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The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354–367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357–360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein. [ABSTRACT FROM AUTHOR]

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. galloprovincialismrp2, 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.

We investigated the activity of the multixenobiotic resistance (MXR) phenotype, a biological defense system in aquatic organisms, in the fish assemblages of two tropical estuaries with different degrees of environmental impacts, the Paraiba River and Mamanguape River Estuaries. The aim of this work was to compare the activity of the MXR phenotype of different fishes to test the hypothesis that each species has an inherent activity level and to use this activity as a bioindicator of aquatic contamination. We assessed the MXR activity of the gills, using rhodamine B (RB) accumulation assay. The results demonstrated a species‐specific difference in the MXR activity of fishes caught in the same estuarine zone. Also, the pelagic species Eucinostomus melanopterus, Eucinostomus argenteus, and Lutjanus jocu had higher RB accumulation, while the demersal species Sphoeroides testudineus and Sphoeroides greeleyi had the lowest RB accumulation, suggesting that the ecological characteristic of fish in the water column exerts an influence on MXR activity. Besides, we demonstrated the potential of using the gill MXR activity of the key estuarine species, the Brazilian silversides Atherinella brasiliensis, as a tool for biomonitoring estuaries. Research Highlight: Fish Assemblages from two tropical estuaries had different species‐specific levels of multixenobiotic resistance (MXR) activity. The gill of the key species Atherinella brasiliensis was the organ with the highest MXR activity. The intraspecific comparison of the MXR activity of Atherinella brasiliensis can be used as a good biomarker of contamination. [ABSTRACT FROM AUTHOR]

The literature indicates that exotic species have a greater tolerance to environmental stressors compared with native species. In recent decades, the introduction of contaminants into the environment has increased as a result of industrialization. The objective of this study was to verify the resistance of bivalve mollusks from freshwater native (Anodontites trapesialis) and exotic (Limnoperna fortunei) species to chemical contamination using an ex vivo/in vitro approach. Gill and muscle tissues were exposed to two different types of environmental stressors, copper (metal), and Roundup Transorb® (herbicide). The tissues were submitted to a cytotoxicity test in which the lysosomal integrity was assessed, from the adaptation of a method to isolated cells, and multixenobiotic resistance (MXR) test which evaluated cellular defense. In the exotic species, only copper at 9000 μg/L and Roundup Transorb® at 5000 μg/L were cytotoxic. In the native species, copper cytotoxicity at 900 and 9000 μg/L and Roundup Transorb® at 50 and 5000 μg/L were observed. Results were the same in both tissues. The MXR, responsible for the extrusion of contaminants (cell defense), was inhibited in both species when exposed to the contaminants, this cell defense system seems to be more inhibited in the native species, when exposed to both pollutants, indicating greater sensitivity. Therefore, cytotoxicity may be related to the lack of capacity of cellular defense. In relation to lysosomal integrity, the native species was more sensitive to cytotoxic pollutants, where a greater number of experimental conditions of metals and herbicide showed cytotoxicity, as well as more experimental situations inhibited its ability to defend itself. [ABSTRACT FROM AUTHOR]

Abstract Environmental impairment resulted from urbanizations can produce damage on freshwater species including strong physiological effects at individual or population level. The multixenobiotic resistance (MXR) is a defence mechanism which has been demonstrated in several aquatic organisms. The key mediators of MXR activity are ATP-binding cassette (ABC) proteins like P-glycoprotein (P-gp). This system protects aquatic organisms against the accumulation of xenobiotics by extruding them from cells in an energy-dependent manner. MXR has been pointed out as relevant in the ecotoxicological context and has been proposed as a biomarker for pollution assessment. Since fish species are common target in freshwater biomonitoring programs, the purpose of the study was to evaluate the MXR mechanism in native Hatcheria macraei (Patagonian catfish) and exotics Salmo trutta (brown trout), Oncorhynchus mykiss (rainbow trout) and Oncorhynchus tshawytscha (Chinook salmon) freshwater fishes widespread in Argentine Patagonia. We characterized the MXR mechanism using a combination of functional assays and Western blot analysis. Our results in different tissues such as liver, gills, muscle and epidermis indicate that the fishes studied have different species-specific levels of MXR activity, being gills and liver the tissues with greater detoxifying activity. Induction of MXR transport activity was also identified in liver tissue from rainbow trout from urban stream suggesting their suitability in the biomonitoring of aquatic environments subjected to urban contaminants. Graphical abstract Unlabelled Image Highlights • MXR mechanism activity is present in fishes inhabiting Patagonian basins. • RB is differently accumulated in liver, gills, lateral muscle and epidermis tissues. • Immunochemical detection of P-gp does not reflect functional RB assays results. • Higher MXR activity is observed in the rainbow trout liver from urban stream. [ABSTRACT FROM AUTHOR]

Neonicotinoids are the most widely used synthetic insecticides in the world. These insecticides are widely distributed in the ecosystem, indicating that more attention should be paid to the potential risks regarding their use in agriculture. Due their intensive use, non-target species in the environment are also exposed to their putative effects. Within acute exposure trials, the time related effect of sublethal dose of the neonicotinoid preparation APACS 50 WG was investigated on swimming behaviour and the multi-xenobiotic resistance system (MXR) activity, as a first line defence pathway of adult Dikerogammarus villosus. Results showed that treated animals manifested an increased swimming activity. Exposed animals were monitored by the rhodamine B accumulation assay, and APACS 50 WG exerted distinct changes in the MXR activity as well. Our results suggested that application of neonicotinoid at a low concentration (3.9 ng/l) contributed to the activation of locomotor activity and at the same concentration range the transmembrane transport mechanisms was altered too. [ABSTRACT FROM AUTHOR]

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Abstract Estuarine osmoconformes rely on their ability to perform tissue and cell water regulation to cope with daily osmotic challenges that occur in the estuary. In addition, these animals currently must deal with pollutants present in the estuarine environment, which can disturb their capacity of water regulation. We collected the mangrove oyster Crassostrea rhizophorae in two tropical estuaries in the Northeast region of Brazil with different degrees of human interference: the Paraíba Estuary (impacted) and the Mamanguape Estuary (preserved). Tissue water content was analyzed after exposure to salinities 12, 24 and 36 for 24 h. Gill cell volume regulation was analyzed in vitro upon hypo- and hyper-osmotic conditions. We also analyzed gill MXR (multi-xenobiotic resistance) mechanism, as reference of environmental pollution. Gill and muscle of oysters from two sites of Paraíba Estuary, and from one site of Mamanguape Estuary were not able to maintain tissue water content upon hypo- and hyper-osmotic conditions. Gill cells of oyster from the same sites exhibited swelling followed by regulatory volume decrease upon hypo-osmotic condition. Gill MXR activity was increased in oysters from these sites. The best tissue and cell water regulation, and the lowest MXR activity, was found in oyster from downstream of Mamanguape Estuary, our reference site and the one most preserved. Tissue and cell water regulation proved to be a sensitive parameter to environmental pollution and could be considered as biomarker of aquatic contamination. Highlights • The water balance of an osmoconformer can be affected by environmental pollution. • Oysters from impacted sites are worse in water regulation than those from preserved sites. • Tissue and cell volume are sensitive to environmental pollution, such as MXR activity. [ABSTRACT FROM AUTHOR]

In order to assess the impact of nanoplastics on marine species, polystyrene nanoparticles (PS NPs) have been largely used as model particles. Here we studied the effects of 50 nm amino-modified PS-NH2 on Mediterranean sea urchin Paracentrotus lividus immune system cells (coelomocytes) in the presence of celomic fluid (CF) and at different NP concentrations (1, 5, 10, and 25 μg mL−1) and experimental conditions (absence or presence of EDTA). PS-NH2 acquired a protein corona once incubated with CF, dominated by the toposome precursor protein (TPP). In short-term cultures, a significant concentration- and time-dependent decrease in lysosomal membrane stability and apoptotic-like nuclear alterations were observed in phagocytes upon exposure to PS-NH2 (10 and 25 µg mL−1) in CF but they resulted abolished in the presence of EDTA confirming the role of TPP in triggering PS-NH2-coelomocytes interaction and toxicity. PS-NH2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH2 to affect pump's efflux activity. Overall results encourage additional studies on positively charged nanoplastics, since the observed effects on sea urchin coelomocytes as well as the TPP corona formation might represent a first step for addressing their impact on sensitive marine species. [ABSTRACT FROM AUTHOR]

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil ( Echinometra lucunter and Crassostrea gasar ) and Rade of Brest; France ( Echinus esculentus and Crassostrea gigas ). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar , suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar . The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette ( ABC ) transporter family is one of the largest gene family in animals, and members of this family are known to be involved in various biological processes due to their ability to transport a wide range of substrates across membranes using ATP cleavage-derived energy. We identified 61 ABC transporters in the genome of the monogonont rotifer Brachionus koreanus , and classified these into eight distinct subfamilies (A–H) by phylogenetic analysis. ABC transporters in the rotifer B. koreanus are comprised of 11 ABCA genes, 19 ABCB genes, 14 ABCC genes, 3 ABCD genes, 1 ABCE gene, 3 ABCF genes, 8 ABCG genes, and 2 ABCH genes. Extensive gene duplication and loss events in synteny were observed in several subfamilies. In particular, massive gene duplications of P-glycoproteins ( P-gps ), multidrug resistance proteins ( MRPs ), and Bk-Abcg-like proteins were observed. The ability of these B. koreanus proteins to function as multixenobiotic resistance (MXR) ABC transporters was validated using specific fluorescence substrates/inhibitors. The ABC transporter superfamily members identified in this study will be useful in future toxicological studies, and will facilitate comparative studies of the evolution of the ABC transporter superfamily in invertebrates. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • MXR activity and P-gp action assessed in native snail Chilina dombeiana and exotic snail Physella acuta. • C. dombeiana and P. acuta from anthropogenic sites showed higher MXR activity than from reference. • The detoxification activity has a differential response by species in relation to land use. • Native species have more restricted ecological requirements appears important for integrated biomonitoring programs. [ABSTRACT FROM AUTHOR]

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions. [ABSTRACT FROM AUTHOR]

Sewage sludge (SS) is a complex organic by-product of wastewater treatment plants. Deposition of large amounts of SS can increase the risk of soil contamination. Therefore, there is an increasing need for fast and accurate assessment of SS toxic potential. Toxic effects of SS were tested on earthworm Eisenia fetida tissue, at the subcellular and biochemical level. Earthworms were exposed to depot sludge (DS) concentration ratio of 30 or 70 %, to undiluted and to 100 and 10 times diluted active sludge (AS). The exposure to DS lasted for 24/48 h (acute exposure), 96 h (semi-acute exposure) and 7/14/28 days (sub-chronic exposure) and 48 h for AS. Toxic effects were tested by the measurements of multixenobiotic resistance mechanism (MXR) activity and lipid peroxidation levels, as well as the observation of morphological alterations and behavioural changes. Biochemical markers confirmed the presence of MXR inhibitors in the tested AS and DS and highlighted the presence of SS-induced oxidative stress. The MXR inhibition and thiobarbituric acid reactive substance (TBARS) concentration in the whole earthworm's body were higher after the exposition to lower concentration of the DS. Furthermore, histopathological changes revealed damage to earthworm body wall tissue layers as well as to the epithelial and chloragogen cells in the typhlosole region. These changes were proportional to SS concentration in tested soils and to exposure duration. Obtained results may contribute to the understanding of SS-induced toxic effects on terrestrial invertebrates exposed through soil contact and to identify defence mechanisms of earthworms. [ABSTRACT FROM AUTHOR]

The economic importance that myticulture is conquering in Santa Catarina State (South of Brazil) explains the crescent search for new coastal sites for farming. Physiological and biochemical studies of the mussel Perna perna are important to the establishment of methodologies for program assessment and environmental monitoring, allowing to infer about site quality and possible influences of xenobiotic agents on coastal areas. In order to evaluate effects caused by lead poisoning (1.21 mumol.L-1), the mussels were maintained at constant temperature (25ºC) and fed with Chaetoceros gracilis for 15 days. The control group was acclimatized in sea water 30?. At the end of this period time, physiological measurements were carried out along with statistic analysis for filtration rates, lead assimilation and overall respiratory activity. The mechanism of multixenobiotic resistance (MXR) was particularly evaluated in standardized gill fragments using rhodamine B accumulation and its quantification under fluorescence optical microscopy. Regarding the control group, results had shown that the mussels maintenance in a lead-poisoned environment caused higher filtration rates (1.04 and 2.3 and L.h-1.g-1; p < 0.05) and lower assimilation rates (71.96% and 54.1%, respectively). Also it was confirmed a lesser rhodamine B accumulation in the assays under influence of lead, suggesting that this metal induces the MXR mechanism expression in mussel P. perna. These results indicate that such physiological and biochemical alterations in the mussels can modify the energy fluxes of its metabolism, resulting in possible problems on the coastal systems used as cultivating sites.

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida ) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies. [ABSTRACT FROM AUTHOR]

Pharmaceutical products are a major group of chemical compounds that are continuously released into the environment. The primary pathway of pharmaceuticals to the aquatic environment is the discharge of wastewater effluents. The Psychiatric hospital of Montpon (Dordogne, France) operates with its wastewater treatment plant. We first evaluated the presence and concentrations of 27 pharmaceuticals compounds in these effluents. All of the 27 compounds were detected in these wastewater effluents at concentrations ranging between 37,500 ng L (paracetamol) and 150 ng L (citalopram). The aim of the study was then to evaluate the exposure effects of the effluents on cytochrome P450, GST, and MXR responses in Corbicula fluminea gills and digestive glands. Experiments on clams exposed during 1, 3, 7 14, and 21 days revealed a strong and continuous overexpression of mdr1 (multidrug resistant 1) gene expression in gills and transitory variations in pi-gst expression and GST activity. EROD activity increased also transitory after 1 day in the digestive gland of exposed clams. These results indicated that in the effluent, some molecules have undergone metabolism of phase 1 and/or phase 2. [ABSTRACT FROM AUTHOR]

The fauna of Lake Baikal in Eastern Siberia, the largest freshwater body on Earth, is characterized by high degrees of biodiversity and endemism. Amphipods, a prominent taxon within the indigenous fauna, occur in an exceptionally high number of endemic species. Considering the specific water chemistry of Lake Baikal with extremely low levels of potentially toxic natural organic compounds, it seems conceivable that certain adaptions to adverse environmental factors are missing in endemic species, such as cellular defense mechanisms mitigating toxic effects of chemicals. The degree to which the endemic fauna is affected by the recently occurring anthropogenic water pollution of Lake Baikal may depend on the existence of such cellular defense mechanisms in those species. We here show that endemic amphipods express transcripts for Abcb1, a major component of the cellular multixenobiotic resistance (MXR) defense against toxic chemicals. Based on a partial abcb1 cDNA sequence from Gammarus lacustris, an amphipod species common across Northern Eurasia but only rarely found in Lake Baikal, respective homologous sequences were cloned from five amphipods endemic to Lake Baikal, Eulimnogammarus verrucosus, E. vittatus, E. cyaneus, E. marituji, and Gmelinoides fasciatus, confirming that abcb1 is transcribed in those species. The effects of thermal (25 °C) and chemical stress (1-2 mg L phenanthrene) in short-term exposures (up to 24 h) on transcript levels of abcb1 and heat shock protein 70 ( hsp70), used as a proxy for cellular stress in the experiments, were exemplarily examined in E. verrucosus, E. cyaneus, and Gammarus lacustris. Whereas increases of abcb1 transcripts upon treatments occurred only in the Baikalian species E. verrucosus and E. cyaneus but not in Gammarus lacustris, changes of hsp70 transcript levels were seen in all three species. At least for species endemic to Lake Baikal, the data thus indicate that regulation of the identified amphipod abcb1 is triggered within the general cellular stress response. This is the first report presenting molecular data on a MXR transporter in amphipods, an ecotoxicologically important but with regard to gene sequence data comparatively little explored taxon. [ABSTRACT FROM AUTHOR]

The species-rich, endemic amphipod fauna of Lake Baikal does not overlap with the common Palearctic fauna; however, the underlying mechanisms for this are poorly understood. Considering that Palearctic lakes have a higher relative input of natural organic compounds with a dominance of humic substances (HSs) than Lake Baikal, we addressed the question whether HSs are candidate factors that affect the different species compositions in these water bodies. We hypothesized that interspecies differences in stress defense might reveal that Baikalian amphipods are inferior to Palearctic amphipods in dealing with HS-mediated stress. In this study, two key mechanisms of general stress response were examined: heat-shock protein 70 (HSP70) and multixenobiotic resistance-associated transporters (ABCB1). The results of quantitative polymerase chain reaction (qPCR) showed that the basal levels (in 3-day acclimated animals) of hsp70 and abcb1 transcripts were lower in Baikalian species ( Eulimnogammarus cyaneus, Eulimnogammarus verrucosus, Eulimnogammarus vittatus-the most typical littoral species) than in the Palearctic amphipod ( Gammarus lacustris-the only Palearctic species distributed in the Baikalian region). In the amphipods, the stress response was induced using HSs at 10 mg L dissolved organic carbon, which was higher than in sampling sites of the studied species, but well within the range (3-10 mg L) in the surrounding water bodies populated by G. lacustris. The results of qPCR and western blotting ( n = 5) showed that HS exposure led to increased hsp70/ abcb1 transcripts and HSP70 protein levels in G. lacustris, whereas these transcript levels remained constant or decreased in the Baikalian species. The decreased level of stress transcripts is probably not able to confer an effective tolerance to Baikalian species against further environmental stressors in conditions with elevated HS levels. Thus, our results suggest a greater robustness of Palearctic amphipods and a higher sensitivity of Baikalian amphipods to HS challenge, which might prevent most endemic species from migrating to habitats outside Lake Baikal. [ABSTRACT FROM AUTHOR]

There are multiple stressors derived from urbanizations that result in frequent disturbances on streams and rivers reducing water quality and threatens aquatic biota. P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) is a defence mechanism analogous to multidrug resistance (MDR), which has been demonstrated in several aquatic organisms. This system protects cells against the entry and the accumulation of xenobiotics and has been proposed as a biomarker for pollution assessment. We conducted a study in a post-urban reach of Esquel stream (Chubut Province) downstream a wastewater treatment plant, in order to assess the presence and activity of MXR in five freshwater macroinvertebrate species ( Helobdella michaelseni, Helobdella simplex, Patagoniobdella variabilis, Hyalella curvispina and Chironomus riparius). We measured the accumulation of the model P-gp substrate rhodamine B (RB) in organisms previously exposed to pollution. Our results described the activity of the MXR system in the three species of leeches suggesting their suitability as the in vivo biomonitoring. We also identified a dependence of the transporter activity with the development stage in H. simplex, highlighting the importance of using organisms of similar size classes since it may affect observed results. Finally, we concluded that benthic freshwater macroinvertebrates possess different species-specific levels of MXR activity possibly influencing their natural distribution as well as their survival. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examine contributions of type B and C ABC activities in mussel hemocyte subtypes. [•] Both transcripts and ABCB/P-glycoprotein are expressed. [•] Treatment with blockers reveals the only role of ABCC/MRP activities. [•] Comparison of control and polluted sampling sites reveals an inducible MRP efflux. [•] MRP transport was mediated mainly by eosinophilic granulocytes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Seabass primary hepatocytes exhibit phase I and III components of detoxification. [•] BaP leads to dose dependent increased mRNA Abcc2 and Cyp1a in seabass hepatocytes. [•] CYP1A in seabass primary hepatocytes is functional and responsive to BaP. [•] Seabass primary hepatocytes are a suitable tool to study detoxification mechanisms. [ABSTRACT FROM AUTHOR]

Highlights: [•] A tissue explant was used to produce a primary cell culture of anemone cells. [•] This is the first use of these cells as a model for ecotoxicological studies. [•] The copper activation of the MXR phenotype is concentration dependent. [•] Low concentrations (μM) of metal is enough to generate DNA damage in anemone cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] We characterized the Daphnia magna multixenobiotic (MXR) system. [•] MXR related ABC transporters are expressed in different developmental stages. [•] D. magna has MXR related transporter activity. [•] ABC transporter genes and activities are inducible by chemical pollutants. [•] Joint toxicity of transporter substrates and inhibitors is synergic in D. magna. [ABSTRACT FROM AUTHOR]

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm's MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) mediated multixenobiotic resistance (MXR) is a mechanism analogous to multidrug resistance (MDR), which has been extensively characterised in mammalian tumours. The expression and function of the MXR mechanism has been demonstrated in numerous aquatic organisms and has been proposed as a biomarker for pollution assessment. The purpose of this study was to evaluate the activity and expression of the MXR mechanism in freshwater snails (Physa acuta) of the Andean–Patagonian region. The accumulation and efflux rates of the model P-gp substrate Rhodamine B in snails previously exposed to pollution were measured. Results confirmed that MXR activity decreased after maintenance in clean water, and that a depuration period of 7 d was long enough to observe a significant deinduction of this system. The results demonstrate the presence of a P-gp-like transport system in the freshwater snailP.acutafrom Patagonia, and suggest its function as a defence system. The results of this study could be used to provide information on the possible use of these snails as bioindicators in toxicological testing. [ABSTRACT FROM AUTHOR]

Background: In mammals, ABCB1 constitutes a cellular "first line of defense" against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results: Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks). Conclusions: We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model. [ABSTRACT FROM AUTHOR]

Highlights: [•] Mammalian Pgp antibodies reacted positively in liver, gills and intestine of Nile tilapia. [•] CYP1A was induced upon exposure to BaP in liver, gills and intestine. [•] CYP1A enzymes metabolize waterborne BaP in liver, gills and intestine. [ABSTRACT FROM AUTHOR]

Multixenobiotic resistance (MXR) in aquatic organisms exposed to natural toxins or anthropogenic contaminants is a phenomenon analogous to multidrug resistance (MDR) in mammalian tumor cell lines tolerant of anti-cancer drugs. Multidrug resistance is commonly due to the elevated expression of transmembrane P-gps which actively transport a wide variety of structurally and functionally diverse compounds. The effect of xenobiotic exposures on P-gp activity and protein titer has been examined in wild and captive populations of aquatic invertebrates and vertebrates. Molecular evolution of P-gp was investigated in teleost fishes, which exhibit remarkable diversity in morphology, behavior, and adaptations. Several phylogenetic analysis were performed using all teleosts P-gp amino acid and complementary DNA (cDNA) sequences present in GenBank. P-gp molecular evolution in teleosts seemed to follow identical evolution pathways to teleost fishes speciation. [ABSTRACT FROM AUTHOR]

In marine organisms the multi xenobiotic resistance (MXR) mechanism via e.g. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) is an important first line of defense against contaminants by pumping contaminants out of the cells. If compounds would impair the MXR mechanism, this could result in increased intracellular levels of other compounds, thereby potentiating their toxicity. A calcein-AM based larval cellular efflux pump inhibition assay (CEPIA) was developed for echinoid ( Psammechinus miliaris) larvae and applied for several contaminants. The larval CEPIA revealed that triclosan (TCS) and the nanoparticles P-85 (P-85) were 124 and 155× more potent inhibitors (IC 0.5 ± 0.05 and 0.4 ± 0.1 μM, respectively) of efflux pumps than the model inhibitor Verapamil (VER). PFOS (heptadecafluorooctane sulfonic acid) and pentachlorophenol also were more potent than VER, 24 and 5×, respectively. Bisphenol A and o, p′-dichlorodiphenyltrichloroethane ( o, p′-DDT) inhibited efflux pumps with a potency 3× greater than VER. In a 48 h early life stage bioassay with P. miliaris, exposure to a non-lethal concentration of the inhibitors TCS, VER, the model MRP inhibitor MK-571, the nanoparticles P-85 and the model P-gp inhibitor PSC-833, increased the toxicity of the toxic model substrate for efflux pumps vinblastine by a factor of 2, 4, 4, 8 and 16, respectively. Our findings show that several contaminants accumulating in the marine environment inhibit cellular efflux pumps, which could potentiate toxic effects of efflux pumps substrates. [ABSTRACT FROM AUTHOR]

Abstract: Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages. [Copyright &y& Elsevier]

Abstract: In fish, some ABC transporters are implicated in a multixenobiotic resistance (MXR) mechanism to deal with the presence of xenobiotics, by effluxing them, or their metabolites, from inside the cells. These efflux transporters have been considered an integral part of cellular detoxification pathways, acting in coordination with phase I and II detoxification enzymes. However, the full characterization of this detoxification system is still incomplete, especially during the developmental stages of aquatic organisms, which are particularly sensitive periods to the presence of anthropogenic contamination. The goal of this study was to evaluate the mRNA expression dynamics of putatively important MXR proteins (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2a) and phase I (CYP1A) and II (GSTα) biotransformation enzymes, during the embryonic and larval developments of the specie Oreochromis niloticus (Nile tilapia). Our results showed that ABCB1b, ABCC1, CYP1A and GSTα transcripts are maternally transmitted. Transcripts for ABCB11, ABCC2 and ABCG2a were only detected after the pharyngula period, which precedes a highly sensitive stage in the embryonic development, the hatching. This study has shown, for the first time, very distinct expression patterns of genes encoding for proteins involved in protection mechanisms against pollutants during the development of Nile tilapia. Moreover, the temporal pattern of gene expression suggests that increased intrinsic protection levels are required at specific developmental stages. [Copyright &y& Elsevier]

Abstract: Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5 > abcg2 > abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2′,7′-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation. [Copyright &y& Elsevier]

Abstract: Coastal environments are often subjected to contamination, whose biological impact is profitably evaluated through sentinel organisms and biomarkers. mRNA profiling was also proposed as a potential biomarker, whose relevance is still under discussion. Indeed, correlation between molecular and cell-organism responses need further investigations, especially under field conditions. In this study, we followed the development of physiological alterations in Mytilus galloprovincialis transplanted into a polluted coastal lagoon for 2, 4, 7, 14 and 30days. Three consolidated biomarkers were measured, i.e. lysosomal membrane stability, lipofuscin and metallothionein contents. In parallel, the expressions of stress-related genes encoding metallothioneins (mt10 and mt20), 70-kDa heat shock proteins (MgHSC70 and MgHSP70), and Multi Xenobiotic Resistance-related transporters (MgPgp, MgMrp2, and MgMvp) were analyzed, to have a greater insight into the time-related evolution of the response. Significant (p <0.05) biomarker responses were induced after 7days of exposure and further increased with time, whereas gene expression profiles were dramatically altered 2days after transplanting. Biomarkers and gene expression profiles indicated a stress syndrome development in mussels, although with different temporal patterns. Their combined application provided insights into the molecular and cellular basis of mussel responses to challenging environments, and may have far-reaching implications for monitoring environmental health. [Copyright &y& Elsevier]

One of the most intriguing defence strategies which aquatic organisms developed through evolution is multixenobiotic resistance (MXR). The key mediators of MXR activity are ATP-binding cassette (ABC) transport proteins. They provide resistance of aquatic organisms by binding xenobiotics and extruding them from cells in an energy-dependent manner. Since Cyprinid fish species are common target in freshwater biomonitoring programs, we have studied the presence of two main MDR/MXR efflux transporters Pglycoprotein (Pgp, Abcb1) and MRP-like protein(s) (Abcc) in the liver of five Cyprinid species: common carp, European chub, sneep, barbel, and silver prussian carp. Their presence was evaluated on the mRNA and protein level. Various pairs of primers were designed to clone homologous fragments of MXR-related genes. At the protein level, we used Western blotting with specific monoclonal antibodies against human Pgp (Abcb1, Ab C219), MRP1 (Abcc1; Ab MRPm6) or MRP2 (Abcc2; Ab M2I-4). Transcripts of both key types of MXR transporters were identified in all species examined and here we provide the phylogenetic analysis of new partial sequences. Immunochemical determinations with mammalian antibodies failed to identify the presence of MRP(s), but Pgp expression was found in all five Cyprinid species. These results support that MXR is a defence system mediated by both Pgp and MRP types of ABC transport proteins. [ABSTRACT FROM AUTHOR]

Abstract: The physiological and biochemical characteristics of tissue samples of the marine mussel, Donax trunculus, from an oil-polluted site (Qiryat Yam) and from a site adjacent to an industrial factory (Frutarom) producing polyvinyl chloride (PVC) were compared with samples from a clean reference site (Akko). All sites were located along the sandy shores of the Israeli Mediterranean Sea. The mussels from the oil-polluted site showed increased activity of the system of active transport of organic anions (SATOA) in the gills and the renocardial organ and also of the multixenobiotic resistance transporter (MXR) in the gills. In contrast, samples collected near the PVC factory showed a decrease in SATOA activity and no increase in the activity of MXR in the gills. Specimens from the reference site demonstrated a redox state of equilibrium between energy production and utilization, while in Donax from both the oil polluted and the PVC-polluted sites, the mitochondrial redox state reflected intensive consumption of energy. No significant changes were found in the activity of reduced glutathione s-transferase (GST) in the cytosolic fraction of the digestive gland of Donax collected from any of the three sites. The data demonstrate a differential increase in the anti-chemical defense systems and an intensification of energy metabolism in the mussels exposed to pollution. [Copyright &y& Elsevier]

The purpose of the study was to evaluate multixenobiotic resistance mechanism expression as a biomarker for contaminant exposure in freshwater fishes. Exposure to a known mammalian inhibitor (verapamil, 10 μM) and inducer (rhodamine 123, 3 μM) on the transport protein P-glycoprotein (Pgp) was investigated in the liver of the western mosquitofish and bluegill sunfish. No differences in the activity or expression of Pgp were measured in either species using a fluorometric accumulation assay and western blot analyses. The preliminary results from this study indicate that this detoxification mechanism may not be a sensitive indicator of contaminant exposure in certain teleost species. [ABSTRACT FROM AUTHOR]

Abstract: Pharmaceuticals represent a diverse collection of thousands of bioactive chemicals used in human and veterinary medicine. The increased consumption, together with the recent development of more sensitive analytical techniques, has identified these as emerging contaminants in the aquatic environment. According to many investigations pharmaceuticals do not cause acute toxic effects in organisms when released in the environment. However, many independent studies agree that chronic exposure and more specific endpoints should be used in risk assessment of these compounds. We thus investigated the effects of exposure to environmentally relevant concentrations of the antiepileptic drug carbamazepine (CBZ) on Mediterranean mussels (Mytilus galloprovincialis) by considering the existing knowledge about the therapeutic and side effects of this drug on humans. To do so we analysed: (a) six consolidated biomarkers related primarily to oxidative stress; (b) cAMP levels and protein kinase A (PKA) activities; (c) mRNA expression of MXR-related genes. MXR proteins are involved both in the cAMP pathway and in the protective response of organisms towards xenobiotics. Mussels exposed to 0.1 or 10μg CBZ per liter water for 7 days showed a 60% and 80% reduction in haemocyte lysosome membrane stability, respectively. Moreover, increased neutral lipid and lipofuscin accumulation in the digestive gland, and lipid peroxidation in gills and mantle/gonads were observed. Also glutathione S-tranferase and catalase activities were increased in digestive gland and mantle, while no increase in primary DNA damage was observed. In agreement with the mode of action of CBZ in humans, exposure resulted in a significant reduction in cAMP levels and PKA activities in digestive gland, gills and mantle/gonads of mussels, and lowered the mRNA expression of genes encoding three different MXR-related transporters in the same tissues. Our data indicate that CBZ, at concentrations found in the environment, affects the Mediterranean mussel by acting on specific biochemical pathways that are evolutionarily conserved. [Copyright &y& Elsevier]

Abstract: Filterfeeders, such as bivalves, are highly affected during toxic cyanobacterial blooms, as they are non-selective and may use the cyanobacteria as main nutrition source. The freshwater mussel Dreissena polymorpha, living in lakes and rivers coexisting with cyanobacteria, was exposed to 100 μg L−1 microcystin-LR (MC-LR) for up to three days. MC-LR concentration in mussel tissue and surrounding media was quantified by HPLC-PDA during uptake and depuration phase, revealing an immediate, continuous uptake, and release of non-metabolized toxin, and occurrence of reincorporation. The involvement of multi-xenobiotic-resistance protein (P-glycoprotein, P-gp) on the excretion of MC-LR was evidenced by efflux and accumulation version of the Rhodamine Assay as well as on P-gp gene expression. P-gp expression was enhanced after 1 h exposure but no changes were detected after longer (72 h) exposure. P-gp enzyme activity showed a significant increase with exposure time, supporting the hypothesis that P-gp is involved in the excretion of MC-LR. Induction of biotransformation enzyme such as pi-class glutathione S-transferase (piGST) and antioxidant enzyme catalase (CAT) was immediately inhibited and returned to control values only after more than 72 h expose time. Heat shock protein 70 (hsp70) and protein phosphatase 2A (PP2A) gene expression was not changed due to the treatment with cyanobacterial toxin MC-LR. [Copyright &y& Elsevier]

The Arctic has become a sink for persistent organic pollutants (POPs) originating from lower latitudes, and relatively high levels have been found in different biota. Recent studies have identified detrimental effects on wildlife including endocrine disruption, impairment of enzyme activity, and reduced immune function. The Arctic spider crab, Hyas araneus, shown interesting potential for its use as sentinel organism in polar ecosystems. This study investigated the effect of 2,2′,4,4′-tetra bromo diphenyl ether (BPDE), bisphenol A (BPA), and diallyl phthalte (DPA) on H. araneus in a three weeks exposure study. Expression of multixenibiotic resistance (MXR) proteins has been studied using the C219 monoclonal antibody which allows identifying an immunoreactive protein of 40kDa in the digestive gland while no such protein could be observed in the gills. Expression of this protein was increased by exposure to DPA (+75%; p <0.05, n =10). All compounds significantly affected muscle acetylcholine esterase (AChE) activity (p <0.05, n =10) with 50μg/L DPA having the strongest effect by lowering the value to 37% of control. The total oxyradical scavenging capacity measured in the digestive gland toward peroxyl, hydroxyl and peroxynitrite was also significantly reduced indicating a decreased resistance to oxidative stress generated by DPA (p <0.05, n =5). These results thus suggest the potential detrimental effects of DPA even at concentration as low as 50μg/L on H. araneus. [Copyright &y& Elsevier]

Abstract: The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 μM doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin resistance points to the fragility of Pgp1-mediated MXR defence mechanism in fish. [Copyright &y& Elsevier]

Abstract: 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 CdCl2 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. [Copyright &y& Elsevier]

The biological defence mechanism called MXR or MXD for multixenobiotic resistance or defence protects cells against the entry and the accumulation of xenobiotics. As the defence is modulated by man made chemicals, MXR is used as a biomarker of organisms’ exposure to environmental contamination. However, to reliably assess and evidence MXR induction, the use of a reference level is required. In this context, we focused on MXR background level in a freshwater bivalve, the zebra mussel Dreissena polymorpha, in order to propose its use as a reference during MXR evaluation. We monitored the MXR transport activity in mussels collected either in a natural population or in a caged population and then transplanted to clean water in the laboratory. The results showed that MXR activity was decreased to its baseline level after an eight to nine day depuration period (13. 1±3. 1; 7±2. 6; 13. 7±3. 9pmolRBmin−1 org−1 after three experiments of laboratory depurations). Moreover, significant MXR induction was measured in depurated zebra mussels exposed to contaminated sites (39. 6±3. 7; 59. 2±20. 3pmolRBmin−1 org−1 after two experiments of field exposure), showing that the laboratory depuration did not affect the induction potential. The MXR responses (decrease as well as increase) occurred in few days and were highly significant, highlighting its reactivity in zebra mussels. Finally, this paper confirms the usefulness of MXR as a tool in biomonitoring studies and provides a protocol for field experiments that enables to establish and use the background level of MXR activity as a reference. [Copyright &y& Elsevier]