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The zebrafish embryo (Danio rerio) has developed into one of the most important nonsentient animal models for the hazard assessments of chemicals, but the processes governing its toxicokinetics (TK) are poorly understood. This study compares the uptake of seven test compounds into the embryonic body and the yolk sac of the zebrafish embryo using TK experiments, a dialysis approach, thermodynamic calculations, and kinetic modeling. Experimental data show that between 95% (4-iodophenol) and 67% (carbamazepine) of the total internal amount in 26 h post fertilization (hpf) embryos and between 80 and 49% in 74 hpf embryos were found in the yolk. Thus, internal concentrations determined for the whole embryo overestimate the internal concentration in the embryonic body: for the compounds of this study, up to a factor of 5. Partition coefficients for the embryonic body and a one-compartment model with diffusive exchange were calculated for the neutral test compounds and agreed reasonably with the experimental data. For prevalently ionic test compounds at exposure pH (bromoxynil, paroxetine), however, the extent and the speed of uptake were low and could not be modeled adequately. A better understanding of the TK of ionizable test compounds is essential to allow assessment of the validity of this organismic test system for ionic test compounds.

Lake Baikal is inhabited by more than 300 endemic amphipod species, which are narrowly adapted to certain thermal niches due to the high interspecific competition. In contrast, the surrounding freshwater fauna is commonly represented by species with large-scale distribution and high phenotypic thermal plasticity. Here, we investigated the thermal plasticity of the energy metabolism in two closely-related endemic amphipod species from Lake Baikal (Eulimnogammarus verrucosus; stenothermal and Eulimnogammarus cyaneus; eurythermal) and the ubiquitous Holarctic amphipod Gammarus lacustris (eurythermal) by exposure to a summer warming scenario (6–23.6 °C; 0.8 °C d−1). In concert with routine metabolic rates, activities of key metabolic enzymes increased strongly with temperature up to 15 °C in E. verrucosus, whereupon they leveled off (except for lactate dehydrogenase). In contrast, exponential increases were seen in E. cyaneus and G. lacustris throughout the thermal trial (Q10-values: 1.6–3.7). Cytochrome-c-oxidase, lactate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase activities were found to be higher in G. lacustris than in E. cyaneus, especially at the highest experimental temperature (23.6 °C). Decreasing gene expression levels revealed some thermal compensation in E. cyaneus but not in G. lacustris. In all species, shifts in enzyme activities favored glycolytic energy generation in the warmth. The congruent temperature-dependencies of enzyme activities and routine metabolism in E. verrucosus indicate a strong feedback-regulation of enzymatic activities by whole organism responses. The species-specific thermal reaction norms reflect the different ecological niches, including the spatial distribution, distinct thermal behavior such as temperature-dependent migration, movement activity, and mating season.

The photomotor response (PMR) of zebrafish embryos, a light pulse-triggered undirected movement, is known to be altered by neuroactive chemicals. Here, we developed an approach for data analysis of the distribution of PMR movement activities along the time axis; differences between treatment and respective controls are expressed by an aggregated value integrating the time-resolved density of the movement parameter as a measure for a chemically elicited PMR effect. Logistic concentration-PMR effect relationships were modeled for neuroactive test compounds with different modes of action (acetylcholinesterase inhibition, activation and inhibition of voltage-gated sodium channels); 50% effect concentrations (EC50) were in the low to medium μM range (EC5010 μM for flucythrinate, esfenvalerate, azinphos-methyl, propoxur; EC5010 μM for tricaine). Modulation of movement activities in different phases of the PMR (i.e., “fingerprint”) by neuroactive test compounds varied across concentrations, showing that mode of action-specific PMR fingerprints are also concentration-dependent. Above concentrations causing 10% lethality (LC10; 48 h), 3,4-dichlo­roaniline caused movement inhibition. This substance presumably is not neuroactive; its effect on the PMR therefore is considered a secondary toxic effect. Quantitative morphological examinations of chemically exposed embryos showed that malformations occurred only above PMR effect concentrations, indicating that changes in the PMR were not due to such indirect effects. The PMR assay will provide a useful measure in ecotoxicological risk assessment of neuroactive chemicals with zebrafish embryos and could potentially be used to infer acute fish toxicity levels from PMR effect concen­trations of neurotoxic compounds.

