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Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) – Transporters and multixenobiotic resistance
Marine Environmental Research 66 (2008) 80–84
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) – Transporters and multixenobiotic resistance P-glycoprotein expression in killifish from the Sydney tar ponds, Nova Scotia, Canada S. Christine Paetzold a, Sara Zucchi b, Robert Richards c, Shannon Mala Bard a,d Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada b Department of Environmental Sciences ‘‘G. Sarfatti”, University of Siena, Siena, Italy c Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada d Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, Canada
a
Abstract We investigated the role of the multidrug resistance transporters P-glycoprotein (MDR1/ABCB1) and sister of P-glycoprotein (SPGP/ ABCB11), both ATP-binding cassette (ABC) proteins, in conferring multixenobiotic resistance in a wild population of killifish (Fundulus heteroclitus) from the highly contaminated Sydney Tar Ponds, Canada. P-glycoproteins act as transmembrane export pumps that prevent cellular accumulation of a wide range of moderately hydrophobic xenobiotics including anthropogenic contaminants. The interaction of P-glycoproteins with phase I and II drug-metabolizing enzymes and other ABC xenobiotic transporters in wild populations are poorly understood. We examined a killifish population living in the estuarine Sydney Tar Ponds that have been contaminated since the early 1900s with polycyclic aromatic hydrocarbons (PAHs) from industrial activities adjacent to the site. PAHs and their phase I metabolites are potential P-glycoprotein substrates and inducers. We investigated whether P-glycoprotein expression was elevated in the liver of killifish from the Sydney Tar Ponds using quantitative reverse-transcription polymerase chain reaction. Cytochrome P450 1A (a phase I metabolizing enzyme) transcript abundance was assessed as indicator of PAH exposure. We also quantified gene expression of the complementary ABC transporters multidrug resistance-associated protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) to understand their interaction with P-glycoproteins. MRP2 and BCRP predominantly transport phase II metabolites but also have some substrate overlap with P-glycoproteins. Gene expression of the phase II enzyme glutathione-Stransferase was also quantified since its metabolites are exported by MRP2 and BCRP. Our aim was to elucidate the cellular and molecular mechanisms that contribute to resistance to PAH contaminants.
0141-1136/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marenvres.2008.02.029
Complexity of multixenobiotic resistance - PGP1 (ABCB1), MRP2 (ABCC2) and BSEP (ABCB11) efflux transporters in primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes Roko Zaja a, Vesna Munic b, Andreja Ambriovi Ristov b, Roberta Sauerborn Klobuèar a, Tvrtko Smital a a Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Bijenicka 54, 10000 Zagreb, Croatia b Laboratory for Genotoxic Agents, Division of Molecular Biology, Rułer Boškovi Institute, Bijenicka 54, 10000 Zagreb, Croatia; GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovica 29, 10000 Zagreb, Croatia
Abstract The extrusion of hydrophobic xenobiotics and their metabolites in fish liver could be mediated by the activities of several types of ABC transport proteins, together contributing to the multidrug/multixenobiotic resistance (MDR/MXR). In order to test this hypothesis the main goal of this study was detection and functional characterization of toxicologically relevant efflux transporters belonging to the ABCB and ABCC sub-family in primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. Using specific pairs of primers, we have identified and cloned three full length nucleotide sequences, which share a high degree of homology with mammalian Pgp1 (ABCB1), MRP2 (ABCC2) and BSEP (ABCB11) efflux transporters, respectively. The expression of identified efflux transporters was quantified in both trout liver tissue and primary hepatocytes using the real-time PCR technique. The levels of all three transcripts were approximately 4 times lower in primary hepatocytes in comparison to intact liver, and the same relative expression pattern (ABCB11>>ABCC2>ABCB1) was determined in both tissues/compartments. In addition, transport activities of identified ABC transporters have been demonstrated using model substrates (rhodamine123, calcein-AM, bodipy-verapamil, dihydrofluorescein diacetate) and inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurochenodeoxycholate, taurocholate) specific for corresponding transport proteins. Taken together these findings show that MDR/MXR is an integral part of the detoxification system in rainbow trout hepatocytes, that is mediated by several ABC transport proteins with different and/or overlapping substrate specificities. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No. 00981510.
Abstracts / Marine Environmental Research 66 (2008) 80–84
Cost, effectiveness, and environmental relevance of multi-drug transporters Bryan Cole, David Epel Hopkins Marine Station, Stanford University, Pacific Grove, CA, USA
Abstract Efflux transporters of the ABCB and ABCC families protect cells from xenobiotics and remove endogenous byproducts of detoxification. We use embryos of the sea urchin, Strongylocentrotus purpuratus, as a model for determining the cost and environmental relevance of this protection system. Analysis of developmental mRNA shows expression of all 30 ABCC genes and all 12 members of the PGP (ABCB) family. We have quantified the cost of efflux transporters as a first-line of defense in terms of activity and infrastructure costs; respectively, these represent the direct ATP cost to power efflux and the indirect ATP cost of synthesizing these proteins. Activity costs are low, utilizing only 0.074% of ATP production, while still reducing toxicant accumulation at micromolar concentrations. Infrastructure costs for the PGP subfamily were estimated by quantitative Western Blot analysis. We found 6.3X108 molecules of PGP-type proteins per embryo, equivalent to approximately 0.9% of total embryonic protein. As a first-line of defense against toxicants, the activity cost of efflux transporters is low. However, the infrastructure cost of synthesizing this amount of protein is comparatively high; infrastructure costs are equivalent to over 32 hours of constant maximal transporter activity. The large number of developmentally expressed transporter genes and the large amount of corresponding protein maternally loaded into the oocyte point towards an increased reliance on these proteins during development. This reliance is especially remarkable considering the high infrastructure costs, and that a female sea urchin produces millions of oocytes, each loaded with such a high amount of toxicant defense proteins. Supported by the California Sea Grant, the National Science Foundation, and Stanford’s Department of Neonatology.
