Comparison of Growth and Chemical Profile of Diatom Skeletonema grevillei in Bioreactor and Incubation-Shaking Cabinet in Two Growth Phases.

Marine microalgae, diatoms, are considered a source of a wide range of high-value compounds, and numerous studies indicate their biotechnological potential in the food and feed industry, cosmetic industry, nanotechnology, pharmaceutical industry, biodiesel production, fertilizers, and wastewater treatment. The aim of this study was to compare the growth, chemical profiles, and antioxidant activity of the diatom Skeletonema grevillei cultivated in a bioreactor and an incubation-shaking cabinet at different growth phases (after 192 and 312 h). Growth was monitored by evaluating cell density with the Sedgewick Rafter chamber, and the collected biomass was extracted with 70% ethanol assisted by ultrasound. Extracts were evaporated to dryness and compounds were identified in derivatized form by gas chromatography and mass spectrometry (GC-MS) analysis, while antioxidant capacity was evaluated by DPPH and ORAC. Significantly faster growth was observed in the bioreactor than in the incubation-shaking cabinet. Oleamide, palmitelaidic acid, glycerol monostearate, myristic acid, cholesterol, eicosapentaenoic acid, 1-monopalmitin, and 24-methylene cholesterol were identified as the major compounds in both systems. Among them, oleamide was the dominant compound in both systems. It is also shown that prolonging the cultivation period had a direct effect on increasing the extract yield. The highest DPPH inhibition (11.4 ± 1%) and ORAC values (93.3 ± 8.4 mM TE) were obtained for the S. grevillei extract recovered from the bioreactor after 312 h. The obtained results contribute to the possibility of using S. grevillei for various biotechnological applications in the future.

Zebrafish (Danio rerio) Oatp2b1 as a functional ortholog of the human OATP2B1 transporter.

OATP2B1 belongs to a highly conserved organic anion transporting polypeptide (OATP) family of transporters, involved in the cellular uptake of both endogenous and exogenous compounds. The reported substrates of human OATP2B1 include steroid conjugates, bile salts, and thyroid hormones, as well as pharmaceuticals. Human OATP2B1 has orthologous genes in other vertebrate species, including zebrafish (Danio rerio), a widely used model organism in biomedical and environmental research. Our previous studies showed that zebrafish Oatp2b1 was phylogenetically closest to mammalian OATP2B1/Oatp2b1 and that it shares a similar tissue expression pattern. In this study, we aimed at discovering whether zebrafish Oatp2b1 could be a functional ortholog of human and rodent OATP2B1. To test this hypothesis, our primary goal was to obtain the first in vitro and in silico insights related to the structure and potential substrate preferences of zebrafish Oatp2b1. We generated cells transiently and stably transfected with zebrafish Oatp2b1 cloned from zebrafish liver, constructed an Oatp2b1 homology model, developed transport activity assays with model fluorescent substrate Lucifer yellow, and finally utilized this assay to analyze the interaction of zebrafish Oatp2b1 with both physiological and xenobiotic substances. Apart from structure similarities, our data revealed the strongest interaction of zebrafish Oatp2b1 with bile acids, steroid sulfates, thyroid hormones, and bilirubin, as well as xenobiotics bromosulfophthalein and sulfasalazine, which indicates its functional orthology with human OATP2B1.

Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio).

