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A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub- lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2), trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters – P- glycoprotein (ABCB1/P-gp) and sulfonylurea receptor like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the 2-cell, as well as an overall decrease during the blastulae stage. Calcein- AM efflux activity assay indicated the activation of multidrug resistance-associated protein (MRP)/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the 2-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI- inducible ABC membrane transporters, coexpressed in the zygote, 2-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil- inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100 nM and 10 μM BPA up to 10 days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50 μg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi- quantitative scoring system. Finally, behavioural changes were detected after only 3 days of exposure to 100 nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses.

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change.

The main goals of this study were (1) to standardize a simple and reliable embryotoxicity test for environmental contaminants and (2) to evaluate the presence and possible protective role of the multidrug resistance-associated (MRP) protein-mediated multixenobiotic defense in two Mediterranean sea urchin species, Paracentrotus lividus and Arbacia lixula. Toxic end-point used was the success of the first cell division in sea urchin embryos. Embryotoxicities of three environmentally relevant contaminants: mercuric chloride (HgCl2, 0.05–6 μM), trybutiltin (TBT, 2.5–500 nM), and oxybenzone (OXI, 0.1–100 μM); as well as seawater samples collected from the polluted and unpolluted locations, were determined and compared. A. lixula embryos were more sensitive to all three toxic compounds, and both P. lividus and A. lixula embryos were highly sensitive to TBT at nanomolar concentrations (EC50 49 ± 5 and 36.8 ± 3 nM, respectively). Inhibition of MRP protein by specific inhibitor MK571 caused significant increase in embryotoxic potency of HgCl2 (EC50 0.697 ± 0.03 and 0.245 ± 0.04 μM, respectively), TBT (EC50 24 ± 3 and 7.4 ± 1 nM, respectively) and polluted seawater sample, but not of OXI or unpolluted, natural seawater. Therefore, our results demonstrated for the first time the protective relevance of MRP proteins in early development of these two Mediterranean sea urchin species. Finally, the embryotoxicity protocol described in this study represents a simple and rapid bioassay for determination of environmentally relevant seawater contamination.

Mussel (Mytilus galloprovincialis (Lamarck, 1819)) is directly exposed to sea water contamination that elicits significant physiological and cellular re- sponse, although its extent mounted in aquaculture- reared in comparison to wild bivalve populations is scarcely known. Therefore, we have compared contam- ination biomarkers in mussels from reared (Marina farm) and wild, anthropogenically affected site (Vranjic Bay). While predictably, the levels of metals (Cu, Cd, Pb, Zn, Fe, and Hg) in whole bivalve tissues determined by atomic absorption spectrophotometry re- sulted in significantly higher concentrations in wild mussels, accompanied by elevated number of apoptotic cells in gills, the activity of multixenobiotic resistance defense mechanism (MXR), measured as the accumulation rate of model substrate rhodamine B (RB) gave contrasting results. The functional RB assay evidenced a lower MXR efflux activity in the gill tissue of wild mussels, indicating two possible scenarios that willneedfurther focus:(1) persistingseawaterpollution increased cell damage of bivalve gill cells and conse- quently led to leakage of the RB into cytoplasm and dysfunctional MXR efflux in wild mussels; or/and (2) a mixture of different toxic compounds present in Vranjic Bay sea water induced oversaturation of MXR efflux, inducing elevated accumulation of the dye. Consequently, it seems that an efficient physiological functioning of MXR in wild mussels is strongly ham- pered by existence of an unknown quantity of sea water pollutants that may endanger intrinsic organismal de- fense system and lead toward the enhancement of toxicity.

Multixenobiotic resistance (MXR) represents an important cellular detoxification mechanism in aquatic organisms as it provides them robustness toward natural and man-made contaminants. Several ABC transporters have major roles in the MXR phenotype – P-gp/ABCB1, MRP1–3/ABCC1–3 and BCRP/ABCG2. In this study, we identified the presence of ABC transporters involved in the MXR mechanism of Arbacia lixula and Paracentrotus lividus. AlABCB1/P-gp, AlABCC3/MRP3, AlABCC9/SUR-like and AlABCG-like transcripts were identified in A. lixula; and PlABCC1/P-gp, PlABCC3/MRP3, PlABCC5/MRP5, and PlABCC9/SUR-like transcripts in P. lividus. For each of the new partial sequences, we performed detailed phylogenetic and identity analysis as a first step toward full characterization and understanding of the ecotoxicological role of these ABC transporters.