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Nucleic acids research : NAR 50(16), 9426-9441 (2022). doi:10.1093/nar/gkac711, Published by Oxford Univ. Press, Oxford

Bioscience reports 42(5), BSR20212489 (2022). doi:10.1042/BSR20212489, Published by Portland Press, Colchester

ADP-ribosylation, a modification of proteins, nucleic acids and metabolites, confers broad functions, including roles in stress responses elicited for example by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases, which transfer ADP-ribose from NAD(+) onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ADP-ribosyltransferases are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as anti-viral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ADP-ribosyltransferases and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ADP-ribosyltransferases that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ADP-ribosyltransferases to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

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.

OATS/Oats are transmembrane proteins that transport a variety of drugs, environmental toxins and endogenous metabolites into the cell. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a–e and Oat3. In this study we specifically address Oat2 (Slc22a7) family. Conserved synteny analysis showed localization of zebrafish oat2 genes on two chromosomes, 11 and 17. All five zebrafish Oats were localized by live cell imaging in membranes of transiently transfected HEK293-T cells, and Oat2a, b, d, and e were confirmed using western blot analysis. Functional studies using the HEK293T cells overexpressing zebrafish Oats revealed two model fluorescent substrates of three Oats: Lucifer yellow for Oat2a and Oat2d (Km 122, and 49.7 μM), and 6- carboxyfluorescein for Oat2b and Oat2d (Km 199.7, and 266.9 μM). The initial screening of a series of diverse endo- and xenobiotics showed interaction with a number of compounds, including cGMP and diclofenac (IC50 27.74, and 19.14 μM) with Oat2a ; estrone-3-sulfate and diclofenac (IC50 30.96, and 12.6 μM) with Oat2b ; and fumarate and indomethacin (IC50 68.24, and 20.41 μM) with Oat2d. This study provides the first comprehensive data set on Oat2 in zebrafish and offers an important basis for more detailed molecular and (eco)toxicological characterizations of these transporters.

Primary hepatocytes are a model for studying various effects of different xenobiotics, including detoxification strategies. In this study we have isolated and cultured European seabass (Dicentrarchus labrax) primary hepatocytes and assessed gene transcription and activity of CYP1A (phase I of cellular detoxification) and ABCC1 and ABCC2 (phase III) transport proteins after exposure to benzo(a)pyrene (BaP). A dose dependent increase in ABCC2 and CYP1A mRNA transcripts was observed in seabass primary hepatocytes upon exposure to BaP. The activity of ABC proteins, as key mediators of the multixenobiotic resistance (MXR), was further confirmed by assessing the accumulation of the model fluorescence substrate rhodamine 123 in the absence and presence of model inhibitors. A weak interaction between BaP and ABC proteins was observed. CYP1A dependent ethoxyresorufin-O-deeethylase (EROD) activity was significantly induced by the presence of BaP. After the 24 h exposure period only 10% of the initial BaP was present in the incubation medium, clearly demonstrating biotransformation potential of primary seabass hepatocytes. Furthermore, the presence of the 3-hydroxybenzo(a)pyrene, a BaP metabolite, in the medium implies its active efflux. In conclusion, we showed that seabass primary hepatocytes do express important elements of the cellular detoxification machinery and may be a useful in vitro model for studying basic cellular detoxification mechanisms and their interaction with environmental contaminants.

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.

ADP-ribosylation is a post-translational protein modification that regulates numerous cellular pathways. An approach involving histone purification, partial filter-aided digestion and ETD mass spectrometry reveals that serine residues in histone proteins are ADP-ribosylated. ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.

Poly(ADP-ribosyl)ation is a post-translational protein modification involved in the regulation of important cellular functions including DNA repair, transcription, mitosis and apoptosis. The amount of poly(ADP-ribosyl)ation (PAR) in cells reflects the balance of synthesis, mediated by the PARP protein family, and degradation, which is catalyzed by a glycohydrolase, PARG. Many of the proteins mediating PAR metabolism possess specialised high affinity PAR-binding modules that allow the efficient sensing or processing of the PAR signal. The identification of four such PAR-binding modules and the characterization of a number of proteins utilising these elements during the last decade has provided important insights into how PAR regulates different cellular activities. The macrodomain represents a unique PAR-binding module which is, in some instances, known to possess enzymatic activity on ADP-ribose derivatives (in addition to PAR-binding). The most recently discovered example for this is the PARG protein, and several available PARG structures have provided an understanding into how the PARG macrodomain evolved into a major enzyme that maintains PAR homeostasis in living cells.

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.