BACKGROUND: SLC22 protein family is a member of the SLC (Solute carriers) superfamily of polyspecific membrane transporters responsible for uptake of a wide range of organic anions and cations, including numerous endo- and xenobiotics. Due to the lack of knowledge on zebrafish Slc22 family, we performed initial characterization of these transporters using a detailed phylogenetic and conserved synteny analysis followed by the tissue specific expression profiling of slc22 transcripts. RESULTS: We identified 20 zebrafish slc22 genes which are organized in the same functional subgroups as human SLC22 members. Orthologies and syntenic relations between zebrafish and other vertebrates revealed consequences of the teleost-specific whole genome duplication as shown through one-to-many orthologies for certain zebrafish slc22 genes. Tissue expression profiles of slc22 transcripts were analyzed using qRT-PCR determinations in nine zebrafish tissues: liver, kidney, intestine, gills, brain, skeletal muscle, eye, heart, and gonads. Our analysis revealed high expression of oct1 in kidney, especially in females, followed by oat3 and oat2c in females, oat2e in males and orctl4 in females. oct1 was also dominant in male liver. oat2d showed the highest expression in intestine with less noticeable gender differences. All slc22 genes showed low expression in gills, and moderate expression in heart and skeletal muscle. Dominant genes in brain were oat1 in females and oct1 in males, while the highest gender differences were determined in gonads, with dominant expression of almost all slc22 genes in testes and the highest expression of oat2a. CONCLUSIONS: Our study offers the first insight into the orthology relationships, gene expression and potential role of Slc22 membrane transporters in zebrafish. Clear orthological relationships of zebrafish slc22 and other vertebrate slc22 genes were established. slc22 members are mostly highly conserved, suggesting their physiological and toxicological importance. One-to-many orthologies and differences in tissue expression patterns of zebrafish slc22 genes in comparison to human orthologs were observed. Our expression data point to partial similarity of zebrafish versus human Slc22 members, with possible compensatory roles of certain zebrafish transporters, whereas higher number of some orthologs implies potentially more diverse and specific roles of these proteins in zebrafish.

Molecular characterization of zebrafish Oatp1d1 (Slco1d1), a novel organic anion-transporting polypeptide.

The organic anion-transporting polypeptide (OATP/Oatp) superfamily includes a group of polyspecific transporters that mediate transport of large amphipathic, mostly anionic molecules across cell membranes of eukaryotes. OATPs/Oatps are involved in the disposition and elimination of numerous physiological and foreign compounds. However, in non-mammalian species, the functional properties of Oatps remain unknown. We aimed to elucidate the role of Oatp1d1 in zebrafish to gain insights into the functional and structural evolution of the OATP1/Oatp1 superfamily. We show that diversification of the OATP1/Oatp1 family occurs after the emergence of jawed fish and that the OATP1A/Oatp1a and OATP1B/Oatp1b subfamilies appeared at the root of tetrapods. The Oatp1d subfamily emerged in teleosts and is absent in tetrapods. The zebrafish Oatp1d1 is similar to mammalian OATP1A/Oatp1a and OATP1B/Oatp1b members, with the main physiological role in transport and balance of steroid hormones. Oatp1d1 activity is dependent upon pH gradient, which could indicate bicarbonate exchange as a mode of transport. Our analysis of evolutionary conservation and structural properties revealed that (i) His-79 in intracellular loop 3 is conserved within OATP1/Oatp1 family and is crucial for the transport activity; (ii) N-glycosylation impacts membrane targeting and is conserved within the OATP1/Oatp1 family with Asn-122, Asn-133, Asn-499, and Asn-512 residues involved; (iii) the evolutionarily conserved cholesterol recognition interaction amino acid consensus motif is important for membrane localization; and (iv) Oatp1d1 is present in dimeric and possibly oligomeric form in the cell membrane. In conclusion, we describe the first detailed characterization of a new Oatp transporter in zebrafish, offering important insights into the functional evolution of the OATP1/Oatp1 family and the physiological role of Oatp1d1.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1).

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towards perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17ß-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species.

Uptake/depuration kinetics, bioaccumulation potential and metabolic transformation of a complex pharmaceutical mixture in zebrafish (Danio rerio).

Uptake and elimination kinetics, bioconcentration factors (BCFs), and metabolic transformation of 20 different pharmaceutically active compounds (PhACs), covering a wide range of therapeutic categories and physico-chemical properties, were studied using zebrafish (Danio rerio). The fish were exposed to the mixture of the selected PhACs at environmentally relevant concentrations similar to 10 µg L-1. The experiments were performed in semi-static conditions and comprised a 7-day uptake period followed by a 7-day depuration period. Most of the PhACs reached a concentration plateau within the 7-day uptake-phase which was followed by an efficient depuration, with the observed uptake (ku) and depuration rate constants (kd,) ranging between 0.002 and 3.752 L kg-1 h-1, and 0.010 to 0.217 h-1, respectively. The investigated PhACs showed low to moderate BCFs. The highest BCFs of 47.8, 28.6 and 47.6 L kg-1 were determined for sertraline, diazepam and desloratadine, respectively. A high contribution of metabolic products to the total internal concentration was observed for some PhACs such as codeine (69%), sulfamethoxazole (51%) and verapamil (87%), which has to be taken into account when assessing the bioconcentration potential. Moreover, most of the metabolites exhibited significantly longer half-lives in zebrafish than their parent compounds and affected the overall depuration kinetics.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos.

