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Research ArticleArticle

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites

Brayden D. Whitlock, Yingze Ma, Gwenaëlle Conseil, Alicia R. O’Brien, Mayukh Banerjee, Diane P. Swanlund, Z. Ping Lin, Yao Wang, X. Chris Le, John D. Schuetz, Susan P.C. Cole and Elaine M. Leslie
Drug Metabolism and Disposition December 2024, 52 (12) 1417-1428; DOI: https://doi.org/10.1124/dmd.124.001852

Abstract

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumor induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette subfamily C (ABCC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the glutathione (GSH) conjugate of the highly toxic monomethylarsonous acid (MMAIII), monomethylarsenic diglutathione [MMA(GS)2], and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in human embryonic kidney (HEK)293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid, or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ∼3-fold more resistant to arsenate than Abcc4(–/–) MEFs; however, further characterization indicated that this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to arsenic toxicokinetic differences between human and mouse.

SIGNIFICANCE STATEMENT Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. Human ATP-binding cassette subfamily C member 4 (hABCC4) is a high-affinity transporter of toxicologically important arsenic metabolites. Here we used multiple approaches to demonstrate that mouse Abcc4 does not protect cells against or transport any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Introduction

Conservative estimates indicate that 94–220 million people worldwide are chronically exposed to the proven human carcinogen arsenic in drinking water at levels above the World Health Organization’ provisional guideline of 10 μg L−1 (IARC, 2012; WHO, 2012; Hubaux et al., 2013; Podgorski and Berg, 2020). Arsenic causes skin, lung, and bladder tumors (IARC, 2012) and is associated with prostate, liver, and kidney cancers (IARC, 2012; Naujokas et al., 2013). Furthermore, chronic arsenic exposure is associated with numerous other deleterious health effects, including diseases of the vascular, respiratory, reproductive, neurologic, and endocrine systems (Rahaman et al., 2021). For most human populations, the primary source of arsenic exposure is naturally contaminated drinking water containing arsenic leached from its complex with other minerals in the ground (Cui and Jing, 2019).

Arsenic can enter cells in vitro in the form of arsenate (AsV, at physiological pH a mixture of H2AsO4– and HAsO42–) through phosphate transporters, including the sodium/phosphate cotransporter Na+/Pi-IIb (SLC34A2) (Villa-Bellosta and Sorribas, 2010; Mukhopadhyay et al., 2014; Roggenbeck et al., 2016). Arsenite [AsIII, As(OH)3 at physiological pH] can enter cells through the glucose transporter 1 (GLUT1, SLC2A1) as well as aquaglyceroporins (AQPs) 3, 7, 9, and 10 (Mukhopadhyay et al., 2014; Roggenbeck et al., 2016; Kaur et al., 2020). Arsenic is metabolized inside cells through methylation, thiolation, and/or conjugation with glutathione (GSH, γ-Glu-Cys-Gly) (Thomas et al., 2007; Wang et al., 2015; Roggenbeck et al., 2016). Although methylation results in an increased rate of arsenic whole-body clearance and reduces acute arsenic toxicity, trivalent methylated forms of arsenic [monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII)] are considered bioactivation products and more potent toxicants than AsIII (Thomas, 2021). Pentavalent forms monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are less toxic.

The human ATP-binding cassette subfamily C (ABCC) transporter members hABCC1 [often referred to as multidrug resistance protein (MRP)1], hABCC2, and hABCC4 have been demonstrated to export multiple methylated and GSH-conjugated arsenic metabolites from cells in an ATP-dependent manner (Roggenbeck et al., 2016; Banerjee et al., 2018; Zhou et al., 2021). This export results in a reduced intracellular arsenic load, reduced cellular toxicity, and potentially increased biliary and/or renal excretion. In vitro transport assays with hABCC4-enriched membrane vesicles have shown that hABCC4 is a high-affinity transporter of the major arsenic metabolite found in human urine, DMAV (K0.5 0.22 μM), as well as the diglutathione conjugate of the highly toxic MMAIII, monomethylarsenic diglutathione [MMA(GS)2] (K0.5 0.7 µM) (Banerjee et al., 2014). Given that arsenic is predominantly methylated in the liver and eliminated in the urine (Cohen et al., 2006) and that hABCC4 is localized to the basolateral surface of hepatocytes and the apical surface of kidney proximal tubule cells (Russel et al., 2008), it is reasonable to consider hABCC4 of potential importance for the urinary elimination of hepatic arsenic metabolites (Banerjee et al., 2014). In support of this idea, several single nucleotide polymorphisms of hABCC4 have been shown to be responsible for changes in arsenic transport in vitro and may thus contribute to interindividual risk of developing arsenic-induced disorders (Banerjee et al., 2016; Sanyal et al., 2024).

In addition to arsenic metabolites, hABCC4 transports a wide range of organic anion solutes, including hormones, signaling molecules, and drugs (Wen et al., 2015). Endogenous solutes include cyclic nucleotides, prostaglandins, thromboxanes, sphingosine 1-phosphate, and 17β-estradiol 17-(β-D-glucuronide) (E217βG) (Chen et al., 2001; Reid et al., 2003; Vogt et al., 2018; Wolf et al., 2022). Drugs and/or their metabolites exported by hABCC4 include several clinically valuable anticancer and antiviral drugs [e.g., 6-mercaptopurine (6-MP), methotrexate, and 9-(2-phosphonylmethoxyethyl)adenine (PMEA)] (Schuetz et al., 1999; Chen et al., 2001). Four recent reports describe multiple hABCC4 structures in complex with (and without) one or more endogenous and exogenous substrates, contributing to a solid foundation for understanding the pleiotropic transport functions of hABCC4 (Bloch et al., 2023; Chen et al., 2023; Huang et al., 2023; Pourmal et al., 2024).

After identification of hABCC4 as a transporter of DMAV and MMA(GS)2, it was a logical next step to explore the role of hABCC4/mAbcc4 in arsenic toxicokinetics using an in vivo model, such as comparing the absorption, distribution, metabolism, and elimination of arsenic species by Abcc4(–/–) with Abcc4(+/+) mice. The hABCC4 and mAbcc4 orthologues have an amino acid identity and similarity of 87% and 92%, respectively (Fig. 1). Despite this extensive sequence conservation, differences in hABCC4 and mAbcc4 substrate selectivity have been reported. For example, hABCC4 was found to be necessary for dendritic cell migration, which is important for normal immune responses, whereas knockout of mAbcc4 had no influence on this process in mice (van de Ven et al., 2008; 2009). hABCC4 and mAbcc4 also differ in their affinities for cGMP, with mAbcc4 having a Km 100-fold higher than hABCC4 for this cyclic nucleotide (de Wolf et al., 2007). Given these marked differences between mAbcc4 and hABCC4, it was important to characterize the relative ability of mAbcc4 to transport and protect cells from inorganic arsenic metabolites in vitro prior to in vivo studies. Thus, the primary objective of the present study was to determine if mAbcc4 protects cells against and/or transports the same arsenic species as hABCC4.

Fig. 1.
Fig. 1.

Schematic diagram of the secondary structure of human ABCC4 highlighting nonconserved amino acid differences with mouse Abcc4. hABCC4 and mAbcc4 are each comprised of 1325 amino acids, 87% of which are identical. Of the 170 (13%) nonidentical amino acids, 93 (7%) are conserved, 36 (2.7%) are semiconserved, and just 41 (3.1%) are not conserved. Approximate positions of the 41 nonconserved amino acids are indicated by red dots. Also indicated are the two N-glycosylation sites in MSD2 (pink) at positions 746 and 754 (Miah et al., 2016) as well as the approximate location of the cytoplasmic regions of the transporter that were used as antigens to raise anti-ABCC4 rat mAb M4I-10 (amino acids 372–431) and pAb ALX-210-856 (amino acids 1249–1268). MSD, membrane spanning domain; NBD, nucleotide binding domain; TM, transmembrane segment.