Development of a behavioural toxicological assay to measure P-glycoprotein activity at the blood-brain barrier and evaluate the neuropathological effects of environmental contaminants Shannon Mala Bard a, Simon Gadbois b Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, B3H 4J1, Canada b Psychology and Neuroscience, Dalhousie University, Halifax, Nova Scotia, B3H 4J1, Canada
a
Abstract The neuroprotective role of P-glycoprotein, a multixenobiotic resistance transporter (ABCB1/MDR1), in the blood-brain barrier in fish was examined using behavioural toxicological assays. P-glycoproteins act as cellular efflux pumps to prevent substrates from accumulating in the brain, including environmental contaminants such as ivermectin, a common aquaculture pesticide and
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mammalian anti-parasitic drug. The behavioural toxicological assays were developed to determine the neuropathological effect of ivermectin in killifish (Fundulus heteroclitus). P-glycoprotein function and thus blood-brain barrier integrity can be compromised by chemosensitizers, such as cyclosporin A, which inhibit transport activity. Fish treated with ivermectin and the P-glycoprotein inhibitor cyclosporin A significantly succumbed more rapidly and were >2-fold as sensitive to tilting, lethargy, slowing of pectoral fin movement and loss of haptic reactivity compared to fish treated with ivermectin only. P-glycoprotein inhibition is associated with significantly earlier onset and increased mortality (~3-fold) in ivermectin-exposed fish. Our results suggest that Pglycoprotein confers resistance against ivermectin-induced behavioural neuropathology and mortality in fish. We then used this assay to evaluate emamectin benzoate, an ivermectin-derivative with fewer side effects and shorter half-life in fish tissue. Compared to ivermectin-treated fish, neuropathological symptoms were of substantially lower severity and mortality was greatly reduced in emamectin benzoate-treated fish. Whether these trends are due to lower inherent toxicity or increased substrate affinity for P-glycoprotein and thus increased elimination is currently under investigation. This assay provides us with a non-invasive tool to study P-glycoprotein function in the blood-brain barrier and evaluate the behavioural effects of potential environmental neurotoxins.
Zebrafish embryos are sensitized towards chemicals by efflux transporter inhibitors Till Luckenbach, Mady Ahnert, Stephan Fischer, Kristin Schirmer UFZ - Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany
Abstract ABC efflux pumps are part of the cellular toxic defensome in many aquatic organisms. Certain environmental contaminants have been shown to antagonize the activity of those xenobiotic transporters. Therefore, there is concern that due to the presence of such chemosensitizers in the environment, susceptibility of cells and organisms could be increased for toxicants that would normally be removed by this mechanism. We used zebrafish (Danio rerio) embryos in order to determine the toxicological impact of chemosensitizers on the organism level. Using RT-PCR we found expression of various xenobiotic transporters from the ABCB, ABCC and ABCG families in zebrafish at stages from 6 to 48 hrs post fertilization. In toxicological experiments the LC50 (48 hrs) for vinblastine, a known toxic substrate of efflux transporters, was 21 % lower (2.8 vs. 2.2 uM) in the presence of the known transporter inhibitor cyclosporine A. Cyclosporine A alone did not show any toxic effects. Similarly, galaxolide significantly enhanced sensitivity of the embryos to vinblastine. Galaxolide, commonly used as a fragrance in hygiene and personal care products, is an environmentally relevant micropollutant with transporter modulating properties. Our experiments show that (1) efflux transporters appear to be an important part of the toxic defense in zebrafish embryos and (2) chemosensitization can be a relevant component in the toxicity of chemical mixtures on zebrafish. Finally, our data further the ecotoxicological relevance of chemosensitisation by environmental contaminants.
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Abstracts / Marine Environmental Research 66 (2008) 80–84
Gene expression of multixenobiotic resistance transporters p-glycoprotein and mpr2 in the antarctic emerald rockcod (Trematomus bernacchii) Sara Zucchi a, Ilaria Corsi a, Shannon Mala Bard b,c, Francesco Regoli d, Robert Richards e, Christine S. Paetzold b, Silvano Focardi a a Department of Environmental Sciences ‘‘G. Sarfatti”, University of Siena, Siena, Italy b Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada c Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, Canada d Istituto di Biologia e Genetica, Ecotossicologia Marina, Università Politecnica delle Marche, Via Ancona, Italy e National Research Council, Institute for Marine Biosciences, Halifax, NS, Canada
Abstract We examined the gene expression of P-glycoprotein (P-gp/MDR1/ ABCB1) and Multidrug Resistance-associated Protein 2 (MRP2/ ABCC2) in Trematomus bernacchii, the Antarctic emerald rockcod, experimentally exposed to organic contaminants and heavy metals. P-gp and MRP2, ATP-binding cassette (ABC) proteins, are xenobiotic transporters which respectively facilitate the transmembrane efflux of moderately hydrophobic substrates and phase II metabolite glutathione conjugates. We analyzed T. bernacchii because this widely distributed benthic feeder is naturally exposed to high levels of cadmium from the local Antarctic geology and is a key sentinel species for monitoring the Antarctic ecosystem. Our main interest is to assess the sensitivity of T. bernacchii to anthropogenic and natural chemicals. In contaminated exposed fish we investigated the expression of the two transporters that play a role in absorption, disposition and elimination of a wide variety of xenobiotics. We evaluated fish (N=8) exposed by intraperitoneal injection to either a single contaminant or pairs of contaminants from the following list: benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, cadmium, copper and lead. Partial sequences of P-gp (410 nucleotides) and MRP2 (750 nucleotides) were obtained and used to design primers to assess hepatic P-gp and MRP2 gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in exposed fish. To determine whether P-gp and MRP2 hepatic gene expression reflects protein expression in the liver, we evaluated protein levels by Western blot. This study provides a preliminary characterization of genes involved in cellular defense mechanisms in the key sentinel species T. bernacchii and evaluates whether anthropogenic and natural compounds could modulate P-gp and MRP2 expression.