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

Identification of P-glycoprotein inhibitors in contaminated freshwater sediments.

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 a two-tiered approach. Resulting nonpolar, 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 noncompetitive 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 subfractions. 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.

The responses of the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity in sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a polluted site: implications for their use in environmental risk assessment.

The present study investigates the response of three hepatic biomarkers in adult sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a wastewater outlet of an oil refinery with fish caged at a pristine site used as controls. The biomarkers that were investigated were the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity. In addition, we have measured the levels of polycyclic aromatic hydrocarbons (PAHs) and selected heavy metals (lead, cadmium, mercury, copper and zinc) in sediment samples at the polluted site. Although the polluted site had high environmental levels of PAHs and heavy metals, there was no difference in hepatic EROD activity and HSI between fish caged at the polluted site and controls. On the other hand, GST activity was significantly lower in fish caged at the polluted site compared to controls. Our results point out that the studied biomarkers have limited use in environmental risk assessment studies, at least when caged adult sea bass is used as the sentinel species and complex toxicant mixtures are involved.

Identification of ABC transporter genes in gonad tissue of two Mediterranean sea urchin species: black, Arbacia lixula L., and rocky, Paracentrotus lividus L.

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.

Environmental contaminants modulate transport activity of zebrafish (Danio rerio) multidrug and toxin extrusion protein 3 (Mate3/Slc47a2.1).

Zebrafish Mate3 is one of six co-orthologs of human multidrug and toxin extrusion proteins. It is highly expressed in the kidneys, intestine, testes, and brain of males. Initial interaction studies showed its interaction with xenobiotic compounds, suggesting a role in the efflux of toxic compounds. In this study, we aimed to test various environmental contaminants for their interaction with zebrafish Mate3. We developed a stable zebrafish Mate3 cell line and optimized a high-throughput screening assay using DAPI and ASP+ as fluorescent model substrates. To gain insight into the structure and function of the Mate3 protein and relate these to the results of the DAPI and ASP+ transport measurements, we predicted its 3D structure using the AlphaFold2 algorithm. A 3D structure with high per residue confidence scores with 13 transmembrane segments (TMs) was obtained, with topology and mutual positioning characteristic of the Mate protein family in a shape open to the extracellular part. Molecular docking methods were used to identify DAPI and ASP+ binding sites on the surface and in the center of the protein cavity. Because our kinetics experiments combined with molecular docking indicated that there may be additional active sites in zebrafish Mate3, additional cytotoxicity experiments were performed and highly potent Mate3 interactors were identified from a set of 55 different environmental contaminants. Our results suggest that some of the identified interactors may be of environmental concern, as their interaction with Mate3 could lead to an impairment of its normal efflux function, making fish more sensitive to harmful substances commonly released into the aquatic environment. Finally, the quality of zebrafish Mate3 structures predicted by the AlphaFold2 algorithm opens up the possibility of successfully using this tool for in silico research on transport preferences of other Mate proteins.

Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

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+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Assessment of toxicological profiles of the municipal wastewater effluents using chemical analyses and bioassays.