Methods

Materials

The anti-hABCC4/mAbcc4 rat monoclonal antibody (mAb) M4I-10 (ab15602) and anti-ABCC1/Abcc1 (MRP1) rat mAb MRPr1 (ab3368) were from Abcam Inc (Cambridge, MA) (Flens et al., 1994; Hipfner et al., 1998; Leggas et al., 2004). The anti-Na+/K+-ATPase rabbit polyclonal antibody (pAb) (H-300, sc-28800) was from Santa Cruz Biotechnology (Dallas, TX). The anti-hABCC4 rabbit pAb ALX-210-856 (ALX-210-856-R200) was from Enzo Life Sciences (Farmingdale, NY) (Schuetz et al., 1999). The horseradish peroxidase (HRP)-conjugated goat anti-rat and anti-rabbit IgG were from Thermo Scientific (Rockford, IL). SuperSignal West Pico Chemiluminescent Substrate was from Pierce Chemicals (Rockford, IL). GSH, ATP, AMP, sucrose, Tris base, AsIII (>99% purity), AsV (>98% purity), MMAV (99.5% purity), DMAV (≥99%), bovine serum albumin (BSA), Tween-20, CaCl2, E217βG, 6-MP, PMEA, and MgCl2 were from Sigma-Aldrich (Oakville, ON). Creatine kinase, glutathione reductase, creatine phosphate, NADPH, X-tremeGENE 9, and protease inhibitor cocktail (PIC) tablets (Complete, Mini, EDTA free) were from Roche Applied Science (Torrance, CA). G418 was from Gibco (Grand Island, NY). The ceefourin-1 inhibitor was from Tocris Bioscience (Toronto, ON) (Cheung et al., 2014). Nitric acid and sulforhodamine B (SRB) were from Fisher Scientific (Ottawa, ON). MMAIII and DMAIII in the form of diiodomethylarsine (CH3AsI2) and iododimethylarsine [(CH3)2AsI], respectively, were synthesized as previously described (Cullen et al., 2016) and were ≥99% pure as confirmed by nuclear magnetic resonance (NMR) analysis. [6,7-3H]E217βG (41.4 Ci mmol−1) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). 73AsV (>5.6 Ci/mmol) was purchased from The Los Alamos National Laboratory (Los Alamos, NM).

Production and Immunoblotting of Immobilized Peptides for Epitope Mapping of the Anti-hABCC4/mAbcc4 mAb M4I-10

An immobilized peptide array was purchased from Kinexus (Vancouver, BC) on a trioxatridecanediamine cellulose membrane. Overlapping 6-mer or 10-mer peptides shifted by one amino acid were synthesized to cover the hABCC4 sequence against which mAb M4I-10 was originally raised (Leggas et al., 2004) (AIERVSEAIVSIRRAQTFLLLDEISQRNRQLPSDGKKMVHVQDFTAFWDKASE TPLQGL; amino acids 372–431). The membranes were wetted with 100% methanol for 10 minutes, washed with Tris-buffered saline (TBS), and blocked with 4% BSA and 5% sucrose in TBS/0.05% Tween 20 for 2 hours at room temperature. After washing with TBS/0.05% Tween 20, the membranes were probed with mAb M4I-10 (1:1,000) in blocking buffer at 4°C overnight and then washed and incubated in HRP-labeled goat anti-rat antibody at 0.1 μg ml−1 in blocking buffer for 2 hours at room temperature, followed by incubation in SuperSignal West Pico Chemiluminescent Substrate for 5 minutes and then exposed to X-ray film (FUJIFILM Corp, Tokyo, Japan) and developed on a Protec OPTIMAX film processor.

Peptide Competition of mAb M4I-10 Binding

The peptide 411HVQDFTA418F of hABCC4 was synthesized (Institute for Biomolecular Design, University of Alberta) and used for peptide competition experiments. Two aliquots (0.5 μg protein) of a single hABCC4-enriched membrane vesicle preparation (described below) were resolved by 6% SDS-PAGE and electrotransferred to a PVDF membrane (0.45 μm; Merck Millipore Ltd., Tullagreen, Ireland), and the membrane was cut into strips with one sample per strip. Prior to probing the strips with mAb M4I-10 (1:30,000), the mAb solution was preincubated with the 411HVQDFTA418F peptide (0, 5, 10, 20, and 50 μg ml−1) at room temperature for 1 hour, and then the strips were incubated overnight at 4°C. The strips were then processed with HRP-labeled secondary antibody and chemiluminescent substrate as above. As a negative control, anti-ABCC4 rabbit pAb, ALX-210-856 (raised against the COOH-proximal hABCC4 peptide 1249SGRLKEYDEPYVLLQNKES1268L) (Schuetz et al., 1999) (1:15,000 dilution) was preincubated with 411HVQDFTA418F (50 μg ml−1) to ensure that the peptide binding was specific for mAb M4I-10. Membranes were then incubated with HRP-labeled secondary antibody and binding detected as before.

Generation of Expression Plasmids for hABCC4 and mAbcc4

The pcDNA3.1/Hygro-ABCC4 vector encoding full-length hABCC4 was a kind gift from Dr. Dietrich Keppler (German Cancer Research Center, Heidelberg, Germany), and was subcloned into pcDNA3.1(+)-neomycin as described (Banerjee et al., 2014). mAbcc4 in pcDNA3.1(+)-neomycin was constructed as follows: Total RNA was extracted from mouse kidney using the TRIzol Reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). cDNA was then generated using SuperScript II reverse transcriptase (Gibco-BRL) according to the manufacturer’s instructions. Subsequently, full-length mAbcc4 was amplified using the following primers: 5′-GCGGGCGAGATGCTGCC-3′ and antisense: 5′-CTGGTGGTCACAATGCTGTTTCAAA-3′. The amplified products were then subcloned into a TOPO TA cloning vector (Invitrogen) according to the manufacturer’s instructions. The isolated plasmid containing the mAbcc4 was digested with EcoRI and subcloned into the pcDNA3.1(+) expression vector. The cloning site and entire mAbcc4 cDNA were then sequenced.

Cell Lines and Maintenance

Human embryonic kidney (HEK)293T and HEK293 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS). HEK293 cells stably expressing empty pcDNA3.1(+) (HEK-V4) and pcDNA3.1(+)-hABCC4 (HEK-hABCC4-1E16 or HEK-hABCC4-1E142) were generated as described (Banerjee et al., 2014) and maintained in DMEM/7.5% FBS with 600 μg ml−1 G418. Two independently derived mouse embryonic fibroblast (MEF) cell line pairs [each pair consisting of a wild-type MEF expressing mAbcc4 (Abcc4+/+) and a MEF with mAbcc4 knocked out (Abcc4–/–)] used in this study were: 1) MEF(1)-Abcc4+/+ and MEF(1)-Abcc4–/– (Sinha et al., 2013) and 2) MEF(2)-Abcc4+/+ and MEF(2)-Abcc4–/– (Lin et al., 2008). All cell lines were cultured in DMEM/10% FBS and maintained at 37°C in a humidified incubator with 95% air/5% CO2. Routine testing of cell lines for mycoplasma contamination was performed using the ATCC Universal Mycoplasma Testing Kit.