Biocomplexity of multidrug resistance related transport in the shore crab Carcinus maenas - job sharing of organs in toxin elimination? Angela Koehler, Sieglinde Bahns Department of Biosciences, Alfred Wegener Institute for Polar and Marine Research, Germany
Abstract MXR transport activity was measured in living gills with CalceinAM and quantified by computerised image analysis. Simultaneously, we detected MXR protein expression with an antibody C 219 detecting evolutionary conserved amino acid sequences of various MXR transporters. Shore crabs adapted to 10ppt and 33ppt seawater according to the natural salinity range were studied for MXR transport activity and for protein expression in gills and digestive glands.
Animals adapted to low salinities had a 30% higher transport efficiency, possibly due to membrane up folding, inferring that these efflux pumps may also be involved in osmo-regulation. Animals were exposed to AAF (0.01M), a specific model inducer of the transmembrane P-glycoprotein involved in MXR transport over a period of 3 days and showed a 50% induction of transport activity in gills at both salinities. Additionally, animals were starved for 4 weeks, re-fed once, and samples taken after 4, 8, 12, 24 and 48 hours according to cell differentiation of the digestive gland. In the digestive glands, MXR proteins appeared in differentiating blister cells 8 hours after re-feeding as immuno-localised with the AB C219. Simultaneously, food supply induced a complete breakdown of MXR transport activity in gills after 8 h. MXR-like transporters, presumably P-gp, appear to exist in shore crab as transport can be specifically induced by a model inducer. Furthermore, gills and hepatopancreas appear to perform a sort of physiological job sharing in which the hepatopancreas overtakes xenobiotic elimination during periods the when gills are involved to serve enhanced oxygen demands during digestion and active ammonia excretion.
Effluents from oil production platforms in the north sea inhibit the function of the abc transporters mdr1 and mrp1 in vitro Branka Pivèevi a, Tvrtko Smital a, Knut-Erik Tollefsen b Laboratory for Molecular Ecotoxicology, Division for EnvironmentalResearch, Rułer Boškovi Institute, Zagreb, Croatia b Norwegian Institute for Water Research (NIVA), Oslo, Norway
a
Marine
and
Abstract Inhibition of multixenobiotic resistance (MXR) defence by various environmental contaminants in aquatic organisms may cause significant increases in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by ABC transport proteins. In this study the inhibition potency of production water samples from North Sea offshore oil platforms on the efflux activity of the ABC transporters Pgp1 (ABCB1) and MRP1 (ABCC1) was examined using an in vitro calcein-AM bioassay with HL60 cells stably transfected with either human ABCB1 or ABCC1 genes. Transfected cells are characterized by an efficient efflux and low intracellular accumulation of calcein-AM, which can be modulated by numerous different inhibitors. The inhibition potency of the samples was expressed as inhibiting equivalents (EQ) of the model inhibitors reversin 205 (REV) for Pgp1 and MK571 for the MRP1 transporters. Production water samples were collected from ten oil and gas production platforms with different chemical composition, extracted by large volume solid phase extraction, and prepared as a particulate/oil fraction and a water soluble fraction. The majority of samples had measurable concentrations of Pgp1 inhibitors (range: 37 – 380 mg/ L REV EQs), and the Pgp1 inhibiting activity was predominantly found in the water fraction of produced water. MRP1 inhibitors were determined in all samples with highest concentration in extracts of particulate/oil fraction of produced water (range: 88 – 1400 mg/L MK571 EQs). These results indicate that there are more inhibitors of MDR1 than of Pgp1 transporter in produced water, and that the active chemicals exhibit different physico-chemical properties that may affect the partition behaviour in the environment. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No. 00981510 and the Norwegian Research Council project No.159113.
Abstracts / Marine Environmental Research 66 (2008) 80–84
Identification and functional characterisation of p-glycoprotein 1 (abcb1) in painter’s mussel (Unio pictorum) Jasna Medvedovi, Roko Zaja and Tvrtko Smital Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia
Abstract The P-glycoprotein 1 (P-gp1; ABCB1) is one of the major mediators of multixenobiotic resistance (MXR) phenotype found in aquatic organisms. Using energy obtained through binding and hydrolysis of ATP, P-gp1 catalyses active efflux of wide range of xenobiotics. Consequently, its inhibition could lead to higher accumulation of toxic compounds inside the cells. Although functional experiments using fluorescent substrates and inhibitors indicate that P-gp1 is frequently present in aquatic invertebrates, there is no clear evidence about the identity of genes involved in MXR of freshwater mussels. In this study we have cloned ABCB1 gene from gill tissue of the freshwater mussel Unio pictorum. Functional experiments using model P-gp inhibitors (cyclosporine, verapamil and reversine 205) and fluorescent substrates (rhodamine B and calcein-AM) demonstrated high P-gp1 like activity in gill tissue. The 23 fold increase in accumulation of both florescent substrates was observed when the gills discs were exposed to lower micromolar concentrations of model inhibitors. In addition, to find out whether some of environmentally relevant pollutants could inhibit P-gp1 activity in mussel, we exposed gill discs to several pharmaceuticals frequently present in freshwater ecosystems. Among tested pharmaceuticals, atrovastatin, tamoxifen and sildenafil resulted in markedly higher accumulation of fluorescent P-gp substrates, with sildenafil being the most potent (EC50 1.9 mM). In conclusion, our study demonstrated that ABCB1 gene and P-glycoprotein 1 activity are expressed in gill tissue of Painter’s mussel. Furthermore, some of the environmentally relevant pharmaceuticals could modulate the P-glycoprotein 1 activity in freshwater mussel. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No 00981510.