The hazardous chemical contamination of untreated wastewater and secondary effluent from the wastewater treatment plant (WWTP) of the city of Zagreb, Croatia was comprehensively characterized using large-volume solid-phase extraction (SPE) and silica gel fractionation, followed by a detailed analysis of the resulting extracts by a combination of chemical and bioassay methods. Over 100 individual contaminants or closely related-contaminant groups were identified by high-resolution gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF). Ecotoxicity profiling of the investigated samples, including cytotoxicity, chronic toxicity and EROD activity; inhibition of the multixenobiotic resistance (MXR), genotoxicity and estrogenic potential, revealed the most significant contribution of toxic compounds to be present in polar fractions. Wastewater treatment using conventional activated sludge process reduced the initial toxicity of raw wastewater to various extents, ranging from 28% for algal toxicity to 73.2% for an estrogenic activity. The most efficient toxicity removal was observed for the polar compounds.

Selective interaction of microcystin congeners with zebrafish (Danio rerio) Oatp1d1 transporter.

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.

Evaluation of cyanobacterial toxicity using different biotests and protein phosphatase inhibition assay.

Cyanobacteria are prolific producers of numerous toxic compounds, among which microcystins (hepatotoxins) are the most frequently found. Cyanobacterial bloom in freshwaters is an increasing problem, and there is still a need for rapid and reliable methods for the detection of toxic cyanobacterial samples. In the present study, the toxicity of crude extracts of 11 cyanobacterial strains from different genera has been assessed on two cell lines (human hepatocellular carcinoma HepG2 and rainbow trout (Oncorhynchus mykiss) liver-derived RTL-W1 cells), crustaceans (Daphnia magna and Artemia salina), and zebrafish (Danio rerio) embryos, as well as by protein phosphatase 1 (PP1) inhibition assay and ELISA test to determine whether the toxicity could be due to the presence of hepatotoxins/microcystins. All the tested strains exhibited toxicity on HepG2 cell line (IC50 from 35 to 702 µg mL-1), including Arthrospira (Spirulina) strains, while toxicity against the RTL-W1 cells was detected only in the positive reference Microcystis PCC 7806 and Nostoc 2S9B. Tested strains expressed higher toxicity to D. magna and zebrafish embryos in comparison to A. salina, whereby Nostoc LC1B and Nostoc S8 belonged to the most toxic strains. The PP1-inhibiting compounds have been detected by PP1 assay only in four strains (Microcystis PCC 7806, Oscillatoria K3, Nostoc LC1B, and Nostoc S8), indicating that their toxic potency can be attributed to these compounds. On the other hand, very low levels of microcystins, as confirmed by ELISA, were insufficient to explain toxicity and different toxic potencies of tested cyanobacteria. Results presented in this study suggested HepG2 cell line as a particularly suitable model for cyanobacterial toxicity assessment. In addition, they highlight terrestrial cyanobacterial strains as potent producers of toxic compounds.

Organic anion transporting polypeptides (OATP) in zebrafish (Danio rerio): Phylogenetic analysis and tissue distribution.

The aim of our study was the initial characterization of Organic anion transporting polypeptides (SLCO gene superfamily) in zebrafish (Danio rerio) as an important model species in biomedical and ecotoxicological research, using phylogenetic analysis, membrane topology prediction and tissue expression profiling. The phylogenetic tree of Oatp superfamily in vertebrates was constructed in Mega 3.1. Software, membrane topology was predicted using HMMTOP algorithm, while qRT-PCR was used to determine tissue-specific gene expression levels. Phylogenetic analysis revealed that Oatp superfamily in zebrafish consists of five families that include 14 SLCO genes. Eight out of 14 transporters do have orthologs or co-orthologs in other vertebrates, while 6 members are found only in fish lineage. Topology analysis showed that all zebrafish Oatps consist of 12 transmembrane domains (TMD) with the large fifth extracellular loop (LP5). Tissue distribution analysis revealed that the expression patterns of Oatp2a1, Oatp2b1 and Oatp3a1 follow tissue distribution patterns of their mammalian (co)orthologs. Expression pattern of a newly identified Oatp1d1 is similar to mouse Oatp1a4, while other new zebrafish Oatps (Oatp1e1, 1f2) do not resemble any of the mammalian Oatps. In summary, the described comprehensive analysis of Oatp superfamily in fish represents a first step towards research on toxicological relevance of uptake transporters in aquatic organisms.

Interaction of organotin compounds with three major glutathione S-transferases in zebrafish.