Stable Cell Line Generation

HEK293 cell lines stably expressing mAbcc4 were generated as previously described (Ito et al., 2001; Leslie et al., 2003; Banerjee et al., 2014). Cells were transfected using X-tremeGENE 9 according to the manufacturer’s instructions and selected in DMEM/10% FBS with 1 mg ml−1 G418 for 2 weeks. Individual surviving colonies were isolated and subcultured in 96-well plates, and clones were screened by immunoblotting of whole cell extracts (Conseil and Cole, 2021) to determine mAbcc4 expression levels (see immunoblotting section above). Positive clones were evaluated by flow cytometry to determine the proportion of cells expressing mAbcc4 (Banerjee et al., 2014), and homogeneous (>90%) populations were derived by limiting dilution cloning. Three independent clones were obtained and designated HEK-mAbcc4-1A6-37, -1C1-1-3, and -1C1-2-62.

Cytotoxicity Testing

The CellTiter 96 tetrazolium-based cell proliferation or sulforhodamine B (SRB) assays were used to measure the toxicity of AsIII, AsV, MMAIII, MMAV, DMAIII, and DMAV in HEK293 cells stably expressing empty vector (HEK-V4), hABCC4, or mAbcc4 (-1A6-37, -1C1-1-3, and -1C1-2-62) as well as in Abcc4(+/+) and Abcc4(–/–) MEF(1) and MEF(2) cells according to the manufacturer’s instructions and previously published work (Skehan et al., 1990; Banerjee et al., 2014, 2018; Orellana and Kasinski, 2016). Briefly, cells were seeded at 10,000 and 3,000 cells per well for HEK and MEF cells, respectively, in 96-well plates. Cells were then exposed to AsIII (0.1–100 μM), AsV (0.3–1000 μM), MMAIII (0.1–100 μM), MMAV (0.001–10 mM), DMAIII (0.1–100 μM), or DMAV (0.003–30 mM). The pH of the MMAV and DMA V solutions was adjusted to 7.4 before being applied to cells. At least one positive control substrate for hABCC4 or mAbcc4 [6-MP (0.1–1000 μM) and/or PMEA (0.3–1000 μM)] was included in all arsenic cytotoxicity assays.

The effect of the hABCC4 inhibitor ceefourin-1 on the toxicity of AsV or PMEA in MEF(1)-Abcc4+/+ and MEF(1)-Abcc4–/– was measured as described previously (Cheung et al., 2014). Briefly, ceefourin-1 (10 μM) or vehicle control (0.1% DMSO) was added to the cells at the same time as AsV or PMEA 24 hours after seeding.

After 48 hours (MEF cells) or 72 hours (HEK cells) of exposure to arsenic or drug or vehicle, cell viability was determined, and EC50 values were calculated using the sigmoidal dose-response equation in GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA). Relative resistance was expressed as the ratio of the EC50 value of the hABCC4- or mAbcc4-expressing cell line to the EC50 value of the HEK-V4 or MEF Abcc4–/– knockout cell lines.

Knockdown of Endogenous hABCC4 in HEK293 Cells with Short Hairpin RNA

The MISSION short hairpin RNA (shRNA) plasmid DNA glycerol stocks were obtained from MilliporeSigma (Oakville, ON) and included shRNA sequences targeting 1) the hABCC4 3’UTR (clone TRCN0000005264 (5264) contained in the TRC1.5 pLKO.1-puro vector, target sequence CCACCAGTTAAATGCCGTCTA); 2) the hABCC4 coding sequence (clones TRCN0000297334 (7334) target sequence GCCTTCTTTAACAAGAGCAAT and TRCN0000279928 (9928) target sequence GCTCCGGTATTATTCTTTGAT) contained in the TRC2 pLKO.1-puro vector; and 3) the nontarget (NT) control contained in the TRC2 pLKO.1-puro vector (sequence CAACAAGATGAAGAGCACCAA). Lentiviral particles were produced using HEK293T cells with the shRNA packaging mix according to the manufacturer’s instructions. HEK-V4 cells were infected 10 days prior to experiments with lentiviral particles containing either shRNA at 1,500 viral particles per cell seeded or untreated. Untreated, NT, and hABCC4-targeted shRNA-treated cells were used in cytotoxicity assays (as described above for AsIII, AsV, MMAIII, and DMAV), and whole cell extracts were prepared before and after each experiment and immunoblotted to verify sustained and substantial hABCC4 knockdown. EC50 values were calculated, and the relative resistance was determined as before.

Transient Transfection and Vesicle Preparation

For membrane vesicle transport assays, HEK293T cells were transiently transfected using the calcium phosphate method as described (Carew and Leslie, 2010). Cells were harvested 72 hours later by scraping into homogenization buffer (250 mM sucrose, 0.25 mM CaCl2, 50 mM Tris, pH 7.4) and collected by centrifugation at 800 g for 10 minutes, and the cell pellets were stored at –80°C until needed. Plasma membrane-enriched vesicles were prepared from the pellets using the nitrogen cavitation method (Carew and Leslie, 2010). hABCC4 and mAbcc4 levels were detected by immunoblotting with mAb M4I-10 (diluted 1:10,000). Bands were visualized after incubation with primary and appropriate HRP-labeled secondary antibodies and chemiluminescent substrate as described above.

MMA(GS)2, DMAV, and E217βG Membrane Vesicle Transport Assays

MMA(GS)2 was freshly prepared prior to each experiment as described previously (Carew et al., 2011). Transport of MMA(GS)2 and DMAV by hABCC4- and mAbcc4-enriched vesicles was measured as described in Carew et al. (2011) (for MMA(GS)2) and Banerjee et al. (2014) (for DMAV) at an initial substrate concentration of 1 μM. Transport of [3H]E217βG was measured as described (Banerjee et al., 2016) at an initial substrate concentration of 400 nM (80 nCi per reaction). Membrane vesicles were incubated with [3H]E217βG-, MMA(GS)2-, or DMAV-containing reaction mixes at 37°C for 1.5, 3, or 5 minutes, respectively, in triplicate. Transport was stopped by rapid dilution in 800 μl ice-cold Tris-sucrose buffer (TSB). For MMA(GS)2 or DMAV, vesicles were then centrifuged at 100,000 g for 20 minutes at 4°C, and pellets were washed twice with TSB and then digested for 48 hours with 250 μl nitric acid. The digested vesicles were diluted 1:1 with deionized water and filtered through a 0.45-μm syringe filter. Arsenic was quantified by inductively coupled plasma mass spectrometry (ICP-MS) using the standard addition method with an Agilent 7500ce as before (Kalivas, 1987; Carew et al., 2011). In the case of [3H]E217βG transport, the reaction mix was vacuum filtered through PerkinElmer UniFilter GF/B filter plates and washed with TSB, and radioactivity was measured with a PerkinElmer Microbeta2 liquid scintillation counter. ATP-dependent transport of all solutes was calculated by subtracting transport in the presence of AMP from that in the presence of ATP.

