transferase class molecular characterization of zebrafish
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Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class
Authors:
Branka Bašicaa, Ivan Mihaljevićb, Nikola Marakovićc, Radmila Kovačevića, Tvrtko Smitalb,*
Affiliations:a University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovica 2, 21000 Novi Sad, Serbiab Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenicka 54, 10000 Zagreb, Croatiac Instiute for Medical Research and Occupational Medicine, Ksaverska cesta 2, 10000 Zagreb, CroatiaThese authors contributed equally to this work
*Corresponding authorTvrtko Smital, PhDLaboratory for Molecular EcotoxicologyDivision for Marine and Environmental ResearchRuđer Bošković InstituteBijenicka 5410 000 Zagreb, CROATIATel.: **385 1 45 61 039E-mail: smital@irb.hr
Highlights
First functional characterization of the teleost-specific Gstr1 in zebrafish (Danio rerio);
Recombinant zebrafish Gstr1 protein used for screening inhibition assay;
New insight into structural characteristics of Gstr1 obtained by molecular docking analyses;
Mechanistic interactions of Gstr1 with GSH and endogenous and exogenous compounds revealed;
Proposed role of Gstr1 in steroidogenesis, metabolism and/or physiological actions of androgens;
Organophosphate insecticides and pharmaceuticals identified as potent Gstr1 inhibitors.
1
1 Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase
2 class
3
4 Authors:
5 Branka Bašicaa, Ivan Mihaljevićb, Nikola Marakovićc, Radmila Kovačevića, Tvrtko Smitalb,*
6
7 Affiliations:
8 a University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovića 2, 21000
9 Novi Sad, Serbia
10 b Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković
11 Institute, Bijenička 54, 10000 Zagreb, Croatia
12 c Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10000 Zagreb, Croatia
13 These authors contributed equally to this work
14
15 *Corresponding author
16 Tvrtko Smital, PhD
17 Laboratory for Molecular Ecotoxicology
18 Division for Marine and Environmental Research
19 Ruđer Bošković Institute
20 Bijenicka 54
21 10 000 Zagreb, CROATIA
22 Tel.: **385 1 45 61 039
23 E-mail: smital@irb.hr
24
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25 Abstract
26 Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes with primary function of
27 glutathione conjugation of various endogenous and exogenous compounds. Teleost-specific Gstr1 in zebrafish
28 (Danio rerio) was previously shown to have high expression in toxicologically relevant tissues and high activity
29 towards model substrates. The aim of this study was a detailed functional characterization of zebrafish Gstr1.
30 Molecular docking analyses were used to get novel insight into structural characteristics of Gstr1 and elucidation
31 the mechanistic interactions with both GSH and various Gstr1 substrates or inhibitors. An initial screening
32 inhibition assay performed by using model fluorescence substrate monochlorobimane (MCB) revealed
33 interactions of different endogenous compounds and environmentally relevant xenobiotics with zebrafish Gstr1.
34 All interacting compounds were further analyzed to determine their inhibition type and Ki values. Our data
35 revealed that pregnenolone, progesterone, testosterone, DHEAS and corticosterone competitively inhibited
36 transformation of MCB by Gstr1 with the calculated Ki values in the range 14-26 μM, implying that these hormones
37 are physiological substrates of zebrafish Gstr1. Estrogens had no effect on Gstr1 activity. Taurochenodeoxycholate
38 (TCDC) expressed lower inhibition potency toward Gstr1 with the Ki value of 33 μM. Among tested xenobiotics
39 tributyltin chloride and rifampicin non-enzymatically bound Gstr1 enzyme (the calculated Ki values are 0.26 μM
40 and 65 μM, respectively) and inhibited its activity, showing that these compounds are reversible noncompetitive
41 inhibitors of zebrafish Gstr1. Insecticide diazinon competitively inhibited Gstr1 activity with calculated Ki value of
42 27 μM, while others Gstr1-interacting insecticides, chlorpyrifos-methyl (CPF-methyl) and malathion, showed
43 allosteric activation-like effect. Among tested pharmaceuticals, tetracycline, erythromycin and methotrexate
44 demonstrated competitive type of inhibition with the calculated Ki values of 17.5, 36.5 and 29 μM, respectively.
45 In summary, we suggest that zebrafish Gstr1 has an important role in steroidogenesis, metabolism and/or
46 physiological actions of androgens, but not estrogens in fish. Finally, our results imply the role of Gstr1 in
47 metabolism of xenobiotics and protection of fish against deleterious environmental contaminants such as
48 organophosphate insecticides and pharmaceuticals.
49
50 Keywords: Glutathione-S-transferase r1; zebrafish; molecular docking, functional characterization, interaction
51 screening, endogenous compounds, xenobiotics.
