verlukast and reversin-205

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.

Topics: Animals; ATP-Binding Cassette Transporters; Base Sequence; DNA, Complementary; Dreissena; Gills; Models, Theoretical; Molecular Sequence Data; Oligopeptides; Phylogeny; Propionates; Quinolines; Sequence Analysis, DNA; Verapamil

This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity of QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl2 at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd(2+) and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters.

Topics: Animals; ATP-Binding Cassette Transporters; Cadmium Compounds; Cell Line; Cell Survival; Cyclosporine; Dogs; Hep G2 Cells; Humans; Kidney; Liver; Madin Darby Canine Kidney Cells; Oligopeptides; Probenecid; Propionates; Quantum Dots; Quinolines; Rifampin; RNA, Messenger; Tellurium

Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8-9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without the model inhibitors verapamil (unspecific ABC inhibitor), reversin 205 (ABCB1 inhibitor) and MK571 (ABCC inhibitor). Juvenile D. magna possessed all examined abcb and abcc transcripts, but only reversin 205 inhibited MXR activity. The MXR activity in L. variegatus was inhibited by MK571, and to a lesser extent by verapamil, whereas reversin 205 seemed to stimulate the transport activity. Whereas calcein-AM worked better as an MXR substrate in D. magna, rhodamine B was a better substrate for L. variegatus MXR activity measurements. This is the first report on MXR activity in the order Lumbriculida, subclass Oligochaeta, and class Clitellata.

Topics: Animals; Arthropod Proteins; ATP-Binding Cassette Transporters; Daphnia; Environmental Monitoring; Environmental Pollutants; Oligochaeta; Oligopeptides; Propionates; Quinolines; Real-Time Polymerase Chain Reaction; Verapamil

Embryos of marine organisms whose development occurs externally are particularly sensitive to ultraviolet (UV) light (bands A and B, respectively, UVA and UVB). ATP-binding cassette (ABC) transporters are the first line of cellular defense against chemical or physical stress. The present work investigated the involvement of ABC transporters on UVA or UVB effects on eggs, spermatozoa, and embryonic cells of the sea urchin Echinometra lucunter. Gametes or embryos were exposed to UVA (3.6-14.4 kJ m(-2)) or UVB (0.112-14.4 kJ m(-2)), and embryonic development was monitored by optical microscopy at different developmental stages in the presence or absence of the ABC-transporter blockers reversin205 (ABCB1 blocker) or MK571 (ABCC1 blocker). E. lucunter eggs, spermatozoa and embryos were resistant to UVA exposure. Resistance to the harmful effects of UVB was strongly associated to ABC transporter activity (embryos > eggs > spermatozoa). ABCB1 or ABCC1 blockage promoted the injurious effects of UVA on spermatozoa. ABCC1 transporter blockage increased UVB-dependent damage in eggs while ABCB1 transporter inhibition increased harmful effects of UVB in embryonic cells. ABC-transporter activity was not, however, affected by UVB exposure. In conclusion, the present study is the first report on the protective role of ABC transporters against harmful effects of UVA and UVB on sea urchin eggs and embryonic cells.

Topics: Analysis of Variance; Animals; ATP-Binding Cassette Transporters; Embryonic Development; Embryonic Stem Cells; Germ Cells; Microscopy; Oligopeptides; Propionates; Quinolines; Sea Urchins; Ultraviolet Rays

Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

Topics: Acrosome; Acrosome Reaction; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Calcium Channel Blockers; Fertilization; Fluoresceins; Leukotriene Antagonists; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; Oligopeptides; Organic Anion Transporters; Propionates; Quinolines; Sea Urchins; Spermatozoa; Verapamil

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1-3d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Cyclosporine; Dreissena; Drug Resistance, Multiple; Drug Tolerance; Embryo, Nonmammalian; Indans; Larva; Oligopeptides; Propionates; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Rhodamines; Risk Assessment; Verapamil; Xenobiotics