ATP binding cassette (ABC) transporter proteins include efflux pumps that confer multixenobiotic resistance to zebrafish embryos, a valuable toxico/pharmacological model. Here, we established an automated microscopy-based rhodamine B dye accumulation assay in which enhanced dye accumulation in live zebrafish embryos indicates inhibition of multixenobiotic efflux transporter activity. Twenty structurally divergent known substrates and/or inhibitors of human ABC transporters and environmentally relevant compounds were examined using this assay and the ATPase activity of recombinant zebrafish Abcb4 as readouts. These two assays confirmed that Abcb4 functions as an efflux transporter in zebrafish, whereas they gave discordant results for some of the tested substances. The dye accumulation assay in zebrafish embryos could be useful to screen environmental pollutants and other chemicals for efflux transporter interaction in a medium-throughput fashion.

The induction of cellular stress response systems, heat shock proteinhsp70/Hsp70 and multixenobiotic transporterabcb1, by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl2, the sensitivities of the different species to CdCl2were ranked (24 hr LC50 in mg/L CdCl2(mean/95% confidence interval)):Gammarus lacustris(1.7/1.3–2.4)

The zebrafish embryo is an important model in ecotoxicology but the spatial distribution of chemicals and the relation to observed effects is not well understood. Quantitative imaging can help to gain insights into the distribution of chemicals in the zebrafish embryo. Laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) is used to quantify the uptake and the uptake kinetics of the bromine (Br) containing organophosphate naled (Dibrom®, dimethyl-1,2-dibromo-2,2-dichloroethylphosphate) and its distribution in zebrafish embryos using Br as the marker element. During exposure, the Br amounts increase in the embryos parallel to the irreversible inhibition of the acetylcholinesterase (AChE). The final amount of Br in the embryo (545 pmol/embryo) corresponds to a 280-fold enrichment of naled from the exposure solution. However, LC-MS/MS analyses showed that the internal concentration of naled remained below the LOD (7.8 fmol/embryo); also the concentration of its known transformation product dichlorvos remained low (0.85 to 2.8 pmol/embryo). These findings indicate the high reactivity and high transformation rate of naled to other products than dichlorvos. 12C normalized intensity distributions of Br in the zebrafish embryo showed an enrichment of Br in its head region. Kernel density estimates of the LA-ICP-MS data were calculated and outline the high reproducibility between replicated and the shift in the Br distribution during exposure. The Br enrichment indicates a preferential debromination or direct covalent reaction of naled with AChE in this region. Graphical abstractᅟ Electronic supplementary material The online version of this article (10.1007/s00216-018-1471-2) contains supplementary material, which is available to authorized users.

The exposome encompasses an individual's exposure to exogenous chemicals, as well as endogenous chemicals that are produced or altered in response to external stressors. While the exposome concept has been established for human health, its principles can be extended to include broader ecological issues. The assessment of exposure is tightly interlinked with hazard assessment. Here, we explore if mechanistic understanding of the causal links between exposure and adverse effects on human health and the environment can be improved by integrating the exposome approach with the adverse outcome pathway (AOP) concept that structures and organizes the sequence of biological events from an initial molecular interaction of a chemical with a biological target to an adverse outcome. Complementing exposome research with the AOP concept may facilitate a mechanistic understanding of stress-induced adverse effects, examine the relative contributions from various components of the exposome, determine the primary risk drivers in complex mixtures, and promote an integrative assessment of chemical risks for both human and environmental health. Keywords: Exposome, AOP, Systems toxicology, Systems biology, Systems chemistry, Risk assessment

