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The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na

The biodegradation of biorecalcitrant opioid drug tramadol (TRAM) was studied in a model biodegradation experiment performed with an enriched activated sludge culture pre-adapted to high concentration of TRAM (20 mg/L). TRAM and its transformation products (TPs) were determined by applying ultrahigh-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS), the sludge culture was characterized using a 16S rRNA gene amplicon sequencing, whereas ecotoxicological evaluation was performed based on determination of toxicity to freshwater algae. Tramadol removal was much faster (t1/2 = 1.3 days) and more efficient in glucose-containing mineral medium (cometabolic conditions) than in a medium without glucose. The elimination of the parent compound resulted in the formation of five TPs, two of which (TP 249 and TP 235) were identified as N-desmethyltramadol (N-DM TRAM) and N,N-didesmethyltramadol (N,N-diDM TRAM). The remaining 3 TPs (TP 277a-c) were isomeric compounds with an elemental composition of protonated molecules C16H24NO3 and a putative structure which involved oxidative modification of the dimethylamino group. Pronounced changes in the taxonomic composition of the activated sludge were observed during the enrichment, especially regarding an enhanced percentage of 8 genera (Bacillus, Mycobacterium, Enterobacter, Methylobacillus, Pedobacter, Xanthobacter, Leadbetterella and Kaistia), which might be related to the observed transformations. The removal of TRAM resulted in proportional reduction of algal toxicity, implying a positive result of the accomplished transformation processes.

The impact of the heavy metal cadmium on the lightly silicified marine diatom Cylindrotheca closterium was studied with regard to growth dynamics, organic matter production and morphological characteristics. C. closterium cultures were able to sustain positive growth in the tested range of 0–1000 μg Cd l–1 with a significant decrease in specific growth rate in the exponential growth phase at the highest tested Cd concentration. The concentrations of both dissolved (DOCcell) and particulate organic carbon per cell (POCcell) in C. closterium cultures exposed to 500 and 1000 µg l–1 Cd more than doubled compared with the control culture. Enhanced organic matter production as a feedback response to Cd exposure may thus contribute to cell detoxification, supported by the electrochemical data showing that Cd was not significantly accumulated by or associated with diatom cells except for the culture exposed to 1000 μg Cd l–1. In order to gain more insight into the effects of cadmium on diatom morphological features, the cell surface of C. closterium was characterized at the nanoscale on a single cell level using atomic force microscopy (AFM). Changes induced by Cd were observed as irregular patterns of silica spheres on more silicified parts of the cell, girdle band and around the raphe. These changes clearly show that beside the physiological response, C. closterium also showed a morphological response which is probably due to the interference of Cd with the diatom biosilification process. This study overall contributes to better understanding of the effects of cadmium on diatoms and shows that morphological characteristics assessed by AFM may be a valuable indicator for metal contamination.

Microcystins (MCs) are the most studied cyanotoxins. The uptake of MCs in cells and tissues of mammals and fish species is mostly mediated by organic anion-transporting polypeptides (OATPs in humans and rodents; Oatps in other species), and the Oatp1d1 appears to be a major transporter for MCs in fish. In this study, six MC congeners of varying physicochemical properties (MC-LR, -RR, -YR, -LW, -LF, -LA) were tested by measuring their effect on the uptake of model Oatp1d1 fluorescent substrate Lucifer yellow (LY) in HEK293T cells transiently or stably overexpressing zebrafish Oatp1d1. MC-LW and -LF showed the strongest interaction resulting in an almost complete inhibition of LY transport with IC50 values of 0.21 and 0.26 μM, while congeners -LR, -YR and -LA showed lower inhibitory effects. To discern between Oatp1d1 substrates and inhibitors, results were complemented by Michaelis–Menten kinetics and chemical analytical determinations of MCs uptake, along with molecular docking studies performed using the developed zebrafish Oatp1d1 homology model. Our study showed that Oatp1d1-mediated transport of MCs could be largely dependent on their basic physicochemical properties, with log POW being the most obvious determinant. Finally, apart from determination of the chemical composition of cynobacterial blooms, a reliable risk assessment should take into account the interaction of identified MC congeners with Oatp1d1 as their primary transporter, and herewith we demonstrated that such a comprehensive approach could be based on the use of highly specific in vitro models, accompanied by chemical assessment and in silico molecular docking studies.