Multidrug resistance-associated protein (MRP) protects sea urchin embryos (Strongylocentrotus purpuratus) against mercuric chloride Ivana Bošnjak a, David Epel b, Ken Johnson c, Jasna Franeki Èoli a, Tvrtko Smital d, Amro M. Hamdoun b a Laboratory for Biology and Microbial Genetics, Department for Biochemical Engineering, University of Food Technology and Biotechnology, Zagreb, Croatia b Hopkins Marine Station, Stanford University, Oceanview Boulevard, Pacific Grove, California USA c Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California,USA d Laboratory for Molecular Ecotoxicology, Department for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia
Abstract Multidrug resistance-associated proteins (MRPs), or ATP-Binding Cassette C (ABCC) family transporters, are unique from other multidrug transporters in that they efflux both organics and heavy metal mercaptide complexes. To investigate the protective capacity of
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these transporters from mercury, a pollutant of major concern in marine environments, we examined the effects of mercuric chloride on sea urchin (Strongylocentrotus purpuratus) embryos, which have high levels of ABCC/MRP efflux activity. We found that mercuric chloride interferes with mitosis (EC50 837 nM) by inducing breakage of astral microtubules, thereby retarding or blocking metaphase and anaphase. Partial inhibition of the ABCC/MRP mediated efflux activity by MK571 (5 mM), a specific inhibitor of MRP efflux transporters, increases this toxicity of mercuric chloride nearly four-fold. Complete inhibition of MRP-mediated efflux activity with 40 mM MK571, in the presence of 500 nM mercuric chloride, results in accumulation of 63.58 mM intracellular mercury in the embryos; 11-fold higher than control (5.73 mM). To determine whether glutathione (GSH) is involved in mercury efflux we exposed embryos to both 500 nM mercuric chloride and 500 nM GSH substrate 1chloro-2,4-dinitrobenzene (CDNB). We observed a four-fold increase in mercury toxicity and a two-fold increase in accumulation suggesting efflux of mercury as a GSH complex. Our results underscore the importance of ABCC/MRP transporters in protection from mercury. Future work will examine the efficacy of ABCC transporters against other forms of mercury, such as methylmercury, and the efficacy of this protective mechanism in the presence of environmentally relevant chemosensitizers. This work was funded by grants NIH F32-HD47136, NSF 0446384 and Ministry of Science, Education and Sports of Republic of Croatia, Projects 058013 and 0098135.
Pharmaceuticals modulate efflux transporters (MDR and MRP proteins) in the fish cell line PLHC-1 Daniel Caminada a,b, Roko Zaja c, Tvrtko Smital c, Karl Fent a,d University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Institute of Ecopreneurship, Muttenz, Switzerland b University of Zürich, Institute of Plant Biology, Division of Limnology, Kilchberg, Switzerland c Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia d Swiss Federal Institute of Technology (ETH), Department of Environmental Sciences, Zürich, Switzerland
a
Abstract The ubiquitous presence of pharmaceuticals in aquatic systems is a challenging problem as their potential effects on aquatic organisms remain largely unknown. The ABC transport proteins contributing to the multidrug/multixenobiotic resistance (MDR/MXR) phenomenon seem to have an important role in the elimination of xenobiotics in aquatic organisms. Modulation of their efflux activities by contaminants may lead to substantial increases in intracellular accumulation and toxic effects of other xenobiotics. The aim of our work was to analyse a series of pharmaceuticals for their potential to modulate the activity of xenobiotic efflux transporters from the ABCB and ABCC sub-family in the PLHC-1 fish cell line. Cellular accumulation of the model fluorescent ABC substrates calcein AM, rhodamine123 and monochlorobimane were used to determine an inhibitory effect on efflux transporters. A number of pharmaceuticals had an inhibitory activity with IC50 values occurring in the lower micromolar to millimolar range. Further, cytotoxic effects of pharmaceuticals were analysed in doxorubicin resistant PLHC-1 cells characterized by an elevated expression of related ABC transporters. Co-exposure of resistant cells to the model MXR inhibitor cyclosporine A and different pharmaceuticals resulted in up to ten times increased cytotoxicity in comparison to cells exposed without inhibitor. Therefore, our work
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revealed significant inhibitory effects of environmentally relevant pharmaceuticals on ABC transporters, demonstrating that the PLHC-1 cells are an appropriate system for the investigation of MDR/MXR mechanisms in fish due to the presence of active and inducible MDR (ABCB) and MRP (ABCC) proteins in this in vitro system. Supported by KTI grant 7114.2 LSPP-LS to K. Fent, Springborn Smithers AG, Novartis International AG, F. Hoffmann-La Roche Ltd.
Selection of p-glycoprotein 1 (P-gp1; ABCB1) mediated high-level resistant fish cell line (PLHC-1) by doxorubicin Roko Zaja a, Jovica Lonèar a, Daniel Caminada b,c, Tvrtko Smital a Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia b University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Institute of Ecopreneurship, Muttenz, Switzerland c University of Zürich, Institute of Plant Biology, Division of Limnology, Kilchberg, Switzerland
a
Abstract The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in
aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in xenobiotic metabolism. Recently we demonstrated the presence of the ABC transport proteins possibly involved in the multixenobiotic resistance (MXR) mechanism. In this study we were able to select highly resistant PLHC-1 cells (PLHC/dox) by culturing them in a gradually increasing doxorubicin concentration. Using real-time PCR, a high expression of Abcb1 gene was measured in PLHC/dox cells compared to non-selected cells (PLHC/wt). Using MTT viability assay we showed that PLHC/dox are approximately 26 fold more resistant to doxorubicin than PLHC/wt. Measured EC50 values were 1 M and 26.1 mM for PLHC/dox cells and PLHC/wt, respectively. The P-glycoprotein 1 (P-gp1) inhibitor cyclosporine A (1 mM) completely reversed resistance of PLHC/dox cells to doxorubincin resulting in EC50 value for doxorubicin similar to the EC50 value found for PLHC/wt (1.4 mM). In contrast, MK571, a specific inhibitor of MRP1-3 (ABCC1-3) type of efflux transporters, did not sensitize PLHC/dox cells to doxorubicin. These data prove that specific, highly inducible P-gp1 mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. Furthermore, the fact that low micromolar concentrations of specific inhibitor may completely reverse a highly expressed doxorubicin resistance points to fragility of P-gp1 mediated (MXR) mechanism in fish. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No 00981510.