The ubiquitous presence of pharmaceuticals in aquatic systems is a challenging problem as their potential chronic effects on aquatic organisms remain largely unknown. The ATP-binding cassette (ABC) transport proteins contributing to the multidrug/multixenobiotic resistance (MDR/MXR) phenomenon seem to have an important role in the elimination of xenobiotics in aquatic organisms. Modulation of their efflux activities by contaminants may lead to substantial increase in intracellular accumulation and toxic effects of other xenobiotics. The aim of our work was to analyse a series of pharmaceuticals for their potential to modulate the activity of xenobiotic efflux transporters from the ABCB and ABCC sub-family in the Poeciliopsis lucida hepatoma cell (PLHC-1) fish cell line (PLHC-1/wt) and a doxorubicin (DOX) resistant PLHC-1 subclone (PLHC-1/dox) characterized by an elevated expression of the P-glycoprotein (ABCB1). Cellular accumulation of the model fluorescent substrates calcein-AM and rhodamine123 were used to determine an inhibitory effect on P-gp1 and/or MRP-like efflux transporters. 18 out of 33 tested pharmaceuticals showed MXR inhibitory activity with IC50 values occurring in the lower micromolar to millimolar range. Further, cytotoxic effects of pharmaceuticals were evaluated in PLHC-1/dox cells. Co-exposure of resistant cells to model P-gp1 inhibitor cyclosporine A (CyA) resulted in up to five times increased cytotoxicity of pharmaceuticals. In addition, some pharmaceuticals lead to a marked increase in cytotoxicity of doxorubicin, a model P-gp1 substrate. The modulation of toxicity by MDR inhibitors indicates their role in influencing cellular toxicity. In conclusion, the results of our study revealed significant inhibitory effects of environmentally relevant pharmaceuticals on P-gp1 and MRP-like transporters in fish. Our findings correspond well with data from mammalian systems indicating that the specificity and roles of the related efflux transporters may be similar in fish. Furthermore, due to the presence of active and inducible ABC transport proteins, the PLHC-1 cells appear to be a reliable in vitro system for the investigation of MDR/MXR mechanisms in fish.

SummaryADP-ribosyltransferases (ARTD1–16) have emerged as major downstream effectors of NAD+ signaling in the cell. Most ARTDs (ARTD7 and 8, 10–12, and 14–17) catalyze the transfer of a single unit of ADP-ribose from NAD+ to target proteins, a process known as mono-ADP-ribosylation (MARylation). Progress in understanding the cellular functions of MARylation has been limited by the inability to identify the direct targets for individual mono-ARTDs. Here, we engineered mono-ARTDs to use an NAD+ analog that is orthogonal to wild-type ARTDs. We profiled the MARylomes of ARTD10 and ARTD11 in vitro, identifying isoform-specific targets and revealing a potential role for ARTD11 in nuclear pore complex biology. We found that ARTD11 targeting is dependent on both its regulatory and catalytic domains, which has important implications for how ARTDs recognize their targets. We anticipate that our chemical genetic strategy will be generalizable to all mono-ARTD family members based on the similarity of the mono-ARTD catalytic domains.

ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5′-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.

The poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD(+) as substrate. Based on the composition of three NAD(+) coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADPr) (PAR) or mono(ADPr) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino-acid targets. In addition, we identify cysteine as a novel amino-acid target for ADP-ribosylation on PARPs.

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 17β-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.

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.

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.

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.

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), genotoxicicity 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.

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.

The aim of our study was initial characterization of Organic anion transporting polypeptides (Oatp ; SLC21 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. Phylogenetic tree of Oatp superfamily in vertebrates was constructed in Mega Software version 3.1., 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 with the large fifth extracellular loop (LP5). Tissue distribution analysis revealed that the expression patterns of Oatp2a1, Oatp2b1 and Oatp3a1 follows 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 and 1f4) do not resemble any of the mammalian Oatps. In summary, the described comprehensive analysis of Oatp superfamily in fish represents the first research step needed for understanding of ecotoxicological relevance of uptake transporters in aquatic organisms.

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.

Fish possess similar mechanisms of billiary excretion of xeno(endo)biotics and their metabolites as found in higher vertebrates and various types of ABC efflux proteins expressed in apical membranes of polarized cells appears to be key mediators of this vectorial transport. To test this hypothesis the main goals of this study were identification and cloning of genes coding for different types of ABC transport proteins, determination of the gene transcript (mRNA) levels, and characterization of the related protein transport activities in primary cultured rainbow trout ( Oncorhynchus mykiss ) hepatocytes. We have cloned one partial and two full gene sequences, which show high degree of identity with mammalian Pgp1 (ABCB1), BSEP (ABCB11) and MRP2 (ABCC2) efflux transporters. Using real-time RT-PCR expression levels of the mRNA of these genes were determined. Identical relative expression patterns of identified efflux transporters (BSEP ≫ MRP2 > Pgp1) were observed for both liver and primary hepatocytes, with expression of all three transporter mRNAs approximately 3–4-fold lower in primary hepatocytes in comparison to intact liver. In addition, the presence of Pgp1-, BSEP- and MRP-like transport activities were indicated using putative specific fluorescent substrates (rhodamine 123, calcein-AM, bodipy-verapamil and dihydrofluorescein diacetat), model inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurocholate and taurochenodeoxycholate) and their combinations. Taken together the results of this study showed that primary trout hepatocytes express critical components of detoxification pathways—phase I and II enzymes, as well as the ABC proteins involved in transport of xenobiotics, affirming this in vitro model as a promising tool in (eco)toxicological research.

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.

As a widely spread cyprinid fish species, the European chub (Leuciscus cephalus) has been used extensively in biomonitoring programs. However, no laboratory dose-response and/or time course studies related to applied biomarkers have been reported on chub yet. In order to address this issue, specimens of juvenile chub caught in September 2005 in the Sava River, Croatia, were laboratory exposed to various (0.25– 50 mg/kg) doses of either model polycyclic aromatic hydrocarbons (PAH)premutagen benzo[a]pyrene (BaP), or -naphthoflavone ( -NF), a well-known model cytochrome 1A (CYP1A) inducer, for 3 (BaP) or 5 d ( -NF). The responses of several hepatic biomarkers were determined in the exposed fish: The hepatic 7-ethoxyresorufin-O-deethylase activity, CYP1A content, glutathione S-transferase (GST) activity, liver bioactivation potential, and the amount of hydroxylated polycyclic aromatic hydrocarbon bile metabolites determined by the fixed wavelength fluorescence and the highperformance liquid chromatography technique. The relevance of determined biomarker responses has been analyzed further and crosscorrelated with the same set of biomarkers, as well as with tissue concentrations of polycyclic aromatic hydrocarbons and polychlorinated biphenyls, determined in chub specimens collected in September 2005 at five different polluted locations along the Sava River. The species-specific upper and lower limits in responses of studied biomarkers were determined and the obtained ranges successfully evaluated in real field situation. With the exception of the GST activity, all other biomarkers determined in chub proved to be valuable indicators of environmental pollution. Finally, the results of the present study demonstrated that the same strategy of laboratory characterization in combination with field evaluation should be used regularly in the selection of optimal biomarkers and indicator species.

The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in the metabolism of xenobiotics. However, the presence of the ABC transport proteins possibly involved in the MultiXenobiotic Resistance (MXR) mechanism as phase III of cellular detoxification has never been described in the PLHC-1 cells. The main goal of this study was the detection and functional characterization of toxicologically relevant xenobiotic efflux transporters from ABCB and ABCC subfamily in the PLHC-1 cells. Using specific primer pairs two PCR products 1769 and 1023bp in length were successfully cloned and sequenced. Subsequent multiple alignment and phylogenetic analysis showed that these sequences share a high degree of homology with the P-glycoprotein (Pgp1; ABCB1) and the MRP3 (ABCC3). Functional experiments with fluorescent model substrates and specific inhibitors were used to verify that transport activities of Pgp- and MRP-related proteins are indeed present in PLHC-1 cells. Accumulation or efflux/retention rates of rhodamine 123, calcein-AM or monochlorbimane were time- and concentration-dependent. Cyclosporine A, MK571, verapamil, reversine 205, indomethacine and probenecid were used as specific inhibitors of Pgp1 and/or MRPs transport activities, resulting in a dose dependent inhibition of related transport activities in PLHC-1 cells. Similar to mammalian systems, the obtained IC(50) values were in the lower micromolar range. Taken together these data demonstrate that: (1) the PLHC-1 cells do express a functional MXR mechanism mediated by toxicologically relevant ABC efflux transporters; and (2) the presence of all three critical phases of cellular detoxification additionally affirms the PLHC-1 cells as a reliable in vitro model in aquatic toxicology.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2981-y) contains supplementary material, which is available to authorized users.

Summary ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability., Graphical Abstract, Highlights • Histone serine ADPr depends on HPF1 • HPF1 promotes PARP-1 and PARP-2 serine specificity • PARP-1 automodifies its serines • Serine ADPr is a widespread modification, Bonfiglio et al. show that serine residues can be ADP-ribosylated by PARP-1 and PARP-2. This serine specificity is conferred by HPF1, a protein that interacts with PARP-1/2. Serine ADP-ribosylation represents a widespread protein modification that targets hundreds of different substrates, including PARP-1 and high-mobility group proteins.