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57 1. Introduction
58 Aquatic environment is under constant pressure of newly emerging environmental contaminants. Large portion
59 of these contaminants persistently stays in the freshwater systems and can cause deleterious effects to aquatic
60 organisms (Murray et al., 2010). Consequently, it is of great importance to have better insight into mechanisms
61 evolutionary developed and present in aquatic organisms as protection against xenobiotic compounds. Cellular
62 uptake, metabolism and elimination of xenobiotics is largely mediated by numerous phase 0 membrane transport
63 proteins, following with the phase I oxidation reactions maintained mainly by the cytochrome P450 enzymes and
64 various conjugation reactions of phase II, and finally extrusion mechanisms mediated by membrane extrusion
65 transporters of the phase III of cellular detoxification. Together, these integral elements form absorption,
66 distribution, metabolism and excretion system (ADME) which critically determine toxicity of environmental
67 contaminants (ADME-Tox) and is directly or indirectly modulated by endo- and xenobiotics (Stegeman et al.,
68 2010).
69 One of critical ADME-Tox elements are glutathione S-transferases (GSTs), multifunctional phase II detoxification
70 enzymes with primary function of glutathione conjugation of various endogenous and exogenous compounds.
71 GSTs are ubiquitously present in most living organisms, from bacteria to humans (Hayes and Pulford, 1995).
72 Sheehan et al. (2001) proposed that GSTs evolved from primordial stress proteins and diverged into several
73 classes. GST classes are organized into three families: soluble cytosolic GSTs, membrane-associated microsomal
74 GSTs also known as MAPEG (membrane-associated proteins involved in eicosanoid and glutathione metabolism),
75 and mitochondrial GSTs comprised of only one Kappa class (Hayes et al., 2005; Sheehan et al., 2001; Oakley, 2011).
76 The most characterized are cytosolic GSTs, divided into six classes: alpha, mu, pi, omega, theta and zeta (Hayes
77 and Pulford, 1995). There are several additional classes reported in non-mammalian species, such as rho class in
78 teleosts and cephalochordates (Glisic et al., 2015).
79 Conjugation of GST substrates by reduced glutathione (γ-L-glutamyl-L-cysteinyl-glycine; GSH) is quantitatively the
80 major reaction of phase II metabolism (Hodgson, 2010). GSTs enzymatic activity primarily consists of ability to
81 bring a substrate to close proximity of GSH using the active site capable of binding both electrophilic substrate
82 and GSH. Second part of GST enzymatic activity is activation of sulfhydryl group of GSH, allowing the nucleophilic
83 attack to electrophilic substrate which results with more water soluble and less reactive compound (Armstrong,
84 1997). Additionally, GSTs are capable of several other enzymatic activities such as isomerization, opening of
85 epoxide rings, nucleophilic aromatic substitutions, reversible Michael additions to α,β-unsaturated aldehydes and
86 ketones, and peroxidase activity (Eaton and Bammler, 1999).
87 GSTs are active in monomeric and dimeric forms. Dimers usually consist of two identical chains, however
88 heterodimers are also found. GST monomers are made of two distinct domains, C-terminal alpha helical domain
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89 and N-terminal thioredoxin-like domain. Within the N-terminal domain there is a specific GSH-binding site, termed
90 G-site, where the GSH cysteinyl side chain gets activated through hydrogen bonding. The C-terminal and N-
91 terminal domain together shape the substrate binding site, termed H-site due to hydrophobic nature of
92 substrates. The variability of structure and chemical properties of H-sites is the main reason for polyspecific
93 interactions of GSTs with substrates (Oakley, 2011).
94 Some of well known deleterious endogenous and exogenous substrates which are conjugated by GSTs are 4-
95 hydroxynonenal (4-HNE), an aldehyde product of lipid peroxidation that can damage proteins and DNA (Singhal
96 et al., 2015), and acrolein, a reactive aldehyde used in various synthesis reactions in organic chemistry and as
97 agricultural biocide. Numerous xenobiotics such as pesticides, pharmaceuticals, polyaromatic hydrocarbons
98 (PAHs), persistent organic pollutants (POPs) and heavy metals are also reported to be substrates of GST
99 conjugating activity (Higgins and Hayes, 2011). GSTs are also involved into detoxification processes of numerous
100 epoxide carcinogens, such as aflatoxin B1, a toxic metabolic product of some Aspergillus species and
101 environmental contaminant present in cereal crops. It is detoxified by GST conjugation and later eliminated
102 through the mercapturic acid pathway (Dohnal et al., 2014). Furthermore, benzo(a)pyrene and trans isomer of
103 stilbene oxide are reported as the GST substrates (Hu et al., 1997; Seidegard et al., 1989).