Crude Membrane Preparations and Immunoblotting for mAbcc4 and mAbcc1 in MEF-Abcc4+/+ and MEF-Abcc4–/–

Crude membrane fractions of the MEF-Abcc4+/+ and MEF-Abcc4–/– cell line pairs were prepared (Almquist et al., 1995), and proteins (30 μg) were resolved on 6% SDS-PAGE and transferred to a PVDF membrane. Membranes were then immunoblotted with mAb M4I-10 (1:2,000), and binding was detected with HRP-labeled secondary antibody followed by application of chemiluminescent substrate as before. Blots were exposed to film or images captured using a ChemiDoc (Bio-Rad). Blots were stripped and probed with the anti-mAbcc1/hABCC1 (Mrp1/MRP1) rat mAb MRPr1 (1:10,000), and binding was detected as described for mAb M4I-10. The blot was stripped again and probed with the anti-Na+/K+-ATPase pAb (1:2,000) as a loading control. Levels of mAbcc1/Mrp1 were quantified using ImageJ Software (National Institutes of Health, Bethesda, MD), normalized to Na+/K+-ATPase levels, and reported as Abcc1/Mrp1 levels in MEF-Abcc4–/– relative to Abcc1/Mrp1 levels in MEF-Abcc4+/+ cells.

Cellular Accumulation of 73AsV

MEF(2)-Abcc4+/+ and MEF(2)-Abcc4–/– were seeded in 12-well plates at 5.5 × 105 cells per well and 24 hours later exposed to 73As-AsV (1 μM, 100 nCi) for 48 hours. Cells were washed three times with ice-cold PBS and lysed in 0.5% Triton X-100. Radioactivity was quantified using the PerkinElmer Liquid Scintillation Analyzer Tri-Carb 2910 TR. Arsenic accumulation was normalized to total protein levels in the lysates, which were determined using a Pierce BCA Assay (Thermo Fisher Scientific).

Statistical Analysis

Cytotoxicity assays were analyzed using GraphPad Prism 6.07 software, and EC50 values were determined using the sigmoidal dose-response fit. Significant differences between EC50 values were determined using a Student’s t test. Relative levels of mAbcc4, mAbcc1, and/or hABCC4 were quantified by densitometry of immunoblots using ImageJ software. Significant differences between levels were tested using a Student’s t test. Significant differences between transport of compounds by empty vector, mAbcc4, and hABCC4 were determined by one-way ANOVA followed by a Tukey multiple comparisons test. An alpha value of <0.05 was used to define significance for all tests.

Results

Mapping the Epitope of Anti-hABCC4/mAbcc4 mAb M4I-10

Rat mAb M4I-10 was raised against the hABCC4 peptide corresponding to amino acids 372–431 in a cytoplasmic region preceding the first nucleotide binding domain fused to maltose binding protein and has been reported to cross-react with ABCC4/Abcc4 from human, rat, and mouse (Leggas et al., 2004). To be confident using mAb M4I-10 as a tool to compare relative hABCC4 and mAbcc4 levels, it was important to determine that its epitope within the two proteins was identical. Thus, the M4I-10 epitope was mapped by probing two peptide arrays, one of 55 overlapping hexapeptides and one of 53 overlapping decapeptides spanning the entire sequence of the segment of hABCC4 against which the mAb was raised. As shown in Fig. 2A, hexa- and decapeptides corresponding to amino acids 410–423 of hABCC4 were detected with mAb M4I-10, with any six consecutive amino acids of 410VHVQDFTA418F being sufficient for binding. The “sufficient” window was defined as the window within which any peptide at the highest resolution used (a hexapeptide) had detectable binding (Fig. 2, A and B). The peptide blots further indicated that amino acids 414D415F were necessary for binding but not sufficient and that maximal binding was observed for the decapeptide 412VQDFTAFWD421K (Fig. 2, A and B).

Fig. 2.
Fig. 2.

Epitope mapping of anti-hABCC4 rat mAb M4I-10. (A) Arrays of overlapping hexa- and decapeptides spanning hABCC4 amino acids 372–431 (the sequence against which mAb M4I-10 was originally raised) (Leggas et al., 2004) were probed with mAb M4I-10 (1:1000). (B) Alignment of amino acids 372–431 of hABCC4 and mAbcc4 with nonidentical amino acids underlined. Boxed residues 411HVQDFTA418F were identified as sufficient, whereas 414D and 415F were necessary for mAb M4I-10 binding. (C) hABCC4-enriched membrane vesicles (0.5 μg protein per lane) were resolved by SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was cut into strips corresponding to each sample lane. (Top panels) Membranes were probed with mAb M4I-10 (1:30,000) or pAb ALX-210-856 (ALX-210, 1:15,000) (negative control) (Schuetz et al., 1999) after preincubation with the synthetic peptide 411HVQDFTA418F at the concentrations indicated. Higher and lower molecular weight bands, indicated by filled and unfilled arrowheads, respectively, correspond to fully mature and core glycosylated hABCC4, respectively. (Bottom panels) Membranes were stripped and probed with anti-Na+/K+-ATPase pAb (H-300) (1:2000) to confirm similar protein loading in each sample lane.

The octapeptide 411HVQDFTA418F containing both the necessary and sufficient amino acids was then synthesized and used at various concentrations in competitive immunoassays to corroborate the location and specificity of the M4I-10 epitope. As shown in Fig. 2C (left), this octapeptide completely inhibited mAb M4I-10 binding to hABCC4 at 50 μg ml−1. In contrast, when hABCC4 was probed with pAb ALX 210-856 (raised against a different ABCC4 epitope in the COOH-proximal half of the transporter), 411HVQDFTAF418 could not compete for binding (Fig. 2C, right). These observations demonstrate that the M4I-10 epitope is located in a region that is 100% conserved between mAbcc4 and hABCC4 and, therefore, mAb M4I-10 can be used with confidence to compare relative hABCC4 and mAbcc4 protein levels. Sequence alignments of multiple species show that the M4I-10 epitope is also completely conserved in Abcc4 from rat, primates, and wild boar but not lower organisms such as Danio rerio, Drosophila melanogaster, or Caenorhabditis elegans (Table 1).

View this table:
TABLE 1

Alignment of the anti-hABCC4 mAb M4I-10 epitope sequence with the comparable region of Abcc4 in other species

mAbcc4 Does Not Confer Resistance to Arsenicals When Stably Expressed in HEK293 Cells

To determine if mAbcc4 could confer protection against inorganic and methylated arsenic species, we generated multiple clonal HEK293 cell lines stably expressing mAbcc4 (HEK-mAbcc4-1A6-37, -1C1-1-3, and -1C1-2-62) and used these together with the previously described HEK-hABCC4 and empty vector control HEK-V4 cell lines (Banerjee et al., 2014). Levels of mAbcc4 in the three independently derived cell lines were similar (Supplemental Fig. 1). Relative levels of hABCC4/mAbcc4 were determined by immunoblotting, and, as shown in Fig. 3, A and B, mAbcc4 levels in the HEK-mAbcc4-1A6-37 cell line were comparable to those of hABCC4 in the HEK-hABCC4 cell line (P > 0.05). HEK-mAbcc4-1A6-37 cells were then exposed to various concentrations of inorganic arsenic and methylated metabolites (AsIII, AsV, MMAIII, MMAV, DMAIII, and DMAV) as well as 6-MP as a positive control. Four of the arsenic species (AsIII, AsV, MMAIII, DMAIII) and 6-MP were also tested on the HEK-mAbcc4-1C1-262 and -1C1-3 cell lines. EC50 values for each arsenic species and 6-MP were determined, and the relative resistance values were calculated from the ratio of the EC50 values for HEK-mAbcc4 and HEK-V4.

Fig. 3.
Fig. 3.