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) – Transporters and multixenobiotic resistance P-glycoprotein expression in killifish from the Sydney tar ponds, Nova Scotia, Canada S. Christine Paetzold a, Sara Zucchi b, Robert Richards c, Shannon Mala Bard a,d Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada b Department of Environmental Sciences ‘‘G. Sarfatti”, University of Siena, Siena, Italy c Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada d Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, Canada
a
Abstract We investigated the role of the multidrug resistance transporters P-glycoprotein (MDR1/ABCB1) and sister of P-glycoprotein (SPGP/ ABCB11), both ATP-binding cassette (ABC) proteins, in conferring multixenobiotic resistance in a wild population of killifish (Fundulus heteroclitus) from the highly contaminated Sydney Tar Ponds, Canada. P-glycoproteins act as transmembrane export pumps that prevent cellular accumulation of a wide range of moderately hydrophobic xenobiotics including anthropogenic contaminants. The interaction of P-glycoproteins with phase I and II drug-metabolizing enzymes and other ABC xenobiotic transporters in wild populations are poorly understood. We examined a killifish population living in the estuarine Sydney Tar Ponds that have been contaminated since the early 1900s with polycyclic aromatic hydrocarbons (PAHs) from industrial activities adjacent to the site. PAHs and their phase I metabolites are potential P-glycoprotein substrates and inducers. We investigated whether P-glycoprotein expression was elevated in the liver of killifish from the Sydney Tar Ponds using quantitative reverse-transcription polymerase chain reaction. Cytochrome P450 1A (a phase I metabolizing enzyme) transcript abundance was assessed as indicator of PAH exposure. We also quantified gene expression of the complementary ABC transporters multidrug resistance-associated protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) to understand their interaction with P-glycoproteins. MRP2 and BCRP predominantly transport phase II metabolites but also have some substrate overlap with P-glycoproteins. Gene expression of the phase II enzyme glutathione-Stransferase was also quantified since its metabolites are exported by MRP2 and BCRP. Our aim was to elucidate the cellular and molecular mechanisms that contribute to resistance to PAH contaminants.
0141-1136/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marenvres.2008.02.029
Complexity of multixenobiotic resistance - PGP1 (ABCB1), MRP2 (ABCC2) and BSEP (ABCB11) efflux transporters in primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes Roko Zaja a, Vesna Munic b, Andreja Ambriovi Ristov b, Roberta Sauerborn Klobuèar a, Tvrtko Smital a a Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Bijenicka 54, 10000 Zagreb, Croatia b Laboratory for Genotoxic Agents, Division of Molecular Biology, Rułer Boškovi Institute, Bijenicka 54, 10000 Zagreb, Croatia; GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovica 29, 10000 Zagreb, Croatia
Abstract The extrusion of hydrophobic xenobiotics and their metabolites in fish liver could be mediated by the activities of several types of ABC transport proteins, together contributing to the multidrug/multixenobiotic resistance (MDR/MXR). In order to test this hypothesis the main goal of this study was detection and functional characterization of toxicologically relevant efflux transporters belonging to the ABCB and ABCC sub-family in primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. Using specific pairs of primers, we have identified and cloned three full length nucleotide sequences, which share a high degree of homology with mammalian Pgp1 (ABCB1), MRP2 (ABCC2) and BSEP (ABCB11) efflux transporters, respectively. The expression of identified efflux transporters was quantified in both trout liver tissue and primary hepatocytes using the real-time PCR technique. The levels of all three transcripts were approximately 4 times lower in primary hepatocytes in comparison to intact liver, and the same relative expression pattern (ABCB11>>ABCC2>ABCB1) was determined in both tissues/compartments. In addition, transport activities of identified ABC transporters have been demonstrated using model substrates (rhodamine123, calcein-AM, bodipy-verapamil, dihydrofluorescein diacetate) and inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurochenodeoxycholate, taurocholate) specific for corresponding transport proteins. Taken together these findings show that MDR/MXR is an integral part of the detoxification system in rainbow trout hepatocytes, that is mediated by several ABC transport proteins with different and/or overlapping substrate specificities. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No. 00981510.
Abstracts / Marine Environmental Research 66 (2008) 80–84
Cost, effectiveness, and environmental relevance of multi-drug transporters Bryan Cole, David Epel Hopkins Marine Station, Stanford University, Pacific Grove, CA, USA
Abstract Efflux transporters of the ABCB and ABCC families protect cells from xenobiotics and remove endogenous byproducts of detoxification. We use embryos of the sea urchin, Strongylocentrotus purpuratus, as a model for determining the cost and environmental relevance of this protection system. Analysis of developmental mRNA shows expression of all 30 ABCC genes and all 12 members of the PGP (ABCB) family. We have quantified the cost of efflux transporters as a first-line of defense in terms of activity and infrastructure costs; respectively, these represent the direct ATP cost to power efflux and the indirect ATP cost of synthesizing these proteins. Activity costs are low, utilizing only 0.074% of ATP production, while still reducing toxicant accumulation at micromolar concentrations. Infrastructure costs for the PGP subfamily were estimated by quantitative Western Blot analysis. We found 6.3X108 molecules of PGP-type proteins per embryo, equivalent to approximately 0.9% of total embryonic protein. As a first-line of defense against toxicants, the activity cost of efflux transporters is low. However, the infrastructure cost of synthesizing this amount of protein is comparatively high; infrastructure costs are equivalent to over 32 hours of constant maximal transporter activity. The large number of developmentally expressed transporter genes and the large amount of corresponding protein maternally loaded into the oocyte point towards an increased reliance on these proteins during development. This reliance is especially remarkable considering the high infrastructure costs, and that a female sea urchin produces millions of oocytes, each loaded with such a high amount of toxicant defense proteins. Supported by the California Sea Grant, the National Science Foundation, and Stanford’s Department of Neonatology.