104 Due to GSTs role in multidrug cancer resistance, high number of GST inhibitors have been developed (Allocati,
105 2018). Additionally, the naturally occurring GST inhibitors in plants have been identified (Harshbarger et al., 2017).
106 Some of the most characterized GST inhibitors are ethacrynic acid, an α,β-unsaturated ketone used as diuretic
107 drug, and its derivatives (Sau et al., 2010). Ethacraplatin is GST inhibitor developed to overcome cisplatin
108 resistance by adding two ethacrynic acid ligands (Johnstone et al., 2016). Some other frequently used GST
109 inhibitors are 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), potent inhibitor of GSTP1-1, and
110 auranofin, gold phosphine used as antiarthritic, with similar anticancer effects as cisplatin (Tentori et al., 2011; De
111 Luca et al., 2013).
112 Recent advances achieved using zebrafish (Danio rerio) as model organism make zebrafish and ideal candidate for
113 investigation of integral elements of ADME processes. In zebrafish, 27 Gst members are distributed within 3 major
114 families, with 7 cytosolic Gst classes showing clear orthology relationships with human GST genes (Glisic et al.,
115 2015). Tissue expression analyses of Gst genes in zebrafish revealed classes Gst Pi, Gstt1a, Gstz1, Gstr1, Mgst3a
116 and Mgst3b as crucial elements of biotransformation of xenobiotics based on their high expression in barrier
117 tissues such as liver, kidney, gills and intestine (Glisic et al., 2015). These GST members are even dominantly
118 expressed in zebrafish embryos (Glisic et al., 2016). Additionally, tissue expression of adult zebrafish GSTs
119 determined on the protein level showed comparable expression profile with mRNA levels (Tierbach et al., 2018).
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120 Functionally, the most potent activities, with high affinities and turnover numbers in reactions with model
121 substrates, were determined for members of Pi class and Gstr1 (Glisic et al., 2015).
122 Consequently, due to high expression of teleost-specific Gstr1 in toxicologically relevant tissues, and high activity
123 it shows towards model substrates, in this study we have characterized zebrafish Gstr1 in more detail. Our major
124 goals were (1) to analyze interactions of zebrafish Gstr1 with different groups of endo- and xenobiotics, and (2) to
125 determine the type of interaction of Gstr1 with tested substances in order to be able to perform initial evaluation
126 of possible protective and/or physiological role of Gstr1. Furthermore, our intention was to provide novel insight
127 into structural characteristics of this teleost-specific GST protein and elucidate the mechanistic interactions with
128 both GSH and various Gstr1 substrates or inhibitors. Therefore, by using recombinant zebrafish Gstr1 protein we
129 performed a screening inhibition assay with different endogenous and exogenous compounds to identify
130 interacting compounds, and to define their inhibition type and Ki values. By molecular docking analyses we
131 obtained data on the mechanistic interactions of Gstr1 with GSH and different endogenous and exogenous
132 compounds.
133
134 2. Materials and methods
135 Chemicals
136 All tested compounds were purchased from Sigma-Aldrich (Taufkirchen, Germany) or Alfa Aesar (Ward Hill, MA,
137 USA) unless stated otherwise.
138 Inhibition assay
139 Gstr1 was cloned and purified as described before by Glisic et al., 2015. The inhibition assay was based on
140 previously described fluorometric assay for GST activity toward monochlorobimane (MCB). The assay was
141 performed at 25 °C in black, flat bottom 96-well plates with final reaction volume of 250 μL. The reaction mixture
142 consisted of phosphate buffer (pH 6.5) in concentration of 100 mM, Gstr1 recombinant protein in final
143 concentration of 1.5 μg per well, inhibitor of desired final concentration, GSH co-substrate in final concentration
144 of 1 mM, and model substrate (MCB) in final concentration of 100 μM. The resulting fluorescence was measured
145 with microplate readers (Infinite M200, Tecan, Salzburg, Austria or Fluoroskan Ascent FL Microplate Reader,
146 Thermo Fisher, Helsinki, Finland) at 355 nm excitation and 460 nm emission wavelengths for 10 min in 15 or 30 s
147 intervals. For blank control the reaction mixture was prepared without Gstr1 recombinant protein with 100 μL of
148 phosphate buffer. Reaction mixture without tested compound was used as positive control, with 100 μL of
149 phosphate buffer.
150 An initial Gstr1 inhibition screening was performed first, using single 100 μM concentration of tested compounds.
151 Then, for compounds which showed the Gstr1 inhibition above 50% the Ki values and type of reversible interaction
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152 were determined. Inhibition assay for determination of Ki values of tested compounds was based on Michaelis-
153 Menten kinetics of MCB, which was determined using MCB in the range of 5 - 600 μM. MCB dose response was
154 inhibited with three concentrations of inhibitors based on the percentage (%) of inhibition observed by single
155 concentration inhibition screening. The strength of inhibition of the tested compounds was considered to be very
156 strong for Ki < 1 μM, strong for Ki 1-20 μM, moderate for Ki 20-40 μM, and weak for Ki > 40 μM.