Organic anion transporters (OATs) are transmembrane proteins which belong to SLC22 subfamily. They are responsible for the uptake of various endo- and xenobiotics into the cells of different organs and tissues. Following our previous work on characterization of zebrafish Oat1 and Oat3, in this study we analyzed interaction of various classes of environmental contaminants with these membrane transporters using the transport activity assay with HEK293 Flp-In cell line stably overexpressing zebrafish Oat1 and Oat3, respectively. Based on the initial screening of a series of 36 environmental contaminants on their ability to interact with zebrafish Oat1 and Oat3, the most potent interactors were selected, their IC50 values calculated and type of interaction determined. Finally, to further confirm the type of interaction and initially evaluate their toxic potential, the cytotoxicity assays were performed. Broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs was confirmed and potent interactors among environmental contaminants identified.

The biotransformation study of difficult-to-degrade opioid analgesic methadone (MTHD) was performed by activated sludge culture adapted to high concentration of methadone (10 mg/L). The study included determination of elimination kinetics of the parent compound, taxonomic characterization of microbial culture, identification of biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation processes. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, whereas the ecotoxicological assessment was made based on determinations of toxicity to freshwater algae. Changes of the adapted sludge culture during the experiment were followed using the 16S rRNA gene amplicon sequencing. Depending on the experimental conditions, the elimination efficiency of methadone (10 mg/L) varied from 9% to 93% with the corresponding half-lives from 11.4 days to 1.5 days. A significantly faster elimination (t1/2 from 1.5 days to 5.8 days) was achieved at cometabolic conditions, using glucose-containing media, as compared to the experiments with MTHD as a single organic carbon source (t1/2 = 11.4 days). Moreover, increased biotransformation rate following the additional supplementation of ammonia, revealed a possible importance of nitrogen availability for the transformation at cometabolic conditions. The elimination of parent compound was associated with the formation of 3 different TPs, two of which were identical to main human metabolites of MTHD, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP). EDDP represented over 90% of the total TP concentration at the end of experiment. The biodegradation of MTHD was associated with a pronounced drop in algal toxicity, confirming a rather positive ecotoxicological outcome of the achieved biotransformation processes.

Zebrafish Mate7 belongs to solute carrier protein superfamily and specifically to subfamily of multidrug and toxin extruders. It is co-orthologous to mammalian Mates, and is ubiquitously expressed in zebrafish tissues with the highest expression in kidney. It has been shown to interact with both endogenous (steroid hormones) and xenobiotic compounds (pharmaceuticals), implying a role in efflux of toxic compounds. The objective of our study was to analyse interaction of environmental contaminants with zebrafish Mate7 using a newly developed high throughput screening (HTS) Mate7 assay. A full-length zebrafish mate7 sequence was obtained from zebrafish cDNA originating from male kidney, and a stable expression of Mate7 in genetically engineered HEK293 Flp-In cells was achieved. Stable Mate7 transfectants were then used for development and optimization of a new HTS cellular uptake protocol, with DAPI and ASP + as model fluorescent substrates. The developed assay was used for identifying zebrafish Mate 7 interactors and discerning the type of interaction. A series of 89 diverse environmental contaminants, including industrial chemicals, pesticides, and pharmaceuticals, was tested and highly effective Mate7 interactors were identified in all of the aforementioned groups. Some of the inhibitors identified could be of environmental concern because they may potentially impair Mate7 efflux function, lowering the fish defence capacity against environmental contaminants, or interfering with transport of yet unidentified physiological substrates. In addition, we found significant differences between zebrafish Mate7 and mammalian Mates’ substrate preferences, a finding that should be taken into consideration when using zebrafish as a model organism in toxicokinetic studies.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2, Izmjenjivač klora i mravlje kiseline (CFEX; SLC26A6) posreduje prijenos oksalata u različitim organima sisavaca. Pokusi u miševa s isključenim genom Cfex upućuju na moguću ulogu toga prijenosnika u razvoju hiperoksalurije i oksalatne urolitijaze, češćih u muških osoba. Štakori su važan model za istraživanje toga patofiziološkoga stanja, ali su spoznaje o lokalizaciji i regulaciji Cfex-a (rCfex) u njihovim organima nedostatne. U ovom radu primijenili smo metode RT-PCR i imunokemije u svrhu istraživanja ekspresije rCfex mRNA i proteina te regulacije istih spolnim hormonima u gušterači, tankom crijevu, jetri i bubrezima u različito tretiranih odraslih i predpubertetskih štakora obaju spolova. rCfex cDNAtransficirane stanice HEK293 uporabljene su za potvrdu specifičnosti komercijalnoga protutijela. Različiti biokemijski parametri izmjereni su u 24-satnom urinu, prikupljenom u metaboličkim kavezima. Ekspresija rCfex mRNA i proteina bila je prisutna u svim ispitanim organima. O spolu neovisna ekspresija prijenosnika rCfex nađena je u apikalnoj domeni interkaliranih kanalića gušterače, četkastoj membrani enterocita u tankom crijevu (duodenum > jejunum > ileum) i kanalikularnoj membrani hepatocita. U bubrezima je a) prijenosnik rCfex imunolokaliziran u četkastoj membrani proksimalnih kanalića sa segment-specifičnim obrascem (S1=S2 ženke) zbog stimulacijskoga učinka androgena nakon puberteta. Međutim, izlučivanje oksalata urinom nije bilo sukladno ekspresiji bubrežnoga prijenosnika rCfex. Dakle, nejasan je učinak povišene ekspresije prijenosnika rCfex u proksimalnim kanalićima mužjaka na izlučivanje oksalata, a postojanje prijenosnika u kanalikularnoj membrani hepatocita mogući je put izlučivanja oksalata putem žuči.