Effect of AsV and 6-MP on the viability of hABCC4 and mAbcc4 stably transfected HEK293 cell lines. (A) Relative levels of mAbcc4 and hABCC4 in whole cell lysates (10 μg protein per lane) prepared from stable clonal cell lines HEK-V4 (empty vector), HEK-hABCC4, and HEK-mAbcc4-1A6-37 were determined by immunoblotting with mAb M4I-10 (1:10,000). Blots were then probed with anti-Na+/K+-ATPase pAb (H-300) (1:2,000) as a loading control. Shown is a representative blot, and similar results were obtained in four additional experiments. The bands shown are from the same blots with irrelevant sample lanes removed. The original blots with all lanes intact are included in Supplemental Fig. 3. (B) Densitometry of the blots described in (A) was performed using ImageJ software. hABCC4 and mAbcc4 levels were normalized for protein loading as needed based on the Na+/K+-ATPase signal, and then protein levels were plotted relative to hABCC4. Bars represent the mean values (+S.D.) obtained from five independent experiments. There were no significant differences in levels of hABCC4 and mAbcc4 (Student’s t test, P = 0.06). (C–F) HEK cells stably expressing empty vector (HEK-V4) (○), mAbcc4 (■), or hABCC4 (●) were incubated in the presence of (C and D) AsV or (E and F) 6-MP for 72 hours. Cell viability was then determined using a tetrazolium-based assay. Data points are means (±S.D.) of quadruplicate determinations in a representative experiment; similar results were obtained in at least three additional experiments. EC50 values for all independent experiments investigating toxicity of the six arsenic species and 6-MP for the HEK-mAbcc4-1A6-3A7 cell line are summarized in Table 2.

As shown in the representative AsV experiment in Fig. 3C and in the summary Table 2 (and Supplemental Tables 1 and 2), mAbcc4 stably expressed in the three clonal cell lines did not confer protection against any of the arsenic species tested. This contrasts with the HEK-hABCC4 stable cell lines, which were resistant to all arsenic species tested except arsenite (Banerjee et al., 2014) (a representative AsV experiment is shown in Fig. 3D). Although mAbcc4 did not confer resistance to any of the arsenicals, it conferred a remarkable 45- to 48-fold resistance to 6-MP in all mAbcc4 expressing cell lines (Fig. 3E; Supplemental Tables 1 and 2; Table 2). This resistance to 6-MP was approximately 9-fold higher than the resistance to 6-MP conferred by hABCC4 in HEK293 (5.4-fold relative resistance) (Banerjee et al., 2014) (Fig. 3F; Table 2). Consistent with Banerjee et al. (2014), others have reported hABCC4 expressed in HEK293 and NIH3T3 cells conferred a 3- to 6-fold relative resistance to 6-MP (Chen et al., 2001; Wielinga et al., 2002), corroborating the conclusion that mAbcc4 confers much higher levels of resistance to 6-MP than hABCC4.

View this table:
TABLE 2

Resistance of HEK293 cells stably expressing mAbcc4 (HEK-mAbcc4-1A6-37), hABCC4 (HEK-hABCC4), or empty vector (HEK-V4) to inorganic and methylated arsenic species

mAbcc4-Enriched Membrane Vesicles Do Not Transport DMAV or MMA(GS)2

Previously, we used a combination of cytotoxicity and membrane vesicle transport assays to show that hABCC4 expressed in HEK293 cells confers protection against AsV, MMAIII, MMAV, DMAIII, and DMAV through the increased export of MMA(GS)2 and DMAV (Banerjee et al., 2014). To determine if mAbcc4 could export MMA(GS)2 and DMAV, mAbcc4- and hABCC4-enriched membrane vesicles were first prepared from HEK293T cells transiently transfected with mAbcc4 and hABCC4, respectively (as well as an empty vector transfected control), and levels of hABCC4 and mAbcc4 determined to be comparable (Fig. 4A). MMA(GS)2 and DMAV transport assays were completed in parallel with transport assays of the well characterized hABCC4 organic anion substrate E217βG (Chen et al., 2001; Zelcer et al., 2003; Banerjee et al., 2016; Miah et al., 2016) included as a positive control. As anticipated, the mAbcc4- and hABCC4-enriched vesicles both exhibited ATP-dependent E217βG transport activity significantly higher than empty vector controls (Fig. 4B). Interestingly, mAbcc4-mediated E217βG transport activity was >3-fold higher than hABCC4-mediated transport despite comparable levels of mAbcc4/hABCC4 proteins. Subsequent preliminary kinetic experiments indicated that this difference between hABCC4 and mAbcc4 E217βG transport was due to a difference in Vmax rather than Km (Supplemental Fig. 2). However, most importantly, although hABCC4-mediated DMAV and MMA(GS)2 transport activities were as expected (Banerjee et al., 2014), transport of these two arsenicals by mAbcc4 was extremely low and comparable to those of the empty vector control (Fig. 4, C and D). Thus, consistent with the inability of mAbcc4 to protect cells from the toxicity of the arsenic species tested (Table 2), mAbcc4 did not transport DMAV or MMA(GS)2.

Fig. 4.
Fig. 4.

Transport of E217βG, MMA(GS)2, and DMAV by hABCC4- and mAbcc4-enriched membranes vesicles from transiently transfected HEK293T cells. (A) hABCC4 and mAbcc4 membrane vesicles (2 μg protein) prepared from HEK293T cells transiently transfected with empty vector pcDNA3.1(+)-neomycin (EV), pcDNA3.1(+)-hABCC4, or pcDNA3.1(+)-mAbcc4 were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with mAb M4I-10 followed by anti-Na+/K+-ATPase pAb (H-300) as a loading control. (B) Vesicles (5 μg protein per experiment) were incubated at 37°C with the prototypic organic anion substrate [3H]E217βG (400 nM, 40 nCi) (positive control) for 1.5 minutes, transport was stopped by rapid dilution, and then vesicles were collected by filtration. ATP-dependent [3H]E217βG uptake was quantified by liquid scintillation counting. (C and D) Arsenic transport was measured using the same batch of membrane vesicles as in (B) (20 μg protein per experiment) and incubating them at 37°C with (C) MMA(GS)2 for 3 minutes or (D) DMAV for 5 minutes (both substrates at 1 μM). Transport was stopped by rapid dilution, and the vesicles were collected by centrifugation and then processed for arsenic quantification by ICP-MS. Bars represent means of at least three independent experiments (±S.D.). Significant differences between bars are indicated as follows: *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA followed by a Tukey multiple comparisons test).

Effect of Endogenous hABCC4 Knockdown on mAbcc4 Resistance to Arsenic

Previously reported hABCC4-mediated protection against arsenicals in HEK cells was low (1.5- to 3-fold) (Banerjee et al., 2014). Thus, the low but detectable levels of endogenous hABCC4 in HEK-V4 cells could potentially mask low levels of protection by transfected mAbcc4 on arsenical toxicity by raising the baseline resistance to these toxicants. The endogenous level of hABCC4 in HEK cells is markedly lower than the transfected mAbcc4 counterparts (Figs. 3A and 4A). Nevertheless, to exclude the possible influence of endogenous hABCC4, hABCC4 in HEK-V4 cells was knocked down, and the effect on arsenic toxicity was measured. Two shRNA sequences (7334, 5264) targeting hABCC4 were identified that almost completely knocked down hABCC4, whereas 9928 was less effective (Fig. 5A). The 5264 sequence was used in subsequent experiments, and a nontarget (NT) shRNA sequence was delivered as a negative control. Knockdown of endogenous hABCC4 in the cell lines was measured before and after all cytotoxicity experiments, and hABCC4 knockdown by shRNA 5264 was determined to be stable (data not shown). HEK-V4 control cells were subjected to three conditions: untreated with shRNA, treated with NT shRNA, and treated with ABCC4-targeted shRNA 5264 [knockdown (KD)]. The EC50 values obtained for the NT and KD conditions were each divided by the EC50 value obtained for the untreated condition to generate a measure of the fold difference in arsenic resistance. As shown in Fig. 5, B–E, almost complete knockdown of endogenous hABCC4 in HEK-V4 cells did not result in any significant differences in resistance to AsIII, AsV, MMAIII, or DMAV compared with the NT shRNA control. These results indicate that the endogenous hABCC4 levels in the HEK-mAbcc4 cell line are not high enough to confer detectable resistance to the arsenic species tested and are thus highly unlikely to mask any resistance conferred by mAbcc4 in this cell line.

