Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio) embryos.
Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.
CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, Coastal and Marine Environmental Toxicology Lab, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal; ICBAS/UP-Institute of Biomedical Sciences Abel Salazar, University of Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal. Electronic address: vfpmc83@gmail.com.
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Cunha
UFZ-Helmholtz-Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstraße 15, 04318 Leipzig, Germany; Technische Universitaet Dresden, Faculty of Environmental Sciences, Institute of Hydrobiology, 01062 Dresden, Germany.
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Burkhardt-Medicke
UFZ-Helmholtz-Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstraße 15, 04318 Leipzig, Germany.
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Wellner
CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, Coastal and Marine Environmental Toxicology Lab, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal; FCUP-Dept of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal; 5IBMC-Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
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Santos
ICBAS/UP-Institute of Biomedical Sciences Abel Salazar, University of Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal; I3S-Institute for Research and Innovation in Health, University of Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; IBMC, Institute for Molecular and Cell Biology, Porto, Portugal.
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Moradas-Ferreira
UFZ-Helmholtz-Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstraße 15, 04318 Leipzig, Germany.
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Luckenbach
CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, Coastal and Marine Environmental Toxicology Lab, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal; School of Marine Studies, Faculty of Science, Technology and Environment, The University of South Pacific, Laucala Bay Road, Suva, Fiji Islands.
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Ferreira
D018528:ATP-Binding Cassette Transporters / Q000187:drug effects*; D000171:Acrolein / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001692:Biological Transport / Q000187:drug effects; D003358:Cosmetics / Q000494:pharmacology*; D004351:Drug Resistance; D004364:Pharmaceutical Preparations; D012235:Rhodamines / Q000378:metabolism; D014700:Verapamil / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D003358:Cosmetics; D004364:Pharmaceutical Preparations; D012235:Rhodamines; C551429:alpha-hexylcinnamaldehyde; D000171:Acrolein; D014700:Verapamil; D000251:Adenosine Triphosphatases
Abcb4; Chemosensitisation; Inhibitors; PCPs; Pharmaceuticals; Substrates