title: Characterization of glutathione-S-transferases in zebrafish (Danio rerio).
abstract: 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.
authors.affiliation: Laboratory for Ecotoxicology, Department of Biology and Ecology, University of Novi Sad, Faculty of Sciences, Novi Sad, Serbia.
authors.forename: Branka
authors.initials: B
authors.lastname: Glisic
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Mihaljevic
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Marta
authors.initials: M
authors.lastname: Popovic
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Roko
authors.initials: R
authors.lastname: Zaja
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Jovica
authors.initials: J
authors.lastname: Loncar
authors.affiliation: University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Muttenz, Switzerland; Swiss Federal Institute of Technology (ETHZ), Department of Environmental System Sciences, Zürich, Switzerland.
authors.forename: Karl
authors.initials: K
authors.lastname: Fent
authors.affiliation: Laboratory for Ecotoxicology, Department of Biology and Ecology, University of Novi Sad, Faculty of Sciences, Novi Sad, Serbia.
authors.forename: Radmila
authors.initials: R
authors.lastname: Kovacevic
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia. Electronic address: smital@irb.hr.
authors.forename: Tvrtko
authors.initials: T
authors.lastname: Smital
mesh_terms: D000818:Animals; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D010802:Phylogeny; D011720:Pyrazoles / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000378:metabolism; D015027:Zebrafish / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004137:Dinitrochlorobenzene; D011720:Pyrazoles; D014874:Water Pollutants, Chemical; C059597:monochlorobimane; D005982:Glutathione Transferase; D005978:Glutathione
keywords: Enzyme kinetics; Functional characterization; Glutathione-S-transferases; Phylogenetic analysis; Zebrafish; mRNA expression