title: Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos.
abstract: BACKGROUND
  In mammals, ABCB1 constitutes a cellular "first line of defense" against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water.
  
  
  RESULTS
  Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks).
  
  
  CONCLUSIONS
  We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model.
authors.affiliation: Department of Bioanalytical Ecotoxicology, UFZ - Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany. till.luckenbach@ufz.de.
authors.forename: Stephan
authors.initials: S
authors.lastname: Fischer
authors.forename: Nils
authors.initials: N
authors.lastname: Klüver
authors.forename: Kathleen
authors.initials: K
authors.lastname: Burkhardt-Medicke
authors.forename: Mirko
authors.initials: M
authors.lastname: Pietsch
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmidt
authors.forename: Peggy
authors.initials: P
authors.lastname: Wellner
authors.forename: Kristin
authors.initials: K
authors.lastname: Schirmer
authors.forename: Till
authors.initials: T
authors.lastname: Luckenbach
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian; D005456:Fluorescent Dyes; D055785:Gene Knockdown Techniques; D026801:Synteny; D018675:Toxicity Tests; D015262:Xenobiotics / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*; D000097202:ATP-Binding Cassette Sub-Family B Member 4
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D005456:Fluorescent Dyes; D015262:Xenobiotics; D029961:Zebrafish Proteins; C000599462:Abcb4 protein, zebrafish; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C095122:multidrug resistance protein 3