Polyaromatic hydrocarbons (PAH) are common pollutants of water ecosystems originating from incineration processes and contamination with mineral oil. Water solubility of PAHs is generally low; for toxicity tests with aquatic organisms, they are therefore usually dissolved in organic solvents. Here we examined the effects of a typical model PAH, phenanthrene, and a solvent, acetone, on amphipods as relevant aquatic invertebrate models. Two of these species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, are common endemics of the oligotrophic and pristine Lake Baikal, while one, Gammarus lacustris, is widespread throughout the Holarctic and inhabits smaller and more eutrophic water bodies in the Baikal area. Neither solvent nor phenanthrene caused mortality at the applied concentrations, but both substances affected gene expression in all species. Differential gene expression was more profound in the species from Lake Baikal than in the Holarctic species. Moreover, in one of the Baikal species, E. cyaneus, we found that many known components of the cellular xenobiotic detoxification system reacted to the treatments. Finally, we detected a negative relationship between changes in transcript abundances in response to the solvent and phenanthrene. This mixture effect, weaker than the impact by a single mixture component, needs further exploration.

Earlier studies have shown that perfluorooctane sulfonate (PFOS) increases the toxicity of other chemicals by enhancing their uptake by cells and tissues. The present study aimed at testing whether the underlying mechanism of enhanced uptake of chemicals by zebrafish (Danio rerio) embryos in the presence of PFOS is by interference of this compound with the cellular efflux transporter Abcb4. Modifications of uptake/clearance and toxicity of two Abcb4 substrates, the fluorescent dye rhodamine B (RhB) and vinblastine, by PFOS were evaluated using 24 and 48h post-fertilization (hpf) embryos. Upon 90min exposure of 24hpf embryos to 1μM RhB and different PFOS concentrations (3-300μM) accumulation of RhB in zebrafish was increased by up to 11.9-fold compared to controls, whereas RhB increases in verapamil treatments were 1.7-fold. Co-administration of PFOS and vinblastine in exposures from 0 to 48hpf resulted in higher vinblastine-caused mortalities in zebrafish embryos indicating increased uptake of this compound. Interference of PFOS with zebrafish Abcb4 activity was further studied using recombinant protein obtained with the baculovirus expression system. PFOS lead to a concentration-dependent decrease of the verapamil-stimulated Abcb4 ATPase activity; at higher PFOS concentrations (250, 500μM), also the basal ATPase activity was lowered indicating PFOS to be an Abcb4 inhibitor. In exposures of 48hpf embryos to a very high RhB concentration (200μM), accumulation of RhB in embryo tissue and adsorption to the chorion were increased in the presence of 50 or 100μM PFOS. In conclusion, the results indicate that PFOS acts as inhibitor of zebrafish Abcb4; however, the exceptionally large PFOS-caused effect amplitude of RhB accumulation in the 1μM RhB experiments and the clear PFOS effects in the experiments with 200μM RhB suggest that an additional mechanism appears to be responsible for the potential of PFOS to enhance uptake of Abcb4 substrates.

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 μg L–1); E. verrucosus: 115 nM (12.9 μg L–1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 μg g–1; E. verrucosus: 0.26 ± 0.07 μg g–1), whereas relative Cd amounts in the biologically detoxified heat stable pr...

Cellular multixenobiotic resistance (MXR) transport proteins enhance the efflux of numerous organic pollutants. However, MXR proteins may be blocked or saturated by xenobiotic compounds, acting as inhibitors - also called chemosensitisers. Although effective on a cellular level, the environmental relevance of chemosensitisers has not been conclusively demonstrated. Since sediments are an important source of bioaccumulating compounds in aquatic ecosystems, sediments and sediment-associated hydrophobic pollutants were investigated for their potential to increase exposure and toxicity in the presence of chemosensitisation. In this study, we address this issue by (1) comparing the net uptake of 17 hydrophobic environmental pollutants by zebrafish (Danio rerio) embryos in the presence and absence of the model chemosensitiser verapamil and (2) investigating the impact of verapamil on the dose-dependent effect on zebrafish embryos exposed to polluted sediment extracts. None of the 17 pollutants showed a reproducible increase in bioaccumulation upon chemosensitisation with verapamil. Instead, internal concentrations were subject to intra-species variation by a factor of approximately two. However, a significant increase in toxicity was observed upon embryo co-exposure to verapamil for one of three sediment extracts. In contrast, another sediment extract exhibited less toxicity when combined with verapamil. In general, the results indicate only a minor impact of verapamil on the uptake of moderately hydrophobic chemicals in zebrafish embryos.

