Identification of multi-drug resistance associated proteins MRP1 (ABCC1) and MRP3 (ABCC3) from rainbow trout (Oncorhynchus mykiss)
The MRPs (multi-drug resistance associated proteins; ABCC subfamily) are members of the ATP binding cassette (ABC) superfamily of transport proteins and act as cellular efflux transporters of a wide variety of substrates, in particular glutathione, glucuronide and sulphate conjugates of diverse compounds. Together with P-gp (P-glycoprotein; MDR1; ABCB1) and BCRP (breast cancer resistance protein, ABCG2) the MRPs are highly important as cellular defense against toxicants and confer multixenobiotic resistance (MXR). In aquatic ecotoxicology MXR research has mostly focussed on P-glycoprotein while the other relevant ABC transporters are widely under-appreciated. We here present complete MRP1 (ABCC1) and partial MRP3 (ABCC3) cDNAs from rainbow trout (Oncorhynchus mykiss). We identified 4398 bp of the MRP1 and 3786 bp of the MRP3 open reading frames (ORF) by screening the NCBI EST database and by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using RNA from RTgill-W1 cells. Identities with human homologs are 56% for MRP1 and 64% for MRP3.
Fischer,S.; Pietsch,M.; Schirmer,K.; Luckenbach,T. (2010) Identification of multi-drug resistance associated proteins MRP1 (ABCC1) and MRP3 (ABCC3) from rainbow trout (Oncorhynchus mykiss), Marine Environmental Research, 69(SUPPL. 1), S7-S10, doi:10.1016/j.marenvres.2009.11.003, Institutional Repository
Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss)
Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11(abcc1-5 > abcg2 > abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2′,7′-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation.
Fischer,S.; Loncar,J.; Zaja,R.; Schnell,S.; Schirmer,K.; Smital,T.; Luckenbach,T. (2011) Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss), Aquatic Toxicology, 101(2), 438-446, doi:10.1016/j.aquatox.2010.11.010, Institutional Repository
Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos
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 of 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 presence of transcripts of both genes throughout the first 48 hrs 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 to mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in 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, i.e., 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.
Fischer,S.; Klüver,N.; Burkhardt-Medicke,K.; Pietsch,M.; Schmidt,A.M.; Wellner,P.; Schirmer,K.; Luckenbach,T. (2013) Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos, BMC Biology, 11, 69 (16 pp), doi:10.1186/1741-7007-11-69, Institutional Repository
Current advances on ABC drug transporters in fish
Most members of the large ATP-binding cassette (ABC) gene family are transporters involved in substrate translocation across biological membranes. In eukaryotes, ABC proteins functioning as drug transporters are located in the plasma membrane and mediate the cellular efflux of a wide range of organic chemicals, with some transporters also transporting certain metals. As the enhanced expression of ABC drug transporters can confer multidrug resistance (MDR) to cancers and multixenobiotic resistance (MXR) to organisms from polluted habitats, these ABC family members are also referred to as MDR or MXR proteins. In mammals, ABC drug transporters show predominant expression in tissues involved in excretion or constituting internal or external body boundaries, where they facilitate the excretion of chemicals and their metabolites, and limit chemical uptake and penetration into “sanctuary” sites of the body. Available knowledge about ABC proteins is still limited in teleost fish, a large vertebrate group of high ecological and economic importance. Using transport activity measurements and immunochemical approaches, early studies demonstrated similarities in the tissue distribution of ABC drug transporters between teleosts and mammals, suggesting conserved roles of the transporters in the biochemical defence against toxicants. Recently, the availability of teleost genome assemblies has stimulated studies of the ABC family in this taxon. This review summarises the current knowledge regarding the genetics, functional properties, physiological function, and ecotoxicological relevance of teleostean ABC transporters. The available literature is reviewed with emphasis on recent studies addressing the tissue distribution, substrate spectrum, regulation, physiological function and phylogenetic origin of teleostean ABC transporters.