157 Modeling the three-dimensional structure of Danio rerio Rho class, Gstr1, and molecular docking studies
158 Biovia Discovery Studio Client v17.2 (Accelrys, San Diego, CA, USA) implemented Build Homology Models protocol
159 was used to construct Gstr1 homology models as based on alignment of the model sequence and the template
160 structure. Build Homology Models protocol uses MODELER (Sali and Blundell, 1993) automodel to build homology
161 models. To build Gstr1 homology model, the input sequence alignment between the model sequence of Gstr1
162 monomer and the sequence of chain A from Gst from Antarctic clam (Laternula elliptica) was used (Fig. 5) (Park
163 et al., 2013). Rest of the parameters in the Parameters Explorer of Build Homology Models protocol were set as
164 described previously (Mihaljević et al., 2017). Ligands to be docked in the homology model of Gstr1 were created
165 with ChemBio3D Ultra 13.0 (PerkinElmer, Inc., Waltham, MA, USA) and minimized using the MMFF94 force field
166 implemented in ChemBio3D Ultra 13.0.
167 Biovia Discovery Studio Client v17.2 implemented Dock Ligands (CDOCKER) protocol was used for the docking
168 study. CDOCKER is a grid based molecular docking method that employs CHARMm force field (Brooks et al., 1983;
169 Momany and Rone, 1992). The model with the highest overlay similarity was used as the rigid receptors while the
170 ligands were allowed to flex during the refinement. Binding site within the homology models was defined by a
171 sphere (r = 13.0 Å) surrounding the amino acids that have been located within the identified G- and H-site. Rest
172 of the parameters included in the CDOCKER protocol were set as described elsewhere (Maraković et al., 2016).
173 The binding affinities of docked ligands in the poses generated by Dock Ligands (CDOCKER) protocol were
174 estimated using the scoring functions as implemented in the Biovia Discovery Studio Client v17.2 Score Ligand
175 Poses protocol. Together with POSE_NUMBER, -CDOCKER_ENERGY, -CDOCKER_INTERACTION_ENERGY, following
176 scoring functions were calculated. LigScore1_Dreiding and LigScore2_Dreiding (Krammer et al., 2005) are fast,
177 simple scoring functions for predicting receptor-ligand binding affinities which are computed in units of pKI (-
178 logKI). PLP1 (Gehlhaar et al., 1995) and PLP2 (Parrill and Reddy, 1999) are fast and simple docking functions that
179 have been shown to correlate well with protein-ligand binding affinities. PLP scores are measured in arbitrary
180 units. Higher PLP scores indicate stronger receptor-ligand binding (larger pKI values). Jain (Jain, 1996) is an
181 empirical scoring function developed through an evaluation of the structures and binding affinities of a series of
182 protein-ligand complexes. The Jain score is a sum of five interaction terms. These terms describe: lipophilic
183 interactions, polar attractive interactions, polar repulsive interactions, solvation of the protein and ligand and an
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184 entropy term for the ligand. The PMF (Muegge and Martin, 1999) and PMF04 (Muegge, 2006) scoring functions
185 were developed based on statistical analysis of the 3D structures of protein-ligand complexes. They were found
186 to correlate well with protein-ligand binding free energies while being fast and simple to calculate. The PMF04
187 score is an updated version of the original PMF score. The PMF scores are reported in arbitrary units. A higher
188 score indicates a stronger receptor-ligand binding affinity.
189 To identify the poses of docked ligands that score high in more than one scoring function, the Biovia Discovery
190 Studio Client v17.2 Consensus Score protocol was used. The Consensus Score protocol calculates the consensus
191 scores of a series of docked ligands for which other scores have been previously computed. For each selected
192 scoring function, the ligands are listed by score in descending order. The consensus score for a ligand is an integer
193 between zero (none of the scores are in the top-ranking percentile) and the total number of scores (all of the
194 scores are in the top-ranking percentile) listed in Input Properties. Thus, in the Parameters Explorer of Consensus
195 Score protocol, the following parameters were set. In Input Properties score properties -PLP2, -PMF, -PMF04, Jain,
196 -CDOCKER_ENERGY, -CDOCKER_INTERACTION_ENERGY, LigScore2_Dreiding, LigScore1_Dreiding, and
197 POSE_NUMBER were chosen to calculate the consensus score. Consensus Percentage was set to 20 to specify the
198 percentage of top molecules to include in the consensus. Use Best Pose only was set to False.