Glutathione S-transferases (GSTs) play an important role in cellular detoxification as enzymatic mediators of glutathione (GSH) conjugation with a wide range of deleterious compounds, enabling their easier extrusion out of the organism. GSTs are shown to interact with organotin compounds (OTCs), known environmental pollutants, either as substrates, serving as electrophilic targets to the nucleophilic attack of GSH, or as noncompetitive inhibitors by binding to GST active sites and disrupting their enzymatic functions. There is a wide range of deleterious biological effects caused by OTCs in low concentration range. Their environmental concentrations, further potentiated by bioaccumulation in aquatic organisms, correspond with inhibitory constants reported for Gsts in zebrafish, which implies their environmental significance. Therefore, our main goal in this study was to analyze interactions of three major zebrafish Gsts - Gstp1, Gstr1, and Gstt1a - with a series of ten environmentally relevant organotin compounds. Using previously developed Gst inhibition assay with recombinant Gst proteins and fluorescent monochlorobimane as a model substrate, we determined Gst inhibitory constants for all tested OCTs. Furthermore, in order to elucidate nature of Gst interactions with OTCs, we determined type of interactions between tested Gsts and the strongest OTC inhibitors. Our results showed that OTCs can interact with zebrafish Gsts as competitive, noncompetitive, or mixed-type inhibitors. Determined types of interactions were additionally confirmed in silico by molecular docking studies of tested OTCs with newly developed Gst models. In silico models were further used to reveal structures of tested Gsts in more detail and identify crucial amino acid residues which interact with OTCs within Gst active sites. Our results revealed more extensive involvement of Gstr1 and Gstp1 in detoxification of numerous tested OTCs, with low inhibitory constants in nanomolar to low micromolar range and different types of inhibition, whereas Gstt1a noncompetitively interacted with only two tested OTCs with significantly higher inhibitory constants.

Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3.

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.

Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class.

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes with primary function of glutathione conjugation of various endogenous and exogenous compounds. Teleost-specific Gstr1 in zebrafish (Danio rerio) was previously shown to have high expression in toxicologically relevant tissues and high activity towards model substrates. The aim of this study was a detailed functional characterization of zebrafish Gstr1. Molecular docking analyses were used to get novel insight into structural characteristics of Gstr1 and elucidation of the mechanistic interactions with both GSH and various Gstr1 substrates or inhibitors. An initial screening inhibition assay performed using model fluorescence substrate monochlorobimane (MCB) revealed interactions of different endogenous compounds and environmentally relevant xenobiotics with zebrafish Gstr1. All interacting compounds were further analyzed to determine their inhibition type and Ki values. Our data revealed that pregnenolone, progesterone, testosterone, DHEAS and corticosterone competitively inhibited transformation of MCB by Gstr1 with the calculated Ki values in the range 14-26 µM, implying that these hormones are physiological substrates of zebrafish Gstr1. Estrogens had no effect on Gstr1 activity. Taurochenodeoxycholate (TCDC) expressed lower inhibition potency toward Gstr1 with the Ki value of 33 µM. Among tested xenobiotics tributyltin chloride and rifampicin non-enzymatically bound Gstr1 enzyme (the calculated Ki values are 0.26 µM and 65 µM, respectively) and inhibited its activity, showing that these compounds are reversible noncompetitive inhibitors of zebrafish Gstr1. Insecticide diazinon competitively inhibited Gstr1 activity with calculated Ki value of 27 µM, while other Gstr1-interacting insecticides, chlorpyrifos-methyl (CPF-methyl) and malathion, showed allosteric activation-like effect. Among tested pharmaceuticals, tetracycline, erythromycin and methotrexate demonstrated competitive type of inhibition with the calculated Ki values of 17.5, 36.5 and 29 µM, respectively. In summary, we suggest that zebrafish Gstr1 has an important role in steroidogenesis, metabolism and/or physiological actions of androgens, but not estrogens in fish. Finally, our results imply the role of Gstr1 in metabolism of xenobiotics and protection of fish against deleterious environmental contaminants such as organophosphate insecticides and pharmaceuticals.

Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds.

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.