Development of a behavioural toxicological assay to measure P-glycoprotein activity at the blood-brain barrier and evaluate the neuropathological effects of environmental contaminants Shannon Mala Bard a, Simon Gadbois b Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, B3H 4J1, Canada b Psychology and Neuroscience, Dalhousie University, Halifax, Nova Scotia, B3H 4J1, Canada
a
Abstract The neuroprotective role of P-glycoprotein, a multixenobiotic resistance transporter (ABCB1/MDR1), in the blood-brain barrier in fish was examined using behavioural toxicological assays. P-glycoproteins act as cellular efflux pumps to prevent substrates from accumulating in the brain, including environmental contaminants such as ivermectin, a common aquaculture pesticide and
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mammalian anti-parasitic drug. The behavioural toxicological assays were developed to determine the neuropathological effect of ivermectin in killifish (Fundulus heteroclitus). P-glycoprotein function and thus blood-brain barrier integrity can be compromised by chemosensitizers, such as cyclosporin A, which inhibit transport activity. Fish treated with ivermectin and the P-glycoprotein inhibitor cyclosporin A significantly succumbed more rapidly and were >2-fold as sensitive to tilting, lethargy, slowing of pectoral fin movement and loss of haptic reactivity compared to fish treated with ivermectin only. P-glycoprotein inhibition is associated with significantly earlier onset and increased mortality (~3-fold) in ivermectin-exposed fish. Our results suggest that Pglycoprotein confers resistance against ivermectin-induced behavioural neuropathology and mortality in fish. We then used this assay to evaluate emamectin benzoate, an ivermectin-derivative with fewer side effects and shorter half-life in fish tissue. Compared to ivermectin-treated fish, neuropathological symptoms were of substantially lower severity and mortality was greatly reduced in emamectin benzoate-treated fish. Whether these trends are due to lower inherent toxicity or increased substrate affinity for P-glycoprotein and thus increased elimination is currently under investigation. This assay provides us with a non-invasive tool to study P-glycoprotein function in the blood-brain barrier and evaluate the behavioural effects of potential environmental neurotoxins.
Zebrafish embryos are sensitized towards chemicals by efflux transporter inhibitors Till Luckenbach, Mady Ahnert, Stephan Fischer, Kristin Schirmer UFZ - Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany
Abstract ABC efflux pumps are part of the cellular toxic defensome in many aquatic organisms. Certain environmental contaminants have been shown to antagonize the activity of those xenobiotic transporters. Therefore, there is concern that due to the presence of such chemosensitizers in the environment, susceptibility of cells and organisms could be increased for toxicants that would normally be removed by this mechanism. We used zebrafish (Danio rerio) embryos in order to determine the toxicological impact of chemosensitizers on the organism level. Using RT-PCR we found expression of various xenobiotic transporters from the ABCB, ABCC and ABCG families in zebrafish at stages from 6 to 48 hrs post fertilization. In toxicological experiments the LC50 (48 hrs) for vinblastine, a known toxic substrate of efflux transporters, was 21 % lower (2.8 vs. 2.2 uM) in the presence of the known transporter inhibitor cyclosporine A. Cyclosporine A alone did not show any toxic effects. Similarly, galaxolide significantly enhanced sensitivity of the embryos to vinblastine. Galaxolide, commonly used as a fragrance in hygiene and personal care products, is an environmentally relevant micropollutant with transporter modulating properties. Our experiments show that (1) efflux transporters appear to be an important part of the toxic defense in zebrafish embryos and (2) chemosensitization can be a relevant component in the toxicity of chemical mixtures on zebrafish. Finally, our data further the ecotoxicological relevance of chemosensitisation by environmental contaminants.
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Gene expression of multixenobiotic resistance transporters p-glycoprotein and mpr2 in the antarctic emerald rockcod (Trematomus bernacchii) Sara Zucchi a, Ilaria Corsi a, Shannon Mala Bard b,c, Francesco Regoli d, Robert Richards e, Christine S. Paetzold b, Silvano Focardi a a Department of Environmental Sciences ‘‘G. Sarfatti”, University of Siena, Siena, Italy b Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada c Environmental Programmes, Dalhousie University, Halifax, Nova Scotia, Canada d Istituto di Biologia e Genetica, Ecotossicologia Marina, Università Politecnica delle Marche, Via Ancona, Italy e National Research Council, Institute for Marine Biosciences, Halifax, NS, Canada
Abstract We examined the gene expression of P-glycoprotein (P-gp/MDR1/ ABCB1) and Multidrug Resistance-associated Protein 2 (MRP2/ ABCC2) in Trematomus bernacchii, the Antarctic emerald rockcod, experimentally exposed to organic contaminants and heavy metals. P-gp and MRP2, ATP-binding cassette (ABC) proteins, are xenobiotic transporters which respectively facilitate the transmembrane efflux of moderately hydrophobic substrates and phase II metabolite glutathione conjugates. We analyzed T. bernacchii because this widely distributed benthic feeder is naturally exposed to high levels of cadmium from the local Antarctic geology and is a key sentinel species for monitoring the Antarctic ecosystem. Our main interest is to assess the sensitivity of T. bernacchii to anthropogenic and natural chemicals. In contaminated exposed fish we investigated the expression of the two transporters that play a role in absorption, disposition and elimination of a wide variety of xenobiotics. We evaluated fish (N=8) exposed by intraperitoneal injection to either a single contaminant or pairs of contaminants from the following list: benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, cadmium, copper and lead. Partial sequences of P-gp (410 nucleotides) and MRP2 (750 nucleotides) were obtained and used to design primers to assess hepatic P-gp and MRP2 gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in exposed fish. To determine whether P-gp and MRP2 hepatic gene expression reflects protein expression in the liver, we evaluated protein levels by Western blot. This study provides a preliminary characterization of genes involved in cellular defense mechanisms in the key sentinel species T. bernacchii and evaluates whether anthropogenic and natural compounds could modulate P-gp and MRP2 expression.
