Ivan Mihaljević

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. 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. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed 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. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest 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. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

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

BackgroundSLC22 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.ResultsWe 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.ConclusionsOur 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.

Biotransformation of macrolide antibiotics using enriched activated sludge culture: Kinetics, transformation routes and ecotoxicological evaluation

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.

Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics

Organic cation transporters (OCTs) serve as uptake transporters of numerous endo- and xenobiotics. They have been in the focus of medical toxicological research for more than a decade due to their key role in absorption, distribution, metabolism and excretion due to their expression on basolateral membranes of various barrier tissues. OCTs belong to the SLC22A family within the SLC (Solute carrier) protein superfamily, with three co-orthologs identified in humans (OCT1, 2 and 3), and two Oct orthologs in zebrafish (Oct1 and Oct2). The structural and functional properties of zebrafish Octs, along with their toxicological relevance, have still not been explored. In this study, we performed a functional characterization of zebrafish Oct1 using transient and stable heterologous expression systems and model fluorescent substrates as the basis for interaction studies with a wide range of endo- and xenobiotics. We also conducted a basic topology analysis and homology modeling to determine the structure and membrane localization of Oct1. Finally, we performed an MTT assay to evaluate the toxic effects of the seven interactors identified - oxaliplatin, cisplatin, berberine, MPP+, prazosin, paraquat and mitoxantrone - in human embryonic kidney cells (HEK293T) stably expressing zebrafish Oct1 (HEK293T-drOct1 cells). Our results show that the zebrafish Oct1 structure consists of 12 transmembrane alpha helices, which form the active region with more than one active site. Five new fluorescent substrates of Oct1 were identified: ASP+ (Km=26μM), rhodamine 123 (Km=103.7nM), berberine (Km=3.96μM), DAPI (Km=780nM), and ethidium bromide (Km=97nM). Interaction studies revealed numerous interactors that inhibited the Oct1-dependent uptake of fluorescent substrates. The identified interactors ranged from physiological compounds (mainly steroid hormones) to different classes of xenobiotics, with IC50 values in nanomolar (e.g., pyrimethamine and prazosin) to millimolar range (e.g., cimetidine). Cytotoxicity experiments with HEK293T-drOct1 cells enabled us to identify berberine, oxaliplatin and MPP+ as substrates of Oct1. The data presented in this study provide the first insights into the functional properties of zebrafish Oct1 and offer an important basis for more detailed molecular and ecotoxicological characterizations of this transporter.

In vitro characterization of zebrafish (Danio rerio) organic anion transporters Oat2a-e

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 49.7μM), and 6-carboxyfluorescein for Oat2b and Oat2d (Km 199.7, and 266.9μM). 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 19.14μM) with Oat2a; estrone-3-sulfate and diclofenac (IC50 30.96, and 12.6μM) with Oat2b; and fumarate and indomethacin (IC50 68.24, and 20.41μM) 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.