Fig. 5.
Fig. 5.

Effect of endogenous hABCC4 knockdown in control HEK-V4 cells on arsenical cytotoxicity. (A) Three shRNA sequences specific for hABCC4 (5264, 7334, 9928) and one nontarget (NT) negative control shRNA were introduced to HEK293 cells expressing empty vector pcDNA3.1(+) (HEK-V4) via lentiviral particles at a ratio of 1,500 viral particles per cell. Total proteins (10 μg) from whole cell lysates were immunoblotted with mAb M4I-10 (1:1,000) followed by anti-Na+/K+-ATPase (1:2,000) as a loading control. (B–E) Cytotoxicity of arsenicals was measured after knockdown of endogenous hABCC4 (KD) with shRNA clone 5264 or after treatment with negative control nontarget shRNA (NT). The EC50 values of (B) AsIII, (C) AsV, (D) MMAIII, or (E) DMAV were determined and compared with cells not treated with shRNA (untreated). The y-axis shows the ratio of the EC50s of NT shRNA and KD shRNA to the EC50 of shRNA untreated cells. Bars show the means (+S.D.) of three independent experiments, each with four replicate determinations. No significant differences in sensitivity to any of the four arsenicals tested were detected between KD and NT control conditions (P > 0.097, Student’s t test).

Abcc4+/+ MEFs Exhibit Selective Resistance to AsV but No Resistance to AsIII, MMAIII, MMAV, DMAIII, or DMAV

To determine if mAbcc4 expression in a mouse cellular environment influences its ability to specifically confer resistance to different arsenicals, the toxicity of AsIII, AsV, MMAIII, MMAV, DMAIII, and DMAV was compared in a paired set of MEF cell lines, wild-type MEF(1)-Abcc4+/+ and mAbcc4 knock out MEF(1)-Abcc4–/– (Sinha et al., 2013) (Fig. 6). Consistent with the results obtained with transfected HEK-mAbcc4 cell lines, MEF(1)-Abcc4+/+ cells did not exhibit significantly different EC50 values for AsIII, MMAIII, MMAV, DMAIII, or DMAV compared with MEF(1)-Abcc4–/– cells (Fig. 6, A–E; Table 3). However, in contrast to HEK-mAbcc4 cells, MEF(1)-Abcc4+/+ cells had a 2.7-fold higher EC50 value for AsV relative to MEF(1)-mAbcc4–/– cells (Fig. 6F; Table 3). This selective resistance to AsV was also observed in a second independently derived pair of MEF cell lines, MEF(2)- mAbcc4–/– and MEF(2)-mAbcc4+/+ (Lin et al., 2008) (Table 3). As observed in the HEK-mAbcc4 cell lines and as expected, both the MEF(1)- and MEF(2)-mAbcc4+/+ cell lines had higher EC50 values for the positive control 6-MP compared with MEF(1)- and MEF(2)-mAbcc4–/–, respectively (Fig. 6G; Table 3). Due to the low endogenous levels of mAbcc4 in the MEF(1)- and MEF(2)-Abcc4+/+ cell lines, their 6-MP EC50 values are much lower than those of the stably overexpressed mAbcc4 in the HEK cell lines (Supplemental Tables 1 and 2; Tables 2 and 3). The above results were somewhat surprising because mAbcc4 has been demonstrated to be functional in HEK293 cells using multiple assays, just not in the transport of, or resistance to, the arsenic species tested. Thus, other differences, in addition to those in mAbcc4 levels, appear to exist between the MEF-Abcc4–/– and MEF-Abcc4+/+ cell lines. One potential difference to explore was the relative levels of mouse Abcc1 (Mrp1), a related ABCC transporter previously shown to confer cellular protection against AsIII in stably transfected HEK293 cells (Stride et al., 1997). Accordingly, mAbcc1 levels were evaluated in the MEF(1) paired cell lines by immunoblotting with mAb MRPr1, which detects a highly conserved epitope in human and mouse ABCC1/Abcc1 (MRP1/Mrp1) (Hipfner et al., 1998). However, as shown in Fig. 6H, Abcc1 (Mrp1) levels are the same in both MEF(1)-Abcc4+/+ and MEF(1)-Abcc4–/– . Similar results were obtained for the MEF(2) pair of cell lines (data not shown).

Fig. 6.
Fig. 6.

Effect of inorganic and methylated arsenic species on the viability of MEF-Abcc4+/+ cells and their relative levels of mAbcc1 (mMrp1). MEF cells expressing endogenous mAbcc4 (MEF-Abcc4+/+, ■) and MEF cells with Abcc4 knocked out (MEF-Abcc4–/–, □) were exposed to (A) AsIII, (B) MMAIII, (C) MMAV, (D) DMAIII, (E) DMAV, (F) AsV, or (G) 6-MP at the concentrations indicated, and viability was measured using a tetrazolium-based cytotoxicity assay. Data points represent the means of four experimental replicates (±S.D.) in a representative experiment. Similar results were obtained in at least two additional experiments, and the results of all experiments are summarized in Table 3. (H) (left panel) Shown is a representative immunoblot comparing endogenous mAbcc4 and mAbcc1 (mMrp1) levels in the MEF(1)-Abcc4–/– and MEF(1)-mAbcc4+/+ cell lines. Crude membrane preparations (30 μg protein) were immunoblotted with anti-ABCC4/Abcc4 mAb M4I-10 (1:2,000), stripped and reprobed with anti-hABCC1/mAbcc1 mAb MRPr1 (1:10,000), and then stripped and reprobed with anti-Na+/K+-ATPase pAb (H-300) (1:2,000) as a loading control. (Right panel) Relative levels of mAbcc1 were quantified by densitometry of immunoblots using ImageJ software. mAbcc1 levels were normalized for protein loading based on the Na+/K+-ATPase signal, and then data were plotted as levels relative to mAbcc1 in the MEF(1)-Abcc4+/+ cell line. Bars represent the mean values (+S.D.) obtained from five independent experiments. No significant differences in mAbcc1 levels in the MEF(1)-Abcc4+/+ and MEF(1)-Abcc4–/– cell lines were detected (Student’s t test, P = 0.5).