Biocomplexity of multidrug resistance related transport in the shore crab Carcinus maenas - job sharing of organs in toxin elimination? Angela Koehler, Sieglinde Bahns Department of Biosciences, Alfred Wegener Institute for Polar and Marine Research, Germany
Abstract MXR transport activity was measured in living gills with CalceinAM and quantified by computerised image analysis. Simultaneously, we detected MXR protein expression with an antibody C 219 detecting evolutionary conserved amino acid sequences of various MXR transporters. Shore crabs adapted to 10ppt and 33ppt seawater according to the natural salinity range were studied for MXR transport activity and for protein expression in gills and digestive glands.
Animals adapted to low salinities had a 30% higher transport efficiency, possibly due to membrane up folding, inferring that these efflux pumps may also be involved in osmo-regulation. Animals were exposed to AAF (0.01M), a specific model inducer of the transmembrane P-glycoprotein involved in MXR transport over a period of 3 days and showed a 50% induction of transport activity in gills at both salinities. Additionally, animals were starved for 4 weeks, re-fed once, and samples taken after 4, 8, 12, 24 and 48 hours according to cell differentiation of the digestive gland. In the digestive glands, MXR proteins appeared in differentiating blister cells 8 hours after re-feeding as immuno-localised with the AB C219. Simultaneously, food supply induced a complete breakdown of MXR transport activity in gills after 8 h. MXR-like transporters, presumably P-gp, appear to exist in shore crab as transport can be specifically induced by a model inducer. Furthermore, gills and hepatopancreas appear to perform a sort of physiological job sharing in which the hepatopancreas overtakes xenobiotic elimination during periods the when gills are involved to serve enhanced oxygen demands during digestion and active ammonia excretion.
Effluents from oil production platforms in the north sea inhibit the function of the abc transporters mdr1 and mrp1 in vitro Branka Pivèevi a, Tvrtko Smital a, Knut-Erik Tollefsen b Laboratory for Molecular Ecotoxicology, Division for EnvironmentalResearch, Rułer Boškovi Institute, Zagreb, Croatia b Norwegian Institute for Water Research (NIVA), Oslo, Norway
a
Marine
and
Abstract Inhibition of multixenobiotic resistance (MXR) defence by various environmental contaminants in aquatic organisms may cause significant increases in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by ABC transport proteins. In this study the inhibition potency of production water samples from North Sea offshore oil platforms on the efflux activity of the ABC transporters Pgp1 (ABCB1) and MRP1 (ABCC1) was examined using an in vitro calcein-AM bioassay with HL60 cells stably transfected with either human ABCB1 or ABCC1 genes. Transfected cells are characterized by an efficient efflux and low intracellular accumulation of calcein-AM, which can be modulated by numerous different inhibitors. The inhibition potency of the samples was expressed as inhibiting equivalents (EQ) of the model inhibitors reversin 205 (REV) for Pgp1 and MK571 for the MRP1 transporters. Production water samples were collected from ten oil and gas production platforms with different chemical composition, extracted by large volume solid phase extraction, and prepared as a particulate/oil fraction and a water soluble fraction. The majority of samples had measurable concentrations of Pgp1 inhibitors (range: 37 – 380 mg/ L REV EQs), and the Pgp1 inhibiting activity was predominantly found in the water fraction of produced water. MRP1 inhibitors were determined in all samples with highest concentration in extracts of particulate/oil fraction of produced water (range: 88 – 1400 mg/L MK571 EQs). These results indicate that there are more inhibitors of MDR1 than of Pgp1 transporter in produced water, and that the active chemicals exhibit different physico-chemical properties that may affect the partition behaviour in the environment. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No. 00981510 and the Norwegian Research Council project No.159113.
Abstracts / Marine Environmental Research 66 (2008) 80–84
Identification and functional characterisation of p-glycoprotein 1 (abcb1) in painter’s mussel (Unio pictorum) Jasna Medvedovi, Roko Zaja and Tvrtko Smital Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia
Abstract The P-glycoprotein 1 (P-gp1; ABCB1) is one of the major mediators of multixenobiotic resistance (MXR) phenotype found in aquatic organisms. Using energy obtained through binding and hydrolysis of ATP, P-gp1 catalyses active efflux of wide range of xenobiotics. Consequently, its inhibition could lead to higher accumulation of toxic compounds inside the cells. Although functional experiments using fluorescent substrates and inhibitors indicate that P-gp1 is frequently present in aquatic invertebrates, there is no clear evidence about the identity of genes involved in MXR of freshwater mussels. In this study we have cloned ABCB1 gene from gill tissue of the freshwater mussel Unio pictorum. Functional experiments using model P-gp inhibitors (cyclosporine, verapamil and reversine 205) and fluorescent substrates (rhodamine B and calcein-AM) demonstrated high P-gp1 like activity in gill tissue. The 23 fold increase in accumulation of both florescent substrates was observed when the gills discs were exposed to lower micromolar concentrations of model inhibitors. In addition, to find out whether some of environmentally relevant pollutants could inhibit P-gp1 activity in mussel, we exposed gill discs to several pharmaceuticals frequently present in freshwater ecosystems. Among tested pharmaceuticals, atrovastatin, tamoxifen and sildenafil resulted in markedly higher accumulation of fluorescent P-gp substrates, with sildenafil being the most potent (EC50 1.9 mM). In conclusion, our study demonstrated that ABCB1 gene and P-glycoprotein 1 activity are expressed in gill tissue of Painter’s mussel. Furthermore, some of the environmentally relevant pharmaceuticals could modulate the P-glycoprotein 1 activity in freshwater mussel. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No 00981510.