Most members of the large ATP-binding cassette (ABC) gene family are transporters involved in substrate translocation across biological membranes. In eukaryotes, ABC proteins functioning as drug transporters are located in the plasma membrane and mediate the cellular efflux of a wide range of organic chemicals, with some transporters also transporting certain metals. As the enhanced expression of ABC drug transporters can confer multidrug resistance (MDR) to cancers and multixenobiotic resistance (MXR) to organisms from polluted habitats, these ABC family members are also referred to as MDR or MXR proteins. In mammals, ABC drug transporters show predominant expression in tissues involved in excretion or constituting internal or external body boundaries, where they facilitate the excretion of chemicals and their metabolites, and limit chemical uptake and penetration into “sanctuary” sites of the body. Available knowledge about ABC proteins is still limited in teleost fish, a large vertebrate group of high ecological and economic importance. Using transport activity measurements and immunochemical approaches, early studies demonstrated similarities in the tissue distribution of ABC drug transporters between teleosts and mammals, suggesting conserved roles of the transporters in the biochemical defence against toxicants. Recently, the availability of teleost genome assemblies has stimulated studies of the ABC family in this taxon. This review summarises the current knowledge regarding the genetics, functional properties, physiological function, and ecotoxicological relevance of teleostean ABC transporters. The available literature is reviewed with emphasis on recent studies addressing the tissue distribution, substrate spectrum, regulation, physiological function and phylogenetic origin of teleostean ABC transporters.

The water flea Daphnia magna is widely used as test species in ecotoxicological bioassays. So far, there is no information available to which extent ATP binding cassette (ABC) transporter based multixenobiotic resistance (MXR) counteracts adverse chemical effects in this species. This, however, would be important for assessing to which extent the bio-active potential of a compound determined with this species depends on this cellular defense. We here present molecular, functional and toxicological studies that provide first evidence for ABC transporter-based MXR in D. magna. We cloned putatively MXR-related partial abcb1, abcc1/3, abcc4 and abcc5 coding sequences; respective transcripts were constitutively expressed in different D. magna life stages. MXR associated efflux activity was monitored in D. magna using the fluorescent substrate dyes rhodamine 123, rhodamine B and calcein-AM combined with inhibitors of human ABCB1 and/or ABCC transporter activities reversin 205, MK571 and cyclosporin A. With inhibitors present, efflux of dye substrates was reduced in D. magna in a concentration-dependent mode, as indicated by elevated accumulation of the dyes in D. magna tissues. In animals pre-exposed to mercury, pentachlorophenol or dacthal applied as inducers of ABC transporter expression, levels of some ABC transporter transcripts were increased in some cases showing that these genes can be chemically induced. Likewise, pre-exposure of animals to these chemicals decreased dye accumulation in tissue, indicating enhanced MXR transporter activity, likely associated with higher transporter protein levels. Toxicity assays with toxic transporter substrates mitoxantrone and chlorambucil that were applied singly and in combination with inhibitors were performed to study the tolerance role of Abcb1 and Abcc efflux transporters in D. magna. Joint toxicities of about half of the binary combinations of test compounds applied (substrate/inhibitor, substrate/substrate, inhibitor/inhibitor) were greater than joint effects predicted with mixture toxicity models, which can be explained by chemosensitization through MXR efflux transporter interference. Our data indicate the presence of an MXR efflux system in D. magna. It needs to be considered when assessing the bioactive potential of test compounds with this species. Further, chemosensitization may explain joint toxicities of compound mixtures to D. magna that are higher than expected.

