform-specific inhibitors of MRPs it was not possible to distinguish between the relative importance of individual isoforms in the transport of avermectins. Importantly, the apparent IC50 values for the 3 avermectins were not significantly different between human MRPs and mouse Mrps (P > 0.05). Nor were there any significant differences in relative affinities of individual avermectins for human MRP or mouse Mrp transporters within the cell lines (P > 0.05). There are currently conflicting data as to whether MRP isoforms play a role in avermectin disposition. In one study, it was shown that ivermectin and selamectin were substrates for MDR1 but that inhibitors of MRPs had no effect upon the flux of ivermectin and selamectin in either mock-transfected MDCKII cells or in MDCKII-MRP1 or MDCK-MRP2 transfected cells (Brayden and Griffin, 2008). Excretion of ivermectin from functionally intact porcine brain endothelial capillaries was mediated by Mdr1 but not Mrp (Nobmann et al., 2001). However, in vitro data from human A459 lung adenocarcinoma cells suggested that ivermectin is a substrate for MRPs. Ivermectin competition of MRP-mediated Calcein efflux gave an apparent IC50 of 1 mM and an apparent IC50 of 2.5 mM for MRP-mediated BCECF efflux in these cells which express high levels of MRP1 (Lespine et al., 2006). The IC50 values for ivermectin-competition for MRPs reported by Lespine are very similar to our IC50 value of 1.58  0.51 mM for ivermectin competition for GSMF dye efflux in human SH-SY5Y cells. Similarly, MRP1- transfected HL60 cells gave an apparent IC50 value of 3.8 mM for ivermectin competition for MRP1-mediated Calcein efflux. Detailed screening of the ability of ivermectin to inhibit MRP-ATPase activity in membrane fractions from cells transfected with either MRP1, MRP2 or MRP3 gave IC50 values of 9  4 mM for MRP1; 18  5 mM for MRP2 and 40  21 mM for MRP3, respectively (Lespine et al., 2006). Our studies extend these observations to report the apparent IC50 values of 2.25  <0.01 mM, 1.87  0.57 mM and 1.58  0.51 mM for abamectin, emamectin and ivermectin competition for human MRPs, together with apparent IC50 values of 1.68  0.63 mM, 2.74  1.01 mM and 1.94  0.75 mM for abamectin, emamectin and ivermectin competition for mouse Mrp isoforms. In this study, we have characterised the kinetics of the interactions of abamectin, emamectin and ivermectin with MDR1/Mdr1a and MRPs/Mrps expressed in human SH-SY5Y or mouse N2a neuroblastoma cell lines. The main conclusions are that competition for H3342 dye efflux by abamectin, emamectin and ivermectin competition for are not significantly different between human MDR1 or mouse Mdr1 with a rank order of affinities ivermectin > emamectin > abamectin in both cell lines. Importantly, no significant species differences in Ki values for the interaction of abamectin, emamectin and ivermectin between human MDR1 or mouse Mdr1a were seen (Table 1); the same was true for IC50 values between human MRPs and mouse Mrps (Table 2); P > 0.05 for all comparisons. In light of data suggesting that there are substantial substrate dependent differences in the handling of molecules between human and mouse homologues of MDR1, the demonstration that there are no differences in the handling of abamectin, emamectin and ivermectin is an important finding. It indicates that these native SH-SY5Y and N2a cell lines (rather than mouse models such as the Mdr1ab (–/–), or CF-1 mice) are good models for understanding avermectin transport, because the native human MDR1, MRP and mouse Mdr1, Mrp transporters behave with similar kinetics. Competition for MDR1/Mdr1 function by ivermectin (Ki 0.24 mM) correlates with peak plasma ivermectin concentrations found in man (0.05–0.1 mM), suggesting that kinetics in these in vitro models may be relevant to in vivo exposure levels (Ottesen and Campbell, 1994; González Canga et al., 2008). Brayden and Griffin (2008), found that there was no

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significant MRP-1- or MRP-2-mediated avermectin transport in the presence of functional MDR1. As with doxorubicin and vincristine, which are substrates for multiple transporters (Fung and Gottesmann, 2009), however, overlapping substrate specificity between MDR1 and MRP isoforms may provide an alternative route of efflux for these avermectins if MDR1/Mdr1 were inhibited. Conflict of interest The authors declare that there are no conflicts of interest.

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