Multidrug resistance-associated protein (MRP) protects sea urchin embryos (Strongylocentrotus purpuratus) against mercuric chloride Ivana Bošnjak a, David Epel b, Ken Johnson c, Jasna Franeki Èoli a, Tvrtko Smital d, Amro M. Hamdoun b a Laboratory for Biology and Microbial Genetics, Department for Biochemical Engineering, University of Food Technology and Biotechnology, Zagreb, Croatia b Hopkins Marine Station, Stanford University, Oceanview Boulevard, Pacific Grove, California USA c Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California,USA d Laboratory for Molecular Ecotoxicology, Department for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia
Abstract Multidrug resistance-associated proteins (MRPs), or ATP-Binding Cassette C (ABCC) family transporters, are unique from other multidrug transporters in that they efflux both organics and heavy metal mercaptide complexes. To investigate the protective capacity of
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these transporters from mercury, a pollutant of major concern in marine environments, we examined the effects of mercuric chloride on sea urchin (Strongylocentrotus purpuratus) embryos, which have high levels of ABCC/MRP efflux activity. We found that mercuric chloride interferes with mitosis (EC50 837 nM) by inducing breakage of astral microtubules, thereby retarding or blocking metaphase and anaphase. Partial inhibition of the ABCC/MRP mediated efflux activity by MK571 (5 mM), a specific inhibitor of MRP efflux transporters, increases this toxicity of mercuric chloride nearly four-fold. Complete inhibition of MRP-mediated efflux activity with 40 mM MK571, in the presence of 500 nM mercuric chloride, results in accumulation of 63.58 mM intracellular mercury in the embryos; 11-fold higher than control (5.73 mM). To determine whether glutathione (GSH) is involved in mercury efflux we exposed embryos to both 500 nM mercuric chloride and 500 nM GSH substrate 1chloro-2,4-dinitrobenzene (CDNB). We observed a four-fold increase in mercury toxicity and a two-fold increase in accumulation suggesting efflux of mercury as a GSH complex. Our results underscore the importance of ABCC/MRP transporters in protection from mercury. Future work will examine the efficacy of ABCC transporters against other forms of mercury, such as methylmercury, and the efficacy of this protective mechanism in the presence of environmentally relevant chemosensitizers. This work was funded by grants NIH F32-HD47136, NSF 0446384 and Ministry of Science, Education and Sports of Republic of Croatia, Projects 058013 and 0098135.
Pharmaceuticals modulate efflux transporters (MDR and MRP proteins) in the fish cell line PLHC-1 Daniel Caminada a,b, Roko Zaja c, Tvrtko Smital c, Karl Fent a,d University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Institute of Ecopreneurship, Muttenz, Switzerland b University of Zürich, Institute of Plant Biology, Division of Limnology, Kilchberg, Switzerland c Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia d Swiss Federal Institute of Technology (ETH), Department of Environmental Sciences, Zürich, Switzerland
a
Abstract The ubiquitous presence of pharmaceuticals in aquatic systems is a challenging problem as their potential effects on aquatic organisms remain largely unknown. The ABC transport proteins contributing to the multidrug/multixenobiotic resistance (MDR/MXR) phenomenon seem to have an important role in the elimination of xenobiotics in aquatic organisms. Modulation of their efflux activities by contaminants may lead to substantial increases in intracellular accumulation and toxic effects of other xenobiotics. The aim of our work was to analyse a series of pharmaceuticals for their potential to modulate the activity of xenobiotic efflux transporters from the ABCB and ABCC sub-family in the PLHC-1 fish cell line. Cellular accumulation of the model fluorescent ABC substrates calcein AM, rhodamine123 and monochlorobimane were used to determine an inhibitory effect on efflux transporters. A number of pharmaceuticals had an inhibitory activity with IC50 values occurring in the lower micromolar to millimolar range. Further, cytotoxic effects of pharmaceuticals were analysed in doxorubicin resistant PLHC-1 cells characterized by an elevated expression of related ABC transporters. Co-exposure of resistant cells to the model MXR inhibitor cyclosporine A and different pharmaceuticals resulted in up to ten times increased cytotoxicity in comparison to cells exposed without inhibitor. Therefore, our work
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revealed significant inhibitory effects of environmentally relevant pharmaceuticals on ABC transporters, demonstrating that the PLHC-1 cells are an appropriate system for the investigation of MDR/MXR mechanisms in fish due to the presence of active and inducible MDR (ABCB) and MRP (ABCC) proteins in this in vitro system. Supported by KTI grant 7114.2 LSPP-LS to K. Fent, Springborn Smithers AG, Novartis International AG, F. Hoffmann-La Roche Ltd.
Selection of p-glycoprotein 1 (P-gp1; ABCB1) mediated high-level resistant fish cell line (PLHC-1) by doxorubicin Roko Zaja a, Jovica Lonèar a, Daniel Caminada b,c, Tvrtko Smital a Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rułer Boškovi Institute, Zagreb, Croatia b University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Institute of Ecopreneurship, Muttenz, Switzerland c University of Zürich, Institute of Plant Biology, Division of Limnology, Kilchberg, Switzerland
a
Abstract The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in
aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in xenobiotic metabolism. Recently we demonstrated the presence of the ABC transport proteins possibly involved in the multixenobiotic resistance (MXR) mechanism. In this study we were able to select highly resistant PLHC-1 cells (PLHC/dox) by culturing them in a gradually increasing doxorubicin concentration. Using real-time PCR, a high expression of Abcb1 gene was measured in PLHC/dox cells compared to non-selected cells (PLHC/wt). Using MTT viability assay we showed that PLHC/dox are approximately 26 fold more resistant to doxorubicin than PLHC/wt. Measured EC50 values were 1 M and 26.1 mM for PLHC/dox cells and PLHC/wt, respectively. The P-glycoprotein 1 (P-gp1) inhibitor cyclosporine A (1 mM) completely reversed resistance of PLHC/dox cells to doxorubincin resulting in EC50 value for doxorubicin similar to the EC50 value found for PLHC/wt (1.4 mM). In contrast, MK571, a specific inhibitor of MRP1-3 (ABCC1-3) type of efflux transporters, did not sensitize PLHC/dox cells to doxorubicin. These data prove that specific, highly inducible P-gp1 mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. Furthermore, the fact that low micromolar concentrations of specific inhibitor may completely reverse a highly expressed doxorubicin resistance points to fragility of P-gp1 mediated (MXR) mechanism in fish. This work has been supported by the Ministry of Science and Technology of the Republic of Croatia, Project No 00981510.