199 The representative poses of docked ligands with the highest consensus score were minimized using Biovia
200 Discovery Studio Client v17.2 Minimization protocol, as described elsewhere (Maraković et al., 2016).
201 Data analysis
202 All assays were performed in 3 independent experiments run in triplicates. Data shown on related figures
203 represent mean ± standard errors of mean (SEM). All calculations were performed using GraphPad Prism 6.00 for
204 Windows (GraphPad Software, San Diego, California, USA) as described below. The kinetic parameters, Km and
205 Vmax values were calculated using the Michaelis-Menten equation (1):
206
207 (1) 𝑉 = 𝑉𝑚𝑎𝑥 × [𝑆]
[𝑆] + 𝐾𝑚
208
209 where V is velocity (fluorescence units per milligram of protein per minute), Vmax is maximal velocity, [S] is
210 substrate concentration and Km is Michaelis–Menten constant.
211 For purpose of Ki determination, measured data were analyzed by nonlinear regression, mixed model enzyme
212 inhibition. The used model also provided the alpha value (α) which was used to determine the type of interaction
213 of tested compounds with Gstr1. Alpha value determines the level to which the inhibitor changes the affinity of
214 the enzyme for substrate. In case of alpha equals 0, the inhibitor does affect the binding of substrate to the
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215 enzyme, which results in noncompetitive inhibition. When alpha equals higher number, binding of inhibitor blocks
216 binding of the substrate resulting in competitive inhibition. If alpha equals less than 1, binding of the inhibitor
217 additionally stimulates binding to the enzyme, which results in uncompetitive inhibition. In addition, Lineweaver-
218 Burk plots (or double reciprocal plots), a graphical representation of the Lineweaver-Burk equation of enzyme
219 kinetics, were used to illustrate the type of interactions. Intersecting lines that converge at the y-axis is diagnostic
220 signature for the competitive inhibition modality. Intersecting lines that converge to the left of the y-axis and on
221 the x-axis is diagnostic signature for noncompetitive (α = 1) inhibition modality (Copeland, 2005).
222
223 3. Results
224 3.1 Inhibitory potential of tested compounds
225 To elucidate possible interaction of different endogenous compounds (Fig. 1) and environmentally relevant
226 xenobiotics (Fig. 2) with zebrafish Gstr1, we performed an initial screening inhibition assay with 100 μM
227 concentration of selected chemicals, except carbaryl and probenecid which were tested in concentration of 50
228 μM due to their low solubility. The selection of xenobiotics was based on the prioritization of trace pollutants in
229 surface waters (Murray et al., 2010). In the second step, all compounds that inhibited Gstr1 enzyme activity at 100
230 µM by more than 50% compared to the control were further analyzed to define their inhibition type and to
231 determine Ki values as it is shown in Table 1. To determine the type of interaction with Gstr1, we compared kinetic
232 parameters (Km and Vmax) of transformation of fluorescence substrate MCB in the absence and in the presence
233 of different interacting compounds using nonlinear regression, and mixed model enzyme inhibition. All of the
234 inhibition curves obtained are given in the Supplementary Data, while representatives of exemplary interactors
235 are shown in Figs. 3 and 4.
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236237
238 Figure 1. Interaction of zebrafish Gstr1 with endogenous compounds. Data are expressed as percentage (%) of the
239 Gstr1 activity toward model fluorescence substrate monochlorobimane (MCB; 100 μM) in presence of each
240 modulator (100 μM) and co-substrate glutathione (GSH; 1 mM) relative to Gstr1 activity in absence of a modulator
241 (control, set to 100%). Abbreviations: dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT),
242 estrone-3-sulfate (E3S), taurochenodeoxycholate (TCDC). Data represent mean ± SEM of triplicates from three
243 independent experiments.
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244245 Figure 2. Interaction of zebrafish Gstr1 and environmental contaminants. Data are expressed as percentage (%)
246 of the Gstr1 activity toward model substrate monochlorobimane (MCB; 100 μM) in presence of each modulator
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247 (100 μM) and co-substrate glutathione (GSH; 1 mM) relative to Gstr1 activity in absence of a modulator (control,
248 set to 100%). Carbaryl and probenecid are tested at 50 μM concentration due to their low solubility. Xenobiotic
249 interactors are divided into four groups: (1) polycyclic aromatic hydrocarbon, (2) industrial chemicals, (3)
250 pesticides, and (4) pharmaceuticals and personal care products (PPCPs). Abbreviations: 3,4-dichlorophenol (DCP),
251 2,4-dichlorophenoxyacetic acid (2,4-D), 3,4-dichloroaniline (DCA), 17α-ethynilestradiol (EE2), acetylsalicylic acid
252 (ASA), bis(2-ethylhexyl) phthalate (DEHP), butylated hydroxytoluene (BHT), chlorpyrifos-methyl (CPF-methyl),
253 dichlorodiphenyldichloroethylene (DDE), N,N-diethyl-meta-toluamide (DEET), perfluorooctane sulphonate
254 (PFOS), perfluorooctanoic acid (PFOA) and tert-Butylhydroquinone (tBHQ). Data represent mean ± SEM of
255 triplicates from three independent experiments.