View this table:
TABLE 3

Resistance of MEF-Abcc4+/+ and MEF-Abcc4–/– cells to inorganic and methylated arsenic species

Ceefourin-1 Reduces Resistance of MEF(1)-Abcc4+/+ Cells to PMEA but Not AsV

To further explore the unexpected observation that MEF-Abcc4+/+ cells were more resistant to AsV than MEF-Abcc4–/– cells, the effect of the hABCC4 inhibitor ceefourin-1 (Cheung et al., 2014) on cytotoxicity was evaluated using the MEF(1) pair of cell lines. As a positive control and to confirm that ceefourin-1 inhibited the function of mAbcc4 at the concentration tested (10 μM), its effect on the cytotoxicity of the known antiviral hABCC4/mAbcc4 substrate PMEA was first evaluated (Fig. 7A; Table 4). In the absence of ceefourin-1, MEF(1)-Abcc4–/– cells had an EC50 value of 32.0 ± 0.9 μM for PMEA whereas the EC50 value of MEF(1)-Abcc4+/+ cells was 2.5-fold higher (81.0 ± 0.9 μM), consistent with previous reports that hABCC4/mAbcc4 protects cells from PMEA (Schuetz et al., 1999; Lin et al., 2008). The addition of ceefourin-1 reduced the PMEA EC50 of MEF(1)-Abcc4+/+ to 39.0 ± 1.2 μM, which was comparable to the PMEA EC50 value for MEF(1)-Abcc4–/– (32.0 ± 2.5 μM), establishing that 10 μM ceefourin-1 can completely inhibit mAbcc4-mediated resistance to PMEA. Next, the influence of ceefourin-1 on AsV toxicity was tested. As expected, MEF(1)-Abcc4+/+ cells had a 2.4-fold higher AsV EC50 value than MEF(1)-Abcc4–/– cells (31 ± 3 μM vs. 13 ± 2 μM) in the absence of ceefourin-1 (Fig. 7B; Table 4). However, in contrast to the results obtained with PMEA, ceefourin-1 did not reduce the AsV EC50 value of the MEF(1)-Abcc4+/+ cells, which remained 2.2-fold higher than the MEF(1)-Abcc4–/– cells (30 ± 5 μM vs. 14 ± 3 μM). These data suggest that AsV resistance in the MEF-Abcc4+/+ cells is independent of mAbcc4.

Fig. 7.
Fig. 7.

Effect of the ABCC4/Abcc4 inhibitor ceefourin-1 on PMEA and AsV toxicity in MEF-Abcc4+/+ and MEF-Abcc4–/– cells and 73As-arsenic accumulation in paired MEF-Abcc4+/+ and MEF-Abcc4–/– cell lines. (A and B) MEF cells expressing endogenous mAbcc4 (MEF(1)-Abcc4+/+) (filled symbols) and MEF cells with Abcc4 knocked out (MEF(1)- Abcc4–/–) (unfilled symbols) were exposed to (A) PMEA or (B) AsV at the concentrations indicated, in the absence (■, □) or presence (▲, Δ) of ceefourin-1 (10 μM) for 48 hours, and then cell viability was determined using the sulforhodamine B cytotoxicity assay. Data points represent the means (±S.D.) of four experimental replicates in a representative experiment. Similar results were obtained in at least two additional experiments, and the results of all experiments are summarized in Table 3. (C) MEF(2)-Abcc4+/+ (filled bars) and MEF(2)-Abcc4–/– (unfilled bars) were incubated with 73As-AsV (1 μM, 100 nCi) for 48 hours. Cells were then collected, and radioactivity was quantified. Bars represent the means of three independent experiments (+S.D.). Significant difference between bars is indicated with **P < 0.01 (Student’s t test).

View this table:
TABLE 4

Resistance of MEF(1)-Abcc4+/+ and MEF(1)-Abcc4–/– cells to arsenate and PMEA in the presence and absence of ceefourin-1 (10 μM)

Abcc4 Does Not Reduce the Accumulation of Arsenate in MEF Cells

To further investigate the Abcc4-independent protection of MEF cells from AsV, accumulation of 73AsV by MEF(2)-Abcc4+/+ was compared with MEF(2)-Abcc4–/–. There was a modest reduction in AsV accumulation in cells lacking Abcc4 (108 and 68 pmol AsV/mg protein for MEF(2)-Abcc4+/+ and MEF(2)-Abcc4–/–, respectively) (Fig. 7C); if AsV protection was Abcc4-mediated, the reduction should have been observed in cells expressing Abcc4.

Discussion

Large variability among mammalian species exists with respect to their sensitivity to arsenic toxicity, with humans having the highest susceptibility to arsenic-induced cancers (IARC, 2012). A considerable body of evidence suggests that these differences in susceptibility are due to species differences in arsenic toxicokinetics and that differing abilities to methylate arsenic contribute substantially to this (Vahter, 1999; Vahter and Concha, 2001; Drobná et al., 2010; Tokar et al., 2010; Douillet et al., 2023). Until now, the impact of transport pathways on species differences in arsenic toxicokinetics, and ultimately susceptibility to disease, has been largely unstudied. In the current investigation, differences in the ability of mAbcc4 and hABCC4 to confer cellular protection against and transport multiple chemical forms of arsenic were explored. We report novel evidence that mAbcc4 does not protect against or transport any of the six arsenic (AsIII, AsV, MMAIII, MMAV, DMAIII, and DMAV) chemical species tested, in multiple cell line models. These results are in stark contrast with hABCC4, which has been firmly established as a high-affinity transporter of DMAV and MMA(GS)2 and, furthermore, affords significant protection of cells against AsV, MMAIII, MMAV, DMAIII, and DMAV (Banerjee et al., 2014).

To compare possible differences in the ability of hABCC4 and mAbcc4 to confer resistance to inorganic and methylated arsenic species when stably expressed in HEK293 cells, the ability to accurately compare the levels of mAbcc4 with hABCC4 was required. For this reason, the epitope of rat mAb M4I-10 was mapped to the level of eight consecutive amino acids located in a region of the two proteins with 100% identity. Subsequently, levels of mAbcc4 and hABCC4 were found to be similar between the stable cell lines. Despite this, mAbcc4 stably expressed in HEK293 cells did not confer protection against the six arsenic chemical species tested. To exclude the possibility that this lack of resistance could be an artifact of a single stable cell clone, two additional, independently derived HEK-mAbcc4 stable clones were tested, and similar results were obtained. Consistent with the lack of cellular protection, ATP-dependent transport of MMA(GS)2 and DMAV [well characterized hABCC4 substrates (Banerjee et al., 2014, 2016)] by mAbcc4-enriched HEK293 membrane vesicles was also not detected.

HEK293 cells are known to express low levels of endogenous hABCC4, raising the possibility that mAbcc4 protection from arsenicals could be masked by this endogenous hABCC4. However, when ABCC4 shRNA knockdown experiments followed by cytotoxicity profiling were performed, no significant differences between HEK293 cells with hABCC4 knocked down and NT-shRNA controls (relative to shRNA free controls) were observed, indicating that endogenous hABCC4 in HEK293 cells is not high enough to influence the cytotoxicity of these arsenic species.

An unexpected finding of the present study was the large activity differences observed between mAbcc4 and hABCC4 for the two prototypic organic anion substrates, 6-MP and E217βG. Thus, despite similar levels of mAbcc4 and hABCC4 in HEK293 cells stably or transiently expressing these proteins, 6-MP resistance was 9-fold higher and E217βG transport was 3-fold higher for mAbcc4 compared with hABCC4. These substantially higher activities confirm that mAbcc4 was fully functional in the assays used to evaluate arsenic toxicity and MMA(GS)2 and DMAV transport. Even more importantly, these observations provide further novel evidence for distinct substrate-selective differences between mAbcc4 and hABCC4 (de Wolf et al., 2007; van de Ven et al., 2008; 2009) and have implications for the cellular handling of 6-MP, a nucleobase analog that is widely used clinically, and E217βG, an important endogenous liver metabolite implicated in cholestasis (Marinelli et al., 2019). Comparative analyses of the hABCC4 and mAbcc4 atomic structures are needed to identify the molecular basis for these intriguing differences in substrate selectivity of these highly conserved orthologs.

