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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.

WOS:000416186800021; International audience; 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 Paraiba 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 \textgreater colorless spherulocytes \textgreater vibrate cells \textgreater 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.

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 fi sh species are common target in freshwater biomonitoring programs, we have studied the presence of two main MDR/MXR effl ux transporters Pglycoprotein (Pgp, Abcb1) and MRP-like protein(s) (Abcc) in the liver of fi ve 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 specifi c 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 identifi ed 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 fi ve Cyprinid species. These results support that MXR is a defence system mediated by both Pgp and MRP types of ABC transport proteins., Mehanizam multiksenobiotičke otpornosti (MXR) stanični je obrambeni sustav odgovoran za svojstvo istovremene otpornosti na različite ksenobiotike, koje se očituje smanjenjem akumulacije, odnosno povećanjem izbacivanja potencijalno toksičnih tvari iz stanica vodenih organizama. MXR-mehanizam pokazuje odlike analogne fenomenu istovremene otpornosti na različite lijekove (engl. multidrug resistance, MDR) prvi put dokazanom u tumorskim stanicama. Posredovan je istim ABC transportnim proteinima kao i MDR. Istraživanja vezana uz MXR vodenih životinja uglavnom su imala težište na određivanju prisutnosti i funkcije P-glikoproteina (Pgp). Budući da se ribe iz reda šaranki često rabe u biomonitoringu slatkovodnih voda, cilj ovog istraživanja bilo je određivanje prisutnosti dvaju glavnih MDR/MXR-tipova proteina - Pgp i MRP-tip proteina - u jetri iz pet vrsta šaranki; šarana (Cyprinus carpio), klena (Sqalius cephalus), mrene (Barbus barbus), babuške (Carassius auratus gibelio) i podusta (Chondrostoma nasus). Njihova prisutnost utvrđena je na razini mRNA te na proteinskoj razini. Različiti parovi početnica dizajnirani su kako bi se identifi cirali homologni fragmenti gena sličnih MXR-u. Detekcija na razini proteina napravljena je putem Western blot analize s pomoću specifi čnih monoklonskih protutijela proizvedenih da prepoznaju konzervirane epitope; Pgp (C219), MRP1 (MRPm6) ili MRP2 (M2I-4) u sisavaca. Transkripti obaju ključnih MXR-transportera identifi cirani su u svim jedinkama, a napravljena je i fi logenetska analiza dobivenih sekvenci. Imunokemijskom detekcijom s protutijelima sisavaca nismo uspjeli detektirati prisutnost MRP-proteina, dok je Pgp-ekspresija potvrđena u svih pet vrsta šaranki. Nove spoznaje da je za MXRmehanizam zaslužno više transportnih proteina zasigurno će pridonijeti potpunijoj karakterizaciji MXR-a kao integralnog dijela detoksikacijskog, odnosno temeljnog obrambenog sustava vodenih organizama te njegovoj znanstvenoj afi rmaciji kao vjerodostojnog pokazatelja kvalitete okoliša.

Multixenobiotic resistance in aquatic organisms exposed to natural toxins or anthropogenic contaminants is a phenomenon analogous to multidrug resistance in mammalian tumor cell lines resistant to anti-cancer drugs. Both mechanisms of resistance are commonly due to the elevated expression of transmembrane P-glycoprotein (P-gp) which actively transport a wide variety of structurally and functionally diverse compounds. Beside some endogenous substrates numerous drugs and other natural products and environmental contaminants are recognized to be processed by this transporter. P-gp functions as an energy-dependent efflux pump that can limit the bioaccumulation of number xenobiotics thereby actively participating in selective cellular barrier. From 1989 a P-gp-like protein was discovered in a variety of aquatic organisms including sponges, mussels, oysters, clams, worms and fish. Both natural products and anthropogenic contaminants found in the aquatic environment appear to be substrates and inducers of multixenobiotic transporter in aquatic organisms. Increased P-gp activity and protein level in aquatic animal populations from polluted habitats suggests that elevated level of xenobiotic transporter is adaptation response, allowing survival and reproduction in spite of the presence of toxic contaminants. This P-gp induction in mussels and clams is used to develop a new membrane biomarker of exposure. Mechanism by which P-gp transports xenobiotics is still unknown, but there is a class of compounds known as chemosensitizers that can inhibit P-gp activity. The role of chemosensitizers as environmental pollutants and the ecotoxicological consequences of P-gp inhibition are highlighted. Recently another transmembrane proteins so-called multidrug resistance-associated proteins (MRPs) has been identified in some aquatic organisms. Like P-gp MRP related proteins function as energy-dependent drug efflux pumps, conferring similar patterns of resistance by decreasing intracellular concentration of xenobiotics, or even expressing overlapping roles in the protection from xenobiotics. This implies that the nature of multixenobiotic resistance in aquatic organisms is likely to be multi-functional. The proper assessment of the expression and xenobiotic extrusion activity of multixenobiotic resistance proteins in various aquatic species is a challenging (eco)toxicological problem.

Recent evidence indicates that inherent presence of multixenobiotic resistance (MXR) mechanism represents a general biological defence mechanism for protection of organisms against both endogenous and environmental toxins. In aquatic organisms exposed to polluted waters, this P-170 glycoprotein pump also removes ‘new’ man-made toxic chemicals out of cells. Many chemical agents may alter the function of this fragile mechanism. A new class of compounds, referred to as ‘chemosensitizers’, deserve a top rank among environmentally-hazardous chemicals, since they block this basic natural defence mechanism. The authors measured the concentration of such MXR-inhibiting substances by two methods: the ‘binding’ assay and the ‘efflux’ assay. The ‘efflux’ assay of water from the polluted Sava and Rhein Rivers and the unpolluted Korona River revealed the presence of 440, 489, and 176 ppb of verapamil equivalents of MXR-inhibitors, respectively. These concentrations of MXR-inhibitors obviously may revert the natural resistance of organisms inhabiting these rivers to a pathobiologic sensitivity.