256
Table 1. Inhibition constants (Ki) determined for a series of endogenous compounds and xenobiotic interactors
of zebrafish Gstr1. Type of interactions, alpha (α) values, 95% confidence intervals and coefficients of
determination are shown. Interactors are divided into four groups separated by doted lines: endogenous
compounds, industrial chemicals, pesticides and pharmaceuticals.
Compound Inhibition type α Ki (μM) 95% c.i. R2
Pregnenolone Competitive α » 1 13.55 ± 1.49 10.60-16.50 0.98Progesterone Competitive α » 1 26.24 ± 2.59 21.10-31.39 0.98DHEASa Competitive α » 1 19.50 ± 1.61 16.31-22.69 0.98Testosterone Competitive α » 1 19.91 ± 2.23 15.54-24.29 0.94Corticosterone Competitive α » 1 20.49 ± 4.89 10.79-30.19 0.89TCDCb Competitive α » 1 32.99 ± 2.74 27.55-38.44 0.98Tributyltin chloride Noncompetitive 1.2 ± 0.6 0.26 ± 0.02 0.21-0.31 0.93Chlorpyrifos-methyl nd 0.1 ± 0.0 nd - -Diazinon Competitive α » 1 27.03 ± 2.06 22.93-31.12 0.98Malathion nd 0.7 nd - -Erythromycin Competitive α » 1 36.47 ± 3.29 29.94-43.00 0.97Methotrexate Competitive α » 1 29.29 ± 3.04 23.25-35.33 0.98Rifampicin Noncompetitive 1.8 ± 0.4 64.83 ± 2.48 59.96-69.70 0.96Tetracycline Competitive α » 1 17.48 ± 0.95 15.59-19.37 0.98nd – not determined.a Dehydroepiandrosterone sulfate;b Taurochenodeoxycholate
257
258 3.1.1 Endogenous compounds
259 Among 10 steroid gonadal hormones screened for their interaction with Gstr1, 4 hormones showed over 50%
260 inhibition of enzyme activity compared to the control (Fig. 1) which implies possible physiological interaction of
261 Gstr1 with those steroids. All of them demonstrated competitive type of inhibition of Gstr1 activity (Table 1, Fig.
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262 3). The calculated Ki values are in the micromolar range (14-26 μM), and the pregnenolone expressed the highest
263 inhibition potency (Ki of 13.55 ± 1.49 μM) and is classified as strong inhibitor. All other endogenous compounds
264 that showed interactions with Gstr1 are classified as moderate inhibitors. Testosterone and DHEAS showed similar
265 potency with Ki (approx. 20 μM, Table 1, Fig. 3, Supplementary Data Fig. S1), while none of the tested estrogenic
266 hormones expressed activity toward Gstr1 (Fig. 1). Corticosterone showed similar inhibition potency as androgens
267 with calculated Ki of 20.49 ± 4.89 μM (Table 1, Fig. 3), while cortisol was without effect. Taurochenodeoxycholate
268 (TCDC) expressed lower inhibition potency toward Gstr1 with Ki value of 33 μM (Table 1, Fig. 3).
269
270271 Figure 3. Competitive inhibition of zebrafish Gstr1 activity by selected endogenous compounds (pregnenolone –
272 the lowest Ki value of tested endogenous compounds; testosterone – the lowest Ki value of tested androgens;
273 corticosterone – representative of glucocorticoids; taurochenodeoxycholate (TCDC) – representative of bile salts).
274 Results are shown as concentration dependence of Gstr1 mediated monochlorobimane (MCB) transformation
275 expressed in fluorescence units normalized to time and protein concentrations (FU/min/mg proteins) over MCB
276 concentration (μM) at 25 °C in presence of 1 mM GSH co-substrate. Inserted figures: Lineweaver-Burk plots that
277 show the type of inhibition. Data were fitted in the GraphPad Prism 6. Mean, SEM and confidence interval (c.i.)
278 were calculated from 4-6 replicates of three independent experiments.
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279
280 3.1.2 Industrial chemicals
281 Among tested industrial chemicals only tributyltin chloride showed a very strong noncompetitive inhibition of
282 Gstr1 activity, with Ki value of 0.26 μM (Table 1, Fig. 4). Phthalates (DEHP, dibutyl phthalate and diethyl phthalate),
283 and perfluorinated compounds (PFOA and PFOS) showed no interaction with Gstr1 enzyme. Other tested
284 industrial chemicals showed limited (inhibition of enzyme activity <50%) to no interaction with Gstr1 enzyme (Fig.