Consistent with the results of cytotoxicity and transport assays using mAbcc4-overexpressing HEK293 cells, MEF cells expressing endogenous mAbcc4 [MEF(1)-Abcc4+/+] had similar EC50 values for AsIII, MMAIII, MMAV, DMAIII, and DMAV as cells with mAbcc4 knocked out [MEF(1)-Abcc4–/–]. Initial studies with two independently derived MEF cell line pairs suggested that mAbcc4 might be selectively protecting MEFs from AsV; however, the pharmacological hABCC4/mAbcc4 inhibitor, ceefourin-1, did not reverse this protection. These results strongly suggest that the low level of protection against AsV was not mAbcc4 mediated but rather occurs through yet-to-be-discovered differences between the MEF-Abcc4–/– and MEF-Abcc4+/+ cell line pair. One possible difference considered was differences in the levels of the related transporter, mAbcc1 (Mrp1), which also confers resistance to inorganic arsenic (Stride et al., 1997); however, this was not the case (Fig. 6H). Furthermore, AsV cellular accumulation experiments revealed no decrease in total arsenic accumulation in MEF(2)-Abcc4+/+ cells compared with MEF(2)-Abcc4–/– cells (Fig. 7C), supporting the conclusion that the cytotoxicity difference observed for AsV was unlikely to be efflux transporter mediated. Thus, further investigation is required to understand the selective differences in AsV sensitivity between these MEF lines, but at present there is no clear evidence that mAbcc4 or mAbcc1 (Mrp1) is involved.

Arsenic toxicokinetic differences between humans and mice are well established, and mice are known to more efficiently methylate and clear arsenic (Vahter, 1999; Cohen et al., 2006; Stýblo et al., 2019). Our findings in the present study are intriguing; since mice are more resistant to arsenic, it would be reasonable to predict that mAbcc4 would be better than hABCC4 at extruding arsenic species into the sinusoid from the liver and into urine from the proximal tubule cell, increasing elimination of arsenicals. However, arsenic toxicokinetics are complex and remain incompletely understood for any individual species. Mice expressing the humanized form of arsenite methyltransferase (As3MT), the enzyme predominantly involved in arsenic methylation, have a similar urinary arsenic chemical species profile to humans (Douillet et al., 2023), suggesting that As3MT is an important contributor to the differences between mice and humans. Differences in expression patterns of transporters between species may also alter arsenic toxicokinetics and contribute to differences in sensitivity. For example, hABCC1 (MRP1) protein is undetectable in human hepatocytes, but mAbcc1 (Mrp1) is found at low basal levels in mouse hepatocytes (Roelofsen et al., 1997; Aleksunes et al., 2006; van de Wetering et al., 2007; Roggenbeck et al., 2015). Therefore, mAbcc1 could compensate for the lack of arsenic metabolite export by mAbcc4 in mice and transport a broader spectrum of arsenic metabolites, resulting in better clearance. hABCC1 (MRP1) is capable of transporting multiple metabolites of arsenic [As(GS)3, MMA(GS)2, and DMAV] (Carew et al., 2011; Shukalek et al., 2016; Banerjee et al., 2018), and although the metabolite selectivity of mAbcc1 has not been investigated, it confers significant resistance against AsIII. Thus, although the distinct mAbcc4/hABCC4 arsenic metabolite selectivity does not directly explain differences in mouse and human sensitivity to arsenic, mAbcc4 likely contributes to arsenic toxicokinetic differences between species and deserves further investigation.

The findings reported here serve as an important foundation for better understanding hABCC4/mAbcc4 function in arsenic detoxification and how ABCC4/Abcc4 may differentially influence arsenic toxicokinetics in mice versus humans. Future work will take advantage of these species differences to provide molecular details of the arsenic metabolite binding sites in hABCC4. Insights provided by the present study are also crucial for the development of future mouse models relevant for studying arsenic transport with application to arsenic-induced human disease.

Acknowledgments

The authors thank Dietrich Keppler (German Cancer Research Centre) for the hABCC4 cDNA. Xiufen Lu is gratefully acknowledged for excellent technical assistance with the ICP-MS. The 73As isotope used in this research was supplied by the US Department of Energy Isotope Program, managed by the Office of Isotope R&D and Production.

Data Availability

The authors declare that all of the data supporting the findings of this study are available within the paper and its Supplemental Material.

Authorship Contributions

Participated in research design: Whitlock, Ma, Conseil, O’Brien, Banerjee, Swanlund, Le, Schuetz, Cole, Leslie.

Conducted experiments: Whitlock, Ma, Conseil, O’Brien, Banerjee, Swanlund, Leslie.

Contributed new reagents or analytic tools: Whitlock, Conseil, Swanlund, Lin, Wang, Le, Schuetz, Cole.

Performed data analysis: Whitlock, Ma, Conseil, O’Brien, Swanlund, Cole, Leslie.

Wrote or contributed to the writing of the manuscript: Whitlock, Conseil, Cole, Leslie.

Footnotes

    • Received August 9, 2024.
    • Accepted September 10, 2024.

  • This work was supported by the Canadian Institutes of Health Research [Grant MOP-272075] (to E.M.L.), [Grant MOP-106513] (to S.P.C.C.), and [Grant PJT-159547] (to E.M.L.); by American Lebanese Syrian Associated Charities (ALSAC) (to J.D.S.); and by National Institutes of Health National Cancer Institute [Grant P30 CA021765], [Grant R01 CA194057], [Grant R01 CA21865], and [Grant R01 CA96832] (to J.D.S.); M.B. was supported by an Alberta Cancer Foundation Cancer Research Postdoctoral Fellowship award. X.C.L. holds the Canada Research Chair in Bioanalytical Technology and Environmental Health.

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • ↵1Current affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

  • ↵2Current affiliation: Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky.

  • dx.doi.org/10.1124/dmd.124.001852.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

ABCC
ATP-binding cassette subfamily C
AsIII
arsenite
AsV
arsenate
DMAIII
dimethylarsinous acid
DMAV
dimethylarsinic acid
DMEM
Dulbecco’s modified Eagle’s medium
E217βG
17β-estradiol 17-(β-D-glucuronide)
GSH
glutathione
HEK
human embryonic kidney
HRP
horseradish peroxidase
ICP-MS
inductively coupled plasma mass spectrometry
KD
knockdown
mAb
monoclonal antibody
MEF
mouse embryonic fibroblast
MMA(GS)2
monomethylarsenic diglutathione
MMAIII
monomethylarsonous acid
MMAV
monomethylarsonic acid
6-MP
6-mercaptopurine
MRP
multidrug resistance protein
NT
nontarget
pAb
polyclonal antibody
PMEA
9-(2-phosphonomethoxyethyl)adenine)
shRNA
short hairpin RNA
TBS
Tris-buffered saline
TSB
Tris-sucrose buffer

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Species Differences in Arsenic Metabolite Transport by ABCC4

Brayden D. Whitlock, Yingze Ma, Gwenaëlle Conseil, Alicia R. O’Brien, Mayukh Banerjee, Diane P. Swanlund, Z. Ping Lin, Yao Wang, X. Chris Le, John D. Schuetz, Susan P.C. Cole and Elaine M. Leslie
Drug Metabolism and Disposition December 1, 2024, 52 (12) 1417-1428; DOI: https://doi.org/10.1124/dmd.124.001852
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