Temperature is the most pervasive abiotic environmental factor for aquatic organisms. Fluctuations in temperature range lead to changes in metabolic performance. Here, we aimed to identify whether surpassing the thermal preference zones is correlated with shifts in universal cellular stress markers of protein integrity, responses to oxidative stress and lactate content, as indicators of anaerobic metabolism. Exposure of the Lake Baikal endemic amphipod species Eulimnogammarus verrucosus (Gerstfeldt, 1858), Ommatogammarus flavus (Dybowski, 1874) and of the Holarctic amphipod Gammarus lacustris Sars 1863 (Amphipoda, Crustacea) to increasing temperatures resulted in elevated heat shock protein 70 (Hsp70) and lactate content, elevated antioxidant enzyme activities (i.e., catalase and peroxidase), and reduced lactate dehydrogenase and glutathione S-transferase activities. Thus, the zone of stability (absence of any significant changes) of the studied molecular and biochemical markers correlated with the behaviorally preferred temperatures. We conclude that the thermal behavioral responses of the studied amphipods are directly related to metabolic processes at the cellular level. Thus, the determined thermal ranges may possibly correspond to the thermal optima. This relationship between species-specific behavioral reactions and stress response metabolism may have significant ecological consequences that result in a thermal zone-specific distribution (i.e., depths, feed spectrum, etc.) of species. As a consequence, by separating species with different temperature preferences, interspecific competition is reduced, which, in turn, increases a species’ Darwinian fitness in its environment.

Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.

Morphology and physiology of fish embryos undergo dramatic changes during their development until the onset of feeding, supplied only by endogenous yolk reserves. For obtaining an insight how these restructuring processes are reflected by body mass related parameters, dry weights (dw), contents of the elements carbon and nitrogen and lipid and protein levels were quantified in different stages within the first four days of embryo development of the zebrafish (Danio rerio). The data show age dependent changes in tissue composition. Dry weights decreased significantly from 79μgdw/egg at 0hours post fertilization (hpf) to 61 μgdw/egg after 96 hpf. The amounts of total carbon fluctuated between 460 mg g-1 and 540 mg g-1 dw, nitrogen was at about 100 mg g-1 dw and total fatty acids were between 48-73 mg g-1 dw. In contrast to these parameters that remained relatively constant, the protein content, which was 240 mg g-1 at 0 hpf, showed an overall increase of about 40%. Comparisons of intact eggs and dechorionated embryos at stages prior to hatching (24, 30, 48 hpf) showed that the differences seen for dry weight and for carbon and nitrogen contents became smaller at more advanced stages, consistent with transition of material from the chorion to embryo tissue. Further, we determined the effect of 2,4-dinitrophenol at a subacutely toxic concentration (14 μM, LC10) as a model chemical challenge on the examined body mass related parameters. The compound caused significant decreases in phospholipid and glycolipid fatty acid contents along with a decrease in the phospholipid fatty acid unsaturation index. No major changes were observed for the other examined parameters. Lipidomic studies as performed here may thus be useful for determining subacute effects of lipophilic organic compounds on lipid metabolism and on cellular membranes of zebrafish embryos.

To determine the interactions between temperature and cadmium on zebrafish ( Danio rerio ) development, fertilized eggs were exposed to combinations of three temperature levels (21 °C, 26 °C, and 33 °C) and six cadmium concentrations (0, 0.25, 0.5, 2.0, 5.0, and 10.0 mg/L). Endpoints used included LC 50 value (48 h), developmental rate, mortality, heart rate, hatching success, liver histopathology, embryo abnormalities, and heat shock protein (hsp) induction. Results showed a significant acceleration in the developmental rate with increasing temperature and irrespective of the presence of cadmium. Data on LC 50 and ELS-test revealed that simultaneous exposure to both cadmium ions and cold stress (21 °C) was highly detrimental to growing embryos, causing a pronounced mortality and a significant reduction in average heart rate and embryo hatchability. In contrast, no similar reactions to cadmium were observed in pre-hatched embryos exposed to both control (26 °C) and high temperature (33 °C), and this can be explained by the significantly higher expression of hsp (hsp70) in embryos at these temperatures. Upon hatching, however, the larvae showed increased sensitivity to cadmium. The severity of malformations in the post-hatched larvae was in the order: hot cadmium stress>cold cadmium stress>cadmium stress alone>no stress at all. Liver histopathology as well as depletion in glycogen reserves exhibited greater severity with increasing cadmium concentration, irrespective of temperature. The present study confirms that temperature effectively confounds cadmium toxicity and needs to be considered for the accurate prediction and assessment of cadmium-induced toxicity in fish.

We studied interactions of nitromusk compounds musk ketone and musk xylene and polycyclic musks Galaxolide trade mark (HHCB), Celestolide trade mark (ADBI), Tetralide trade mark (AHTN), and Traseolide trade mark (AITI) with multixenobiotic resistance (mxr) transporters in gill tissue of the marine mussel Mytilus californianus (Conrad, 1837). A competitive substrate transport test with rhodamine B was used to assay modulation of transport activity by musks. All tested musks inhibited the transport activity in the low microm range as indicated by increased accumulation of rhodamine B in the tissue. Compared to known substrates of mxr transporters, the effective concentration range was similar to quinidine and about 100 times higher than verapamil. Musk ketone and musk xylene also inhibited efflux of rhodamine B from gill tissue which was loaded with the dye and subsequently incubated with these compounds. Synthetic musk compounds are persistent environmental pollutants in aquatic environments with a high potential to bioaccumulate. As potent inhibitors of mxr transporters they may also play a role as chemosensitizers that enable toxic mxr substrates to accumulate in cells of aquatic organisms.

Brown trout (Salmo trutta f. fario L.) early life stages were studied for physiological effects caused by chronic exposure to sub-acute levels of unionised ammonia, a mixture of PCP and PAHs, and a combination of ammonia and the mixture of organics during the entire embryonic development. Nominal concentrations of tested compounds were based on field data. Accumulation data for PAHs and PCP in trout tissue reflected respective water concentrations of PCP and PAHs. Physiological responses were studied by early life stage tests (ELST) and by the analysis of the 70 kDa stress protein (hsp70). Endpoint responses in the ELST were: accelerated development, pre-hatching, and increased heart rates. For these endpoints, response levels were highest in the ammonia treatment, followed by the exposure to the PCP/PAH mixture. Weight was reduced in embryos treated with the PCP/PAH mixture, but not in the group treated with this mixture combined with ammonia. Induction of hsp70 by the test agents was found to be stage-specific with increased response levels at advanced developmental stages. In both the ELST and hsp70 analysis, response levels were lower in the combined ammonia/PCP/PAH treatment than in groups treated with either ammonia or the PCP/PAH mixture alone.

Early life stage (ELS) studies with brown trout(Salmo trutta f. fario L.) andstone loach (Barbatula barbatula L.)were performed between 1995 and 2000 toevaluate embryotoxic potentials in twodifferently polluted streams in southwestGermany. With both species, semistatic exposureexperiments with water samples and sedimenteluates were conducted in the laboratory.Additionally, brown trout ELS tests wereperformed in flow-through systems in thesemi-field and in the field. Thus, differentlevels of complexity of environmentalconditions were addressed which allowed thestudy of effects of xenobiotic contamination,temperature, and sediments on the success ofembryonic development. Additionally, effects ofwater from the polluted stream on fertilizationof brown trout eggs were determined. In themore polluted stream, xenobiotics caused anembryotoxic potential for both brown trout andstone loach, and viability of exposed browntrout eggs was drastically reduced by suspendedsolids in the water which covered the eggs.Additionally, fertilization rates of browntrout eggs were significantly decreased inwater of the more polluted stream. In the lesspolluted stream, low water temperature andinfestations by protozoic ectoparasites causedmortality of embryos. In this stream, pollutionand sediment effects were not observed. Resultsmade evident that in the more polluted streamrecruitment of brown trout was drasticallyimpaired.

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1–3 d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.

Aquatic organisms and, in particular, filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and, presumably, require adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP-binding cassette (ABC) transporters protects mussels against toxicants, but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression, and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiotic transporter types in the gills, where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-glycoprotein and ABCC/MRP-type transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to the ABCB inhibitor PSC-833 and the ABCC inhibitor MK-571. This activity profile is supported by our cloning of the complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity of the amino acid sequences with corresponding homologs from other organisms was 38–50% (ABCB) and 27–44% (ABCC). C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills, providing protection against environmental xenotoxicants.

In his letter on our recent article in EHP (Luckenbach and Epel 2005), Salvito raises important questions about effects of the synthetic musk fragrances regarding a) human and environmental health effects, b) environmental concentrations of the musks, and c) uniqueness of inhibition of efflux transporters to the musks, the effect we described in our article. a) Regarding health issues, we agree with Salvito that the available evidence indicates minimal direct affects of most synthetic musks on the health of humans and aquatic organisms. However, our data expand the definition of toxicity and detrimental effects to indirect and unanticipated consequences of these chemicals, even if the chemical itself might be nontoxic. The major point of our article (Luckenbach and Epel 2005) was that the musks inhibit efflux (drug) transporters, which act as first lines of defense to pump potentially toxic substances out of cells. These efflux transporters are ubiquitous and are found in bacteria, fungi, plants, and animals, including humans. The transporters have wide substrate specificity, and this binding to many compounds can result in inhibition of activity by competing substrates. As a consequence of transporter inhibition, cells and organisms can therefore become exposed to toxicants normally kept out of their cells. An unexpected finding was not only that the musks inhibit these transporters in marine mussels but that the effect is long-term and persists up to 24–48 hr after removal of the musk compounds. These indirect and long-term toxicity effects are of particular concern because these chemicals are stable and bioaccumulate; for example, musk xylene has a half-life of 70 days in human tissue (Riedel and Dekant 1999). Effects on human transporters by the musks cannot be inferred from our results, but they do point to the possibility of an interaction, considering the general property of the transporters to recognize a wide array of substrates. Therefore—and in light of accumulation of the musks in human tissue—research is needed to determine if the musks similarly inhibit the human efflux transporters, thereby compromising this defense against toxicants. b) The musks are of environmental concern because they enter the water column from incomplete degradation in sewage plants. We agree with Salvito that the reported levels in surface waters are extremely low (picomolar range) but disagree with his conclusion that such levels indicate that musks are not a problem. In spite of these low environmental levels, there is significant bioaccumulation of these chemicals in tissues of mussels and fish, and just several months ago Nakata (2005) reported significant bioaccumulation in cetaceans. The concentrations in aquatic organisms can become quite high, being on the order of nanograms per gram fresh weight, which translates to about 0.1 μM final concentration in tissue (Nakata 2005; Rimkus 1999; Yamagishi et al. 1983). According to Salvito, worldwide production of synthetic musks are only about one-half of the amount we cite. These lower numbers are even more worrisome because because this means that the potency of the musks to bioaccumulate is even higher. c) Salvito points out that the inhibition of transporters is not unique to the musks. We agree and note that the observed inhibition of efflux transporter activity by the musks may be the tip of the iceberg. As with the musks, there may be many chemicals that by themselves are not toxic but similarly inhibit the efflux transporters and thereby expose the organism to normally excluded toxicants. In summary, the available data suggest that efflux transporter inhibition could be a significant indirect, negative, and unappreciated effect of environmental chemicals. Several questions need to be answered: Do these chemicals inhibit human transporters? Are there other anthropogenic and natural products that inhibit these transporters in aquatic organisms and also in humans? Should anthropogenic chemicals be screened for inhibitory activity? If so, should there be voluntary or governmental regulations to ensure that such chemicals do not affect the health of exposed populations through these indirect actions?

A recent article by Luckenbach and Epel (2005) on in vitro observations of inhibitory properties exhibited by certain nitromusk and polycyclic musk fragrance ingredients on mussel cells raised some concerns regarding potential environmental risks and safety to humans that may be associated with nitromusk and polycyclic musk compounds. The Research Institute for Fragrance Materials would like to address several points that may help readers more clearly understand the meaning and context of the reported research. The tonnages of musk compounds reported by Luckenbach and Epel (2005) in their article (7,000–8,000 tons) are higher than the industry-reported global tonnage of these materials. From 1995 to 2000, the total worldwide usage declined from 300 tons to 200 tons for musk xylene and musk ketone combined. The 2000 worldwide use of polycyclic musks is approximately 4,000 tons. Measured concentrations of these compounds in the environment are less than the effects concentrations reported by Luckenbach and Epel (2005). In a review of measured environmental concentrations, Rimkus (1999) stated that the highest reported measurement of hexahydrohexamethyl-cyclopenta (γ)-2-benzopyran (HHCB) in surface water was 12.5 μg/L (0.048 μM). The IC50 (concentration that inhibits 50%) reported for polycyclic musks was 2.34 μM. Overall, measured environmental concentrations were 2–6 orders of magnitude lower than the effects concentrations reported by Luckenbach and Epel (2005). The data reported by Luckenbach and Epel (2005) reflect a method under development. There are many steps between the observation of an in vitro effect and effects on whole organisms, ecosystems, and humans. In vivo studies in mussels and studies linking mussel gill tissue to undefined tissues in mammals and humans are some of the research necessary to conclude that these higher level effects may exist. These effects would then need to be placed into a risk-based context by comparing them to exposure concentrations. The safety of nitromusk and polycyclic musk compounds for humans has been extensively tested and affirmed by numerous regulatory agencies and academic scientists around the world [Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) 2002a, 2002b]. The trace environmental levels of the musks continue to be investigated, and environmental safety and monitoring studies are ongoing so that the public can be assured of their safety. Regarding the environmental effects of synthetic musks, the IC10 (concentration that inhibits 10%) values should be compared to no observed effect concentrations (NOECs). The IC10 values of the synthetic musks are around the level of the lowest in vivo NOECs observed for aquatic organisms. Table 1 shows that the in vitro multi-xenobiotic resistance (MXR) transporter activity in mussel gill is of the same sensitivity as the effects observed in the standard toxicity tests with aquatic organisms. Thus, at the exposure level where the protective transporter efflux is decreased, rendering the cell more accessible to other potential toxicants, the effects of the synthetic musks are also indicated in other end points, such as development and growth. Table 1 Intensity of in vitro MXR transporter activity in mussel gill. The observed effects are not limited to the nitromusk and polycyclic musk compounds. For example, the other chemicals used by Luckenbach and Epel (2005)—verapamil and quinidine—also produced the phenomenon. In the case of verapamil, the IC10 was reported at 1–2 orders of magnitude below the nitromusks and polycyclic musks. I look forward to continued discussions with the Luckenbach and Epel to determine the relevance of the results of this study.

The objective of the present study was to review the current knowledge regarding the bioaccumulation potential of ionizable organic compounds (IOCs), with a focus on the availability of empirical data for fish. Aspects of the bioaccumulation potential of IOCs in fish that can be characterized relatively well include the pH dependence of gill uptake and elimination, uptake in the gut, and sorption to phospholipids (membrane-water partitioning). Key challenges include the lack of empirical data for biotransformation and binding in plasma. Fish possess a diverse array of proteins that may transport IOCs across cell membranes. Except in a few cases, however, the significance of this transport for uptake and accumulation of environmental contaminants is unknown. Two case studies are presented. The first describes modeled effects of pH and biotransformation on the bioconcentration of organic acids and bases, while the second employs an updated model to investigate factors responsible for accumulation of perfluorinated alkyl acids. The perfluorinated alkyl acid case study is notable insofar as it illustrates the likely importance of membrane transporters in the kidney and highlights the potential value of read-across approaches. Recognizing the current need to perform bioaccumulation hazard assessments and ecological and exposure risk assessment for IOCs, the authors provide a tiered strategy that progresses (as needed) from conservative assumptions (models and associated data) to more sophisticated models requiring chemical-specific information. Environ Toxicol Chem 2017;36:882-897. © 2016 SETAC.

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., BMC Biology, 11, ISSN:1741-7007