285 2).
286
287288 Figure 4. Inhibition of zebrafish Gstr1 activity by industrial chemical (tributyltin chloride – the lowest Ki value of all
289 tested compounds), pesticide representative (diazinon) and pharmaceuticals (rifampicin – as a noncompetitive
290 inhibitor; tetracycline – the lowest Ki value among tested pharmaceuticals). Concentration dependence of Gstr1
291 mediated monochlorobimane (MCB) transformation is expressed as fluorescence unit normalized to time and
292 protein concentrations (FU/min/mg proteins) over MCB concentration (μM) at 25 °C in presence of 1 mM GSH co-
293 substrate. Inserted figures: Lineweaver-Burk plots that show type of inhibition. Data were fitted in the GraphPad
294 Prism 6. Mean, SEM and confidence interval (c.i.) were calculated from 4-6 replicates of three independent
295 experiments.
673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728
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296 3.1.3 Pesticides
297 Gstr1 showed interaction with insecticides chlorpyrifos-methyl (CPF-methyl), diazinon and malathion, and
298 inhibition of enzyme activity was in the range of 49-66% (Fig. 2). Diazinon expressed moderate competitive type
299 of inhibition with calculated Ki value of 27 μM (Table 1, Fig. 4). However, CPF-methyl and malathion showed
300 allosteric activation-like effect. In fact, in the presence of the lowest concentration of both pesticides the enzyme
301 activity was increased in comparison to control, while in the presence of the highest concentration (100 μM) the
302 enzyme activity was similar to control values (Supplementary Data, Fig. S2). Other tested pesticides showed no
303 interaction with Gstr1 enzyme (Fig. 2).
304
305 3.1.4 Pharmaceuticals and personal care products (PPCPs)
306 Initial screening inhibition assay showed that pharmaceuticals erythromycin, methotrexate, rifampicin and
307 tetracycline inhibited Gstr1 activity in the range of 47-72% (Fig. 2). Among them tetracycline demonstrated strong
308 competitive type of inhibition with the lowest calculated Ki value of 17.5 μM (Table 1, Fig. 4), while erythromycin
309 and methotrexate, moderate competitive inhibitors, have higher Ki values of 36.5 and 29.29 μM, respectively
310 (Table 1, Supplementary Data, Fig. S3). On the contrary, rifampicin showed weak noncompetitive inhibition of
311 Gstr1 activity with Ki values of 65 μM (Table 1, Fig. 4). Other tested PPCPs showed limited (inhibition of enzyme
312 activity <25%) to no interaction with Gstr1 enzyme (Fig. 2).
313
314 3.2 In silico analysis of zebrafish Gstr1 structure and binding sites
315 3.2.1 Homology modeling
316 Prior to the docking studies, the three-dimensional model structure of Danio rerio Rho class Gstr1 was constructed
317 by homology modeling. For template structure used in homology modeling, crystal structure of a glutathione S-
318 transferase from Antarctic clam Laternula elliptica, chain A (PDB ID: 3QAV) (Park et al., 2013; Espinoza et al., 2013)
319 was chosen based on high indexes of sequence identity (39.1) and sequence similarity (63.9), and sequence
320 alignment was carried out (Fig. 5). To further improve the obtained model structure, energy minimization was
321 performed as described in the Materials and methods section. However, prior to defining and editing binding site
322 of the Gstr1 model structure we needed to obtain the full dimer of model structure, as it is known for binding site
323 to be outlined with amino acid residues from counterpart B monomer to considerable extent. This was done
324 through structural alignment of two copies of generated Gstr1 model structure with crystal structure of dimer of
325 human class pi glutathione S-transferase P1-1 (hGSTP1-1) (PDB ID: 1MD3), followed by manual rotation of one
326 monomer of modeled structure until the same symmetry between the two monomers was obtained as the one
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327 observed in crystal structure of hGSTP1-1. Finally, binding site was defined and edited using software feature that
328 automatically identifies cavities within the receptor and knowledge of different GSTs active sites.
329
330331332 Figure 5. Sequence alignment between 3QAV and Gstr1.333
334 3.2.2 Molecular docking
335 To confirm our experimental data that showed different modes of interaction between substrates and inhibitors
336 of rho class GST, binding studies were carried with GSH and compounds identified as substrates (testosterone and
337 corticosterone) or inhibitors (tributyltin chloride and rifampicin) using molecular docking approach. Since amino
338 acid residues that constitute glutathione-binding site (G-site) and hydrophobic substrate binding site (H-site) are
339 largely conserved through different classes of GSTs, by sequence alignment of zebrafish Gstr1 with different GSTs
340 (GSTP1, GSTP2, 6GST, GST4_4) the residues forming G-site (Tyr8, Trp9, Ser13, Pro15, Lys40, Glu42, His43, Glu47,
341 Lys49, Gln56, Leu57, Glu69, and Ser70) and H-site (Thr11, Pro14, Cys16, Glu107, Gln111, Lys112, Tyr114, Glu115,
342 Val116, Phe118, Pro124, Gly219, and Glu220) were identified. Also, to obtain starting structure for docking studies
343 with GSH occupying G-site, structural alignment between model structure of Gstr1 and crystal structure of a
344 glutathione S-transferase from Antarctic clam Laternula elliptica in a complex with GSH (PDB ID: 3QAW) was
345 conducted to hard dock the GSH to the G-site of Gstr1 model structure. Moreover, hard docking of GSH was used
346 to confirm the positions of G-site forming amino acid residues above identified via sequence alignment. Indeed,
347 GSH was found to be in close proximity to Ser13, Pro15, His43, Gln56, Leu57, Glu69, and Ser70 (Fig. 6).
348
785786787788789790791792793794795796797798799800801802803804805806807808809810811812813814815816817818819820821822823824825826827828829830831832833834835836837838839840
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349350351 Figure 6. Final 3D structure of zebrafish Gstr1 with docked GSH in each monomer G-site.352353
354 Using molecular docking of compounds previously established as substrates (testosterone or corticosterone) we
355 were able not only to confirm its susceptibility to nucleophilic addition of the GSH, but also to confirm the positions
356 of above identified H-site forming amino acid residues. From the 30 generated conformations of docked ligand
357 the representative pose was chosen based on the shortest distance between the thiol sulfur of GSH and β-carbon
358 atom from α,β-unsaturated carbonyl group of ligand. Namely, testosterone and corticosterone are supposed to
359 undergo conjugate addition of GSH to their α,β-unsaturated carbonyl group. Figure 7 depicts model complex
360 between corticosterone and GSH-Gstr1 obtained via molecular docking. The distance between thiol sulfur and β-
361 carbon atom of corticosterone is estimated to be 4.43 Å, a distance close enough for reactivating groups in pre-
362 reactionary complex for nucleophilic addition to proceed. Another important feature predicted by model complex
363 is the 3.41 Å distance between thiol sulfur and hydrogen atom of the hydroxyl group of Ser13, a highly conserved
364 residue known to catalyze nucleophilic addition of the GSH to substrates electrophilic groups, which is sufficient
365 for hydrogen bond to occur between these two atoms. Also, corticosterone is found to be in close proximity of
366 Tyr114 and Glu115, amino acid residues above identified to form H-site, but also in close proximity of Gln110,
367 pointing at this residue as another H-site forming residue. Interestingly, corticosterone is found to be forming
368 alkyl-type hydrophobic interaction with Val116, another H-site forming residue, but from the counterpart B
841842843844845846847848849850851852853854855856857858859860861862863864865866867868869870871872873874875876877878879880881882883884885886887888889890891892893894895896
17
369 monomer. Other H-site residues from the B monomer in close proximity of corticosterone include Gln111 and
370 Glu115. This finding emphasizes the importance of using dimeric model structures of GSTs for more accurate
371 binding studies.
372
373374375 Figure 7. Final docking pose of the corticosterone-GSH-Gstr1 complex, and close-up view of the active site 376 residues.377378 On the other hand, with molecular docking of compounds established as reversible noncompetitive inhibitors
379 (tributyltin chloride or rifampicin) we were able to elucidate the binding mode responsible for its inhibitory mode
380 of action. The representative pose was chosen based on the highest score when implementing Consenus Score
381 protocol from Biovia Discovery Studio Client v17.2 (for more details, please see Materials and methods section).
382 Indeed, as can be seen in Figure 8, binding studies suggest that known reversible inhibitor rifampicin occupies G-
383 site, thus blocking the approach of GSH. In doing so, rifampicin interacts with neighboring residues via different
384 types of non-covalent interactions.
897898899900901902903904905906907908909910911912913914915916917918919920921922923924925926927928929930931932933934935936937938939940941942943944945946947948949950951952
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385
386 387
388 Figure 8. Final docking pose of the rifampicin-GSH-Gstr1 complex, and close-up view of the active site residues.
389 Dotted lines represent non-covalent interactions.
390
391 4. Discussion
392 Zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology. In recent years it has
393 emerged as a popular model for use in pharmacological and toxicological studies. To understand the disposition
394 of endo- and xenobiotic compounds in zebrafish, it is important to identify and characterize in detail critical
395 elements of the absorption, distribution, metabolