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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]

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]

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]

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]

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]

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]

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]

Copyright of Archives of Industrial Hygiene & Toxicology / Arhiv za Higijenu Rada I Toksikologiju is the property of Sciendo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

Copyright of Archives of Industrial Hygiene & Toxicology / Arhiv za Higijenu Rada I Toksikologiju is the property of Sciendo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

Synthetic musk compounds, widely used as fragrances in consumer products, have been detected in human tissue and, surprisingly, in aquatic organisms such as fish and mollusks. Although their persistence and potential to bioaccumulate are of concern, the toxicity and environmental risks of these chemicals are generally regarded as low. Here, however, we show that nitromusks and polycyclic musks inhibit the activity of multidrug efflux transporters responsible for multixenobiotic resistance (MXR) in gills of the marine mussel Mytilus californianus. The IC10 (concentration that inhibits 10%) values for the different classes of musks were in the range of 0.09–0.39 μM, and IC50 values were 0.74–2.56 μM. The immediate consequence of inhibition of efflux transporters is that normally excluded xenobiotics will now be able to enter the cell. Remarkably, the inhibitory effects of a brief 2-hr exposure to musks were only partially reversed after a 24- to 48-hr recovery period in clean seawater. This unexpected consequence of synthetic musks—a long-term loss of efflux transport activity—will result in continued accumulation of normally excluded toxicants even after direct exposure to the musk has ended. These findings also point to the need to determine whether other environmental chemicals have similar long-term effects on these transporters. The results are relevant to human health because they raise the possibility that exposure to common xenobiotics and pharmaceuticals could cause similar long-term inhibition of these transporters and lead to increased exposure to normally excluded toxicants. [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. [ABSTRACT FROM AUTHOR]