The biotransformation of three prominent macrolide antibiotics (azithromycin, clarithromycin and erythromycin) by an activated sludge culture, which was adapted to high concentrations of azithromycin (10 mg/L) was investigated. The study included determination of removal kinetics of the parent compounds, identification of their major biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, which enabled a tentative identification of TPs formed during the experiments. The ecotoxicological evaluation included two end-points, residual antibiotic activity and toxicity to freshwater algae. The enriched activated sludge culture was capable of degrading all studied macrolide compounds with high removal efficiencies (> 99%) of the parent compounds at elevated concentrations (10 mg/L). The elimination of all three macrolide antibiotics was associated with the formation of different TPs, including several novel compounds previously unreported in the literature. Some of the TPs were rather abundant and contributed significantly to the overall mass balance at the end of the biodegradation experiments. Biodegradation of all investigated macrolides was associated with a pronounced reduction of the residual antibiotic activity and algal toxicity, indicating a rather positive ecotoxicological outcome of the biotransformation processes achieved by the enriched sludge culture.

In order to be metabolized and excreted form the organism, xenobiotics need to pass through four phases of the cell metabolism. Detoxification process starts with the phase 0 - initial uptake of xeno- and endobiotics across the cell membrane by polyspecific membrane transporters. After the entrance, the xenobiotics pass through phase I of cell metabolism, which is characterized with enzymatic bioactivation of initial compounds through oxidation-reduction reactions. The phase II encompasses enzymes that mediate conjugation of phase I metabolites or initial compounds to the water soluble moieties, thus increasing the bioavailability of metabolized compounds. Finally, phase III refers to the efflux of parent compounds or metabolites by membrane transporters. Glutathione-S- transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The goal of our research was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as a valuable vertebrate model species frequently used in various fields of biology and biomedicine. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes: seven classes of cytosolic Gsts, mitochondrial genes within the kappa class, and microsomal genes within the MAPEG class. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. The results showed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. Functional characterization was performed on 9 representatives of cytosolic Gst classes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured using GSH as co-substrate and a series of model substrates. The functional characterization revealed specific interactions with model GST substrates and enabled the comparison of kinetic properties with human and fish orthologs. Finally, we conclude that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes.

Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endogenous compounds and xenobiotics across the plasma membrane. They are conserved from bacteria to mammals, with different numbers of genes within groups. Here, we present the first data on identification and functional characterization of Mate proteins in zebrafish (Danio rerio). Phylogenetic analysis revealed six Mates in teleost fish, annotated as Mate3–8, which form a distinct cluster separated from the tetrapod MATEs/Mates. Synteny analysis showed that zebrafish mate genes are orthologous to human MATEs. Gene expression analysis revealed that all the mate transcripts were constitutively and differentially expressed during embryonic development, followed by pronounced and tissue-specific expression in adults. Functional analyses were performed using transport activity assays with model substrates after heterologous overexpression of five zebrafish Mates in HEK293T cells. The results showed that zebrafish Mates interact with both physiological and xenobiotic substances but also substantially differ with respect to the interacting compounds and interaction strength in comparison to mammalian MATEs/Mates. Taken together, our data clearly indicate a potentially important role for zebrafish Mate transporters in zebrafish embryos and adults and provide a basis for detailed functional characterizations of single zebrafish Mate transporters.

ABCG2 (BCRP - breast cancer resistance protein) belongs to the ATP-binding cassette (ABC) superfamily. It plays an important role in the disposition and elimination of xeno- and endobiotics and/or their metabolites in mammals. Likewise, the protective role of ABC transporters, including Abcg2, has been reported for aquatic organisms. In our previous study we have cloned the full gene sequence of rainbow trout (Oncorhynchus mykiss) Abcg2a and showed its high expression in liver and primary hepatocytes. Based on those insights, the main goal of this study was to perform a detailed functional characterization of trout Abcg2a using insect ovary cells (Spodoptera frugiperda, Sf9) as a heterologous expression system. Membrane vesicles preparations from Sf9 cells were used for the ATPase assay determinations and basic biochemical properties of fish Abcg2a versus human ABCG2 have been compared. A series of 39 physiologically and/or environmentally relevant substances was then tested on interaction with trout Abcg2a and human ABCG2. Correlation analysis reveals highly similar pattern of activation and inhibition. Significant activation of trout Abcg2a ATPase was observed for prazosin, doxorubicine, sildenafil, furosemid, propranolol, fenofibrate and pheophorbide. Pesticides showed either a weak activation (malathione) or strong (endosulfan) to weak (chlorpyrifos, fenoxycarb, DDE) inhibition of trout Abcg2a ATPase while the highest activation was obtained for benzo(a)pyrene, curcumine and testosterone. In conclusion, data from this study offer the first characterization of fish Abcg2a, reveal potent interactors among physiologically or environmentally relevant substances and point to similarities regarding strengths and interactor preferences between human ABCG2 and fish Abcg2a.

The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

Važnost kvalitete i rokova izrade, nameće potrebu stalnog poboljšanja uvjeta proizvodnje i tehnološkog razvoja. Isto tako nameće se i uvođenje tehnoloških inovacija u postojećim procesima ili usvajanje potpuno novih tehnologija. Obrada vodenim i abrazivnim mlazom jedan je od procesa kojim se mogu ostvariti predhodno navedeni tehnološki zahtijevi i koji je prilagodljiv novim trendovima obrade materijala. Postupak obrade materijala vodenim mlazom spada među mehaničke postupke obrade, kao i obrada abrazivnim vodenim mlazom, pri kojem čestice abraziva velikom brzinom napuštaju mlaznicu u reznoj glavi i udaraju o obradak., The importance of quality and making deadlines, imposed by the need to continuously improve the conditions of production and technological development. It also imposes the introduction of technology and innovation in existing processes or adopting a completely new technology. Treatment of water and abrasive blasting is one of the processes by which they can achieve the previously mentioned technological requirements and which is adaptable to new trends in material processing. The method of processing materials waterjet is among the mechanical processing operations as well as processing abrasive waterjet in which abrasive particles rapidly leave the nozzle in the cutting head and hitting the workpiece.

Abch1 is a novel ATP binding cassette (ABC) transporter found in fish. This study represents an initial characterisation of this transporter using phylogenetic analyses, membrane topology prediction and determination of its tissue expression pattern in adult zebrafish (Danio rerio). Blast search showed that Abch1 orthologs are not present in genomes of other vertebrate taxa and similar genes are found only in invertebrate genomes. Abch1 is most closely related to the ABCG subfamily, although it shares only 12-14% of amino acid sequence identity with ABCG subfamily members. Topology analysis indicated that Abch1 is a half transporter that consists of six transmembrane domains with reverse domain arrangement (NBD-TMD), like ABCG subfamily members, but with differences in loop organization. Tissue distribution pattern revealed the highest Abch1 expression in brain, gills and kidney, followed by lower expression in intestine, gonads, skeletal muscle and liver. Considering moderate similarity in topology and tissue distribution pattern between Abch1 and ABCG subfamily members, we speculate that Abch1 is either involved in sterol transport similar to ABCG1, or is a part of the multidrug/multixenobiotic defence like ABCG2. These hypotheses remain to be addressed in further research.

P-glycoprotein (P-gp, ABCB1) is an important part of the multixenobiotic resistance (MXR) defense system in aquatic organisms. The main goal of this study was identification of P-gp inhibitors in contaminated sediments using the effect-directed analysis (EDA) approach. The samples were collected from the Gorjak creek (Zagreb, Croatia), a recipient of wastewater effluents from the pharmaceutical industry. Sediment samples were extracted and fractionated using two-tiered approach. Resulting non-polar, medium polar and polar fractions were tested on the inhibition of P-gp activity using P-gp overexpressing PLHC-1/dox cells and calcein-AM as model substrate. The obtained EC50 values - up to 757 μg/g, expressed in toxicity equivalents of model P-gp inhibitor cyclosporine A - revealed high inhibitory potential of polar fractions of investigated sediments and clearly reflected the impact of pharmaceutical wastewater. P-gp specific ATPase assay and the cytotoxicity modulation experiments with colchicine indicated that most of the observed P-gp inhibition was due to the presence of non-competitive inhibitors. A detailed chemical analysis by ultrahigh-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QToFMS) revealed nonionic surfactants, including alcohol polyethoxylates (LAEOs) and polypropylene glycols (PPGs), as the major components of the most active sub-fractions. Testing of several LAEO and PPG commercial mixtures confirmed their potential to inhibit the fish P-glycoprotein and modulate toxicity of other xenobiotics present in complex environmental samples.

Region-specific contaminant prioritisation is an important prerequisite for sustainable and cost- effective monitoring due to the high number of different contaminants that may be present. Surface water and sediment samples from the Sava River, Croatia, were collected at four locations covering a 150-km-long river section characterised by well-defined pollution gradients. Analysis of contaminant profiles along the pollution gradients was performed by combining toxicity screening using a battery of small-scale or in vitro bioassays, which covered different modes of action, with detailed chemical characterisation based on gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time- of-flight mass spectrometry (LC-QTOF-MS). A large number of contaminants, belonging to different toxicant classes, were identified in both analysed matrices. Analyses of water samples showed that contaminants having polar character occurred in the highest concentrations, while in sediments, contributions from both non-polar and amphiphilic contaminants should be taken into account. Estimated contributions of individual contaminant classes to the overall toxicity indicated that, besides the classical pollutants, a number of emerging contaminants, including surfactants, pharmaceuticals, personal care products and plasticizers, should be taken into consideration in future monitoring activities. This work demonstrates the importance of the integrated chemical and bioanalytical approach for a systematic region-specific pollutant prioritisation. Finally, the results presented in this study confirm that hazard assessment in complex environmental matrices should be directed towards identification of key pollutants, rather than focusing on a priori selected contaminants alone.

P-glycoprotein (P-gp; abcb1) is one of the major ABC transport proteins that mediates multixenobiotic resistance (MXR) defense in fish. In order to offer a sound evaluation of its ecotoxicological relevance it is critical to characterize substrate specificity of fish P-gp. Measurement of the ATPase activity is a reliable approach often used to discern type of interaction of various drugs with mammalian P-gp. A similar assay has never been used for characterization of P-gp in aquatic organisms and the main goal of this study was to develop a specific ATPase assay for characterization of fish P-gp. For this purpose we have used P-gp enriched membrane vesicles isolated from fish hepatoma PLHC-1/dox cells characterized by high overexpression of P-gp. As additional demonstration of a P-gp specific phenotype, we have quantified transcript expression of a series of eight ABC efflux transporter genes constitutively expressed in PLHC-1 wild type and PLHC-1/dox cells. Transcript expression analysis confirmed high and specific P-gp transcript overexpression in PLHC-1/dox cells. Provided that the transcript abundance is translated to protein, the development of ATPase assay is enabled. Using this model we determined Km(ATP) of 0.4mM, baseline ATPase activity from 35-50nmol/mg(PROT)/min, and maximal activation of ATPase activity obtained for fish P-gp in our system was 1.8-2.5-fold over baseline. All these values were in good agreement with data previously reported for mammalian P-gp. In order to perform a more detailed characterization of fish P-gp substrate specificity, in the next step of our study we used the developed ATPase assay to test 50 different compounds for their interaction with fish P-gp. The same set of compounds was also tested with calcein-AM (Ca-AM) transport activity assay both using PLHC-1/dox cells and NIH 3T3/MDR1 fibroblast cells overexpressing human P-gp. Our results showed that there is a clear difference for some substances-five compounds specifically interacted only with fish P-gp, while seven compounds exhibited interaction with human P-gp only. Most of the compounds tested in this study showed similar behavior in respect to fish or human P-gp and relatively high correlation in the interaction potency was found between fish and human P-gp. In summary, the described results represent the first in depth insight into substrate specificity of an important xenobiotic efflux transporter in fish. In addition, our study showed that combination of Ca-AM assay and the developed ATPase assay using inside/out vesicles isolated from PLHC-1/dox cells, offers a high-throughput and reliable approach for identification of environmentally relevant pollutants that interact with fish P-gp.

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1 ; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5 > abcg2 > abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes H33342 and 2', 7'- dichlorodihydrofluorescein diacetate were basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation.

In this study we examined gene expression of a series of ABC efflux transporters in various rainbow trout (Oncorhynchus mykiss) tissues. Based on their reported toxicological relevance, we have used quantitative real time PCR SYBR green quantification methodology, with combination of absolute and relative approach, to quantify RNA expression of eight ABC transporters from three different families: abcb1 and abcb11, abcc1-5 and abcg2. Level of mRNA transcripts was measured in seven tissues: liver, brain, gonads, kidney, gills, proximal intestine and distal intestine, and the obtained expression profiles were compared with data available for related mammalian tissues. Most of the analyzed genes showed similar gene expression pattern as the ones found in mammals, with some notable exceptions. E.g., gills were in our study characterized by extremely low expression of all analyzed ABC genes ; and despite the pronounced role of ABCC1 (MRP1) in protection of mammalian cells from chemical toxins, we found low expression of this transporter in trout tissues. Taken together, our study offers the first thorough insight into distribution pattern of (eco)toxicologically relevant ABC transporters, serving as a necessary base for further studies directed to better understanding of physiological and/or protective role of ABC transporters in fish.

The use of in vitro biotests in combination with chemical determination of priority pollutants is considered a promising approach in environmental risk assessment. The main goal of this study was to evaluate the relationship between the CYP1A induction potential and the concentration of priority pollutants (PAHs, PCBs and heavy metals) in contaminated marine sediments. Six sediment samples characterized by different types of pollution were collected from the Bay of Kvarner, Croatia. CYP1A induction potency was determined in vitro by the measurement of ethoxyresorufin-O-deethylase (EROD) activity in PLHC-1 fish hepatoma cells. The results were compared to the potency of the model CYP1A inducer 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and expressed in 2, 3, 7, 8-TCDD equivalents. All of the tested sediment samples were able to induce CYP1A activity in a dose-dependent manner. On a general scale, there was a good correlation between CYP1A induction and the concentration of priority pollutants in the tested samples. However, some samples, which had relatively low levels of priority pollutants, exhibited a strong CYP1A induction response. Therefore, apart from the confirmed usability and sensitivity of the EROD determination in the PLHC-1 cells as a suitable in vitro model in ecotoxicology, the results of this study indicate that the list of priority pollutants usually determined in the attempt to evaluate the risk of adverse effects to marine wildlife should be reconsidered.

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