Multixenobiotic Resistance System Research Articles

Multixenobiotic defence mechanism in native and exotic freshwater snails as a biomarker for land uses-changes

Human activities such as agriculture and urbanization generate a large number of substances like personal care products, pharmaceutical compounds, and pesticides, which often reach aquatic environments and represent a threat to biodiversity. Many organisms have developed different evolutionary strategies to remove pervasive substances from their bodies, allowing them to persist even in polluted environments, and one of these is the multixenobiotic resistance (MXR) mechanism associated with the expression of membrane proteins like P-glycoprotein (P-gp). Numerous chemical compounds with diverse functions and structures can modulate this mechanism, which can be employed as a pollution biomarker. We examined the MXR activity in two species of snails that inhabit Patagonian freshwaters. Functional assay measurements of MXR were conducted on the native Chilina dombeiana and the exotic Physella acuta in stream reaches affected by anthropogenic impacts. Results indicated that at agricultural sites, C. dombeiana snails had a more active MXR system than organisms sampled at reference and moderately disturbed urban sites, whereas P. acuta snails from agricultural and highly disturbed urban sites showed better detoxifying activity than organisms from reference sites. Only in exotic snails, part of this activity was due to the action of P-gp. The most important environmental variables explaining MXR activity were ammonium, nitrate and nitrite, phosphates, and electrical conductivity. These results show the promise of measuring MXR activity in native and exotic snails, as a biomarker in the environmental monitoring of Patagonian freshwaters.

Expression Analyses of Genes Related to Multixenobiotic Resistance in Mytilus galloprovincialis after Exposure to Okadaic Acid-Producing Dinophysis acuminata.

The mussel Mytilus galloprovincialis is one of the most important aquaculture species in Europe. Its main production problem is the accumulation of toxins during coastal blooms, which prevents mussel commercialization. P-glycoprotein (ABCB1/MDR1/P-gp) is part of the multixenobiotic resistance system in aquatic organisms, and okadaic acid, the main DSP toxin, is probably a substrate of the P-gp-mediated efflux. In this study, the presence and possible role of P-gp in the okadaic acid detoxification process was studied in M. galloprovincialis. We identified, cloned, and characterized two complete cDNAs of mdr1 and mdr2 genes. MgMDR1 and MgMDR2 predicted proteins had the structure organization of ABCB full transporters, and were identified as P-gp/MDR/ABCB proteins. Furthermore, the expression of mdr genes was monitored in gills, digestive gland, and mantle during a cycle of accumulation-elimination of okadaic acid. Mdr1 significantly increased its expression in the digestive gland and gills, supporting the idea of an important role of the MDR1 protein in okadaic acid efflux out of cells in these tissues. The expression of M. galloprovincialis mrp2, a multidrug associated protein (MRP/ABCC), was also monitored. As in the case of mdr1, there was a significant induction in the expression of mrp2 in the digestive gland, as the content of okadaic acid increased. Thus, P-gp and MRP might constitute a functional defense network against xenobiotics, and might be involved in the resistance mechanisms to DSP toxins.

Measurement of multixenobiotic resistance activity in enchytraeids as a tool in soil ecotoxicology

The multixenobiotic resistance (MXR) mechanism is the first defense line against xenobiotics. Enchytraeids, a model organism in soil ecotoxicology, are often exposed to various xenobiotics, some of which may influence MXR activity. Since MXR activity has not been studied in these organisms, the aim of this paper was to establish a methodology for the implementation of the dye assay in enchytraeids. Enchytraeus albidus and Enchytraeus crypticus were exposed to model chemosensitizers: cyclosporine A (CA), dexamethasone (DEX), ivermectin (IVM), rifampicin (RIF), verapamil (VER), and fungicide propiconazole (PCZ). Thereafter, a dye assay with specific fluorescent dyes rhodamine B and rhodamine 123 was performed. Changes in MXR activity caused by variations in dye accumulation were measured fluorometrically. CA, IVM, and VER were found to inhibit the MXR system and increase the fluorescence 2.2-fold, while DEX and RIF induced the MXR system and decreased the fluorescence. CA was the strongest inhibitor in both E. albidus (IC50 5.48 ± 1.25 μM) and E. crypticus (IC50 5.20 ± 3.10 μM). In the validation experiment, PCZ was found to inhibit the MXR system. The IC50 varied between species and exposure substrates: water (E. albidus – IC50 0.74 ± 0.24 mg/L; E. crypticus – 1.31 ± 0.24 mg/L) or soil (E. albidus – 1.79 ± 0.42 mg/kg; E. crypticus – 1.79 ± 0.17 mg/kg). In conclusion, the tested compounds changed the MXR activity, which confirms the applicability of this method as a valuable complementary biomarker in soil ecotoxicology.

Both quiescent and proliferating cells circulate in the blood of the invasive apple snail Pomacea canaliculata

Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients’ circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψm disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06–0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.

Effect of imidacloprid on the behavior, antioxidant system, multixenobiotic resistance, and histopathology of Asian freshwater clams (Corbicula fluminea)

In the current study, to investigate the effect of imidacloprid on benthic bivalves, adult Asian clams (Corbicula fluminea) were exposed to 0, 20, 200, and 2000 μg/L imidacloprid for 30 d. Imidacloprid significantly inhibited the siphoning and burrowing behaviour (p < 0.05) of the clams. Significant histopathological changes were associated with degeneration of the cilium, the contraction and adhesion of the lymphocyte, and the swelling of epithelium cells in gills, and there was notable degeneration in the digestive tubules, haemolytic infiltration in the connective tissue and epithelial cell necrosis in the digestive glands in the 2000 μg/L treatment group. The activity of AChE in the digestive glands was significantly inhibited at all treatment levels, whereas this inhibition was observed in gills only in the 2000 μg/L treatment (p < 0.05). Additionally, indicators of the antioxidant system (e.g., SOD, CAT, and GST activity) and MDA content were significantly increased in the gills and digestive glands with all treatments (p < 0.05). Moreover, the mRNA expression levels of Hsp genes (hsp 22, hsp 40, hsp 60, hsp 70, hsp 90) and multixenobiotic resistance (MXR) system-related genes (abcb1, abcc1) were significantly downregulated (p < 0.05). Therefore, our results suggest that imidacloprid changes the oxidative stress, cellular detoxification, and MXR system of C. fluminea. Our findings provide new insights into the effects of neonicotinoids on benthic bivalves such as C. fluminea.

The Multixenobiotic resistance system as a possible protective response triggered by microplastic ingestion in Mediterranean mussels (Mytilus galloprovincialis): Larvae and adult stages

The emerging paradigm on plastic pollution in marine environments is that microsize particles (MPs) have far more subtle effects than bigger fragments, given their size range overlapping with that of particles ingested by filter-feeders. The impacts include gut blockage, altered feeding and energy allocation, with knock-on effects on widespread physiological processes. This study investigated whether ingestion of polystyrene MPs (PS-MPs) triggers protective processes in marine mussels. The Multixenobiotic resistance (MXR) system is a cytoprotective mechanism acting as an active barrier against harmful xenobiotics and a route of metabolite detoxification. Both larvae and adults were employed in laboratory experiments with different concentrations of 3-μm PS-MPs (larvae), and 3-μm and 45-μm PS-MPs (adults) matching size range of planktonic food through the mussel lifecycle. Embryos grown in the presence of 3-μm PS-MPs showed significant reduction of MXR activity and down-regulation of ABCB and ABCC transcripts encoding the two main MXR-related transporters P-glycoprotein and the Multidrug resistance-related protein, respectively. In adults, effects of PS-MPs were assessed in haemocytes and gills, which showed different modulation of MXR activity and ABCB/ABCC expression according to MP size (haemocyte and gills) or particle concentration (haemocyte). These data showed that modulation of MXR activity is part of a generalized response triggered by particle ingestion.

Neonicotinoid insecticides are potential substrates of the multixenobiotic resistance (MXR) mechanism in the non-target invertebrate, Dreissena sp.

Mussels are among the most frequently used invertebrate animals in aquatic toxicology to detect toxic exposure in the environment. The presence and activity of a cellular defence system, the multixenobiotic resistance (MXR) mechanism, was also established in these organisms.In isolated gill tissues of dreissenid mussels (D. bugensis) the MXR activity was assayed after treatment by commercially available insecticides (formulated products) which contain neonicotinoids as their active ingredients: Actara (thiamethoxam), Apacs (clothianidin), Calypso (thiacloprid) and Kohinor (imidacloprid), respectively. While applying the accumulation assay method, 0.5 μM rhodamine B was used as model substrate and 20 μM verapamil as model inhibitor of the MXR mechanism.In acute (in vitro) experiments when isolated gills were co-incubated in graded concentrations of insecticides and rhodamine B simultaneously, Calypso and Kohinor treatment resulted increasing rhodamine accumulation. Chemical analysis of gills in vitro incubated in insecticides demonstrated higher tissue concentrations of thiamethoxam, clothianidin and thiacloprid in the presence of verapamil suggesting that the active ingredients of Actara, Apacs and Calypso are potential substrates of the MXR mediated cellular efflux. In contrast, verapamil did significantly alter the accumulated imidacloprid concentrations in gills, suggesting that the active component of Kohinor is not transported by the MXR mechanism.Chronic (in vivo) exposures of the intact animals in lower, 1, 10 mg/L concentration of neonicotinoid products, resulted in a decreased level of both rhodamine accumulation and verapamil inhibition by the 12th–14th days of treatment. These results suggest an enhancement of MXR activity (chemostimulation), building up gradually in the animals exposed to Actara, Apacs and Kohinor, respectively.Neonicotinoid-type insecticides are generally considered as selective neurotoxins for insects, targeting the nicotinic type acetylcholine receptors (nAChRs) in their central nervous system. Our present results provide the first evidences that neonicotinoid insecticides are also able to alter the transmembrane transport mechanisms related to the MXR system.

Styrene impairs normal embryo development in the Mediterranean mussel (Mytilus galloprovincialis)

This study analysed the effects of styrene, a main monomer in plastic manufacturing and acknowledged to be amongst the most common plastic leachates, on early embryo development of the Mediterranean mussel. Embryotoxicity tests showed that styrene impaired normal embryo development at concentrations (0.01 μg/L–1 mg/L) encompassing the environmental range. Occurrence of normal D-veligers was significantly reduced up to 40% of the total, and larval size was reduced of about 20%. D-veligers grown in the presence of styrene (0.1 and 10 μg/L) showed significant reduction of total Multixenobiotic resistance (MXR) efflux activity that was not apparently related to transcriptional expression of genes encoding P-glycoprotein (ABCB) and Mrp (ABCC), the two main ABC transporters of embryonal MXR system. Indeed, ABCB transcription was not affected by styrene, while ABCC was up-regulated. At these same concentrations, transcriptional profiles of 15 genes underlying key biological functions in embryo development and potential targets of adverse effects of styrene were analysed. Main transcriptional effects were observed for genes involved in shell biogenesis and lysosomal responses (down-regulation), and in neuroendocrine signaling and immune responses (up-regulation). On the whole, results indicate that styrene may affect mussel early development through dysregulation of gene transcription and suggest the possible conservation of styrene mode of action across bivalve life cycle and between bivalves and humans, as well as through unpredicted impacts on protective systems and on shell biogenesis.

Halogen-free organophosphorus flame retardants caused oxidative stress and multixenobiotic resistance in Asian freshwater clams (Corbicula fluminea)

Halogen-free organophosphorus flame retardants are widespread in aquatic environments. Although it has been documented that they affect the behavior and reproduction of aquatic species, researches investigating cellular detoxification and the defense system in bivalves are scarce. In this study, adult Asian clams (C. fluminea) were exposed to tris (2-butoxyethyl) phosphate (TBEP) and tributyl phosphate (TBP) at 20, 200, and 2000 μg/L for 28 d. The results showed no noticeable difference in siphoning behavior. However, the siphoning behavior displayed a trend toward a slight decrease in the treatment groups. GR activity was markedly reduced compared with the control groups, whereas the levels of cyp4 significantly increased following the 2000 μg/L TBP treatments (p < 0.05). Moreover, the levels of gsts1 and gstm1 significantly decreased following all TBEP treatments and were significantly inhibited by 20 μg/L TBP (p < 0.05). The adverse effects on antioxidant enzymes suggested that C. fluminea mainly relies on the antioxidant system to reduce damage without an increase in MDA levels following exposure to a low concentration. Moreover, mRNA expression levels of heat shock proteins (hsp 22, 40, 60, 70, and 90) were significantly down-regulated with TBEP and TBP treatments lower than 200 μg/L (p < 0.05), whereas significant up-regulations were observed for hsp 22 and hsp 70 in response to 2000 μg/L TBP treatment (p < 0.05). Up-regulation of ATP-binding cassette (ABC) transporter genes (abcb1 and abcc1) showed that TBEP and TBP could activate the multixenobiotic resistance (MXR) system to discharge xenobiotics in C. fluminea, which kept its shell closed at high concentrations to prevent xenobiotic entry. Our results provide a new insight into the different mechanisms of cellular detoxification and the MXR system of C. fluminea in response to low and high concentrations of TBEP and TBP.

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.

Organic UV filters inhibit multixenobiotic resistance (MXR) activity in Tetrahymena thermophila: investigations by the Rhodamine 123 accumulation assay and molecular docking.

Multixenobiotic resistance (MXR) transporters, which belong to ATP-binding cassette (ABC) family proteins, are present in living organisms as a first line of defense system against xenobiotics and environmental contaminants. The effects of six organic UV filters (4-methyl -benzylidene camphor, 4-MBC; benzophenone-3, BP-3; butyl methoxydibenzoyl-methane, BM-DBM; ethylhexyl methoxy cinnamate, EHMC; octocrylene, OC and homosalate, HMS) on multixenobiotic resistance (MXR) in Tetrahymena thermophila were investigated in this study. It was found that 4-MBC, BP-3 and BM-DBM could significantly inhibit activity of the MXR system, causing concentration dependent accumulation of rhodamine 123; while EHMC, OC and HMS had weak MXR inhibition. The IC50 (50% inhibition concentration) values of 4-MBC, BP-3 and BM-DBM were 23.54, 40.59 and 26.37μM, respectively, with inhibitory potentials of 23.1, 13.4 and 20.6% relative to verapamil (VER, a model inhibitor of P-glycoprotein). Our results firstly provide the evidence for UV filters inhibition effect on MXR in aquatic organisms. In addition, it was revealed by molecular docking analysis that the selected six UV filters can occupy the same binding site on T. thermophila P-gp as VER does; and form H-bonds with residues Ser 328 and/or Asn 281. This study raises the awareness of aquatic ecological risk from the organic UV filters exposure, as they would be involved in potentiating toxic effects by chemosensitizing.

Effects of fluoxetine on behavior, antioxidant enzyme systems, and multixenobiotic resistance in the Asian clam Corbicula fluminea

Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is widespread in aquatic environments. Despite its reported effects on behavior and reproduction in aquatic species, little is known about the effects of FLX on cellular detoxification and defense system in bivalves. Here, the adult Asian clam (Corbicula fluminea) was exposed to 0.5, 5, and 50μgL−1 FLX for 30d. Siphoning behavior was inhibited by treatment with 50μgL−1 FLX. Additionally, superoxide dismutase (SOD) activity in the gills and digestive glands significantly decreased (p<0.05) with 5 and 50μgL−1 FLX treatments, whereas catalase (CAT) activity and malondialdehyde (MDA) content markedly increased (p<0.05). Moreover, transcription of thioredoxin reductase (TR), glutathione peroxidase (GPx) and Glutathione S-transferase pi class (GSTpi) was significantly upregulated (p<0.05), whereas glutathione reductase (GR) was markedly downregulated (p<0.05). These findings suggest that FLX affects behavior and induces oxidative stress in C. fluminea. The downregulation of ATP-binding cassette (ABC) transportor genes (ABCB1, ABCC1 and ABCG2) transporter genes indicated that FLX might suppress the multixenobiotic resistance (MXR) system in C. fluminea. Our results provide new insights into the adverse effects of FLX on the cellular detoxification and MXR system of C. fluminea.

Cyclic-AMP Mediated Regulation of ABCB mRNA Expression in Mussel Haemocytes

BackgroundThe multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified.Methodology/Principal FindingscAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89.ConclusionsThis study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

PKA-mediated transcriptional regulation of the Mediterranean mussel ABCB1 gene involved in the Multixenobiotic Resistance system

PKA-mediated transcriptional regulation of the Mediterranean mussel ABCB1 gene involved in the Multixenobiotic Resistance system

Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids.

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.

Amphiphilic Copolymer for Delivery of Xenobiotics: In Vivo Studies in a Freshwater Invertebrate, a Mesostominae Flatworm

The synthesis of an amphiphilic polymethacrylate copolymer containing cholesterol hydrophobic moieties and rhodamine as a fluorescent probe, the formation of microspheres, and the uptake of these microspheres in an invertebrate are reported. The cholesterol-derived methacryloyl monomer, which was prepared via a one-step synthesis, was copolymerized with methacrylic acid and methacryloxyethyl thiocarbamoyl rhodamine B in the presence of AIBN as initiator. The obtained dye-labeled copolymer was characterized by (1)H NMR and UV-vis spectroscopy. Fluorescence and TEM microscopies studies show that this amphiphilic copolymer aggregates to give microspheres with diameters ranging from approximately 3 to 11 microm. The in vivo study in a freshwater invertebrate, a Mesostominae flatworm (Rhabdocoela, Thyphloplanidae), indicates that the microspheres enter the cells by endocytosis. The data collected demonstrate that the rhodamine B covalently attached to the amphiphilic copolymers is bioaccumulated without being translocated out of the cell by the multixenobiotic resistance (MXR) transporters. As the MXR system is similar to the multidrug resistance (MDR) first observed in tumor cell lines resistant to anticancer drugs, the present data confirm the significant role that amphiphilic copolymers can play in the ongoing development of drug delivery strategies to overcome multidrug resistance. These investigations illustrate a promising approach for the development of new medical and ecotoxicological tools that can deliver specific molecules within cells.

Effect of temperature on the expression of P-glycoprotein in the zebra mussel, Dreissena polymorpha

The multixenobiotic resistance (MXR) system is a defence mechanism which protects living organisms by actively extruding xenobiotics from cells. Membrane transporters responsible for this phenotype can be induced by numerous compounds, but also by physical stressors such as temperature. In order to better understand the contribution of temperature in MXR expression, the MXR system was studied in the freshwater bivalve, Dreissena polymorpha. Results showed that the zebra mussel possesses a gene closely related to the ATP-binding cassette (ABC) subfamily B, which is expressed as a 120-kDa protein. Moreover, gill cells can transport the model substrate Rhodamine B in a verapamil-sensitive manner, which is a good indication of MXR protein activity such as P-glycoprotein (Pgp). When bivalves were slowly acclimated to temperatures ranging from 4 to 20 °C (maximum temperature variation, 2–3 °C/day), no significant change in MXR protein expression could be immunochemically measured. Furthermore, the MXR transport activity did not show significant variation in this temperature range. Conversely, incubation of zebra mussel specimens in 20 μM Dacthal dramatically enhanced both protein expression and transport activity, thus indicating that temperature is not a major determinant in zebra mussel gill Pgp expression.

Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus

Based on the chemical features of natural organic matter (NOM) with its variety of functional groups, we hypothesized that NOM will modify the multixenobiotic-resistance (MXR) of an organism as xenobiotic chemicals do. The MXR system is a general first rather non-specific line of defense against environmental contaminants. The aim of this study was to compare the impacts on MXR activity in amphipod species ( Eulimnogammarus cyaneus and E. verrucosus, from Lake Baikal) stressed by cadmium chloride or dissolved NOM for 24 h. NOM exposure concentrations were environmentally realistic. MXR activity was assessed based on rhodamine B efflux; its specificity was proven by a verapamil inhibition assay. It was shown that both NOM and CdCl 2 lead to substantial reduction of the rhodamine B efflux. This suggests that NOM may be regarded as a chemosensor which is able to reduce the efficiency of the MXR system. Possible mechanisms of direct NOM impact on MXR processes are discussed, such as peroxidation of the membranes (including P-glycoproteins) or internal blockage of the MXR pump by bioconcentrated NOM. In general, our results show that well-developed depuration pathways of freshwater organisms in contaminated environments may be impaired by strong chemical stressors and, more important, by natural biogeochemical matrices such as humic substances — humic substances are present in all freshwater systems.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.

Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot ( Scophthalmus maximus)

Pleiotropic resistance driven by transport proteins constitutes a very ubiquitous protection mechanism against natural or synthetic toxic compounds. The multidrug (MDR) or multixenobiotic (MXR) system has been identified in many different species, and may be used as a biomarker for pollution assessment. Here we report the existence of a gene encoding a MXR-related protein in a benthic fish species, the turbot Scophthalmus maximus, and its constitutive expression in several tissues. A 433bp cDNA fragment has been cloned by RT-PCR. The deduced amino-acid sequence shares close to 80% homology with class I or class II mammalian MDR proteins. This cDNA corresponds to a major mRNA of 5.6 kb and encodes a protein having an apparent molecular weight of 83 kDa. Constitutive expression levels assessed by semi-quantitative RT-PCR and Western blot, revealed that the kidney and the brain, and to a lesser extent, the heart, gills and intestine, are the organs which contain the highest amount of both MXR mRNAs or proteins. This tissue specific expression suggests a role for the identified mechanism in protection against endogenous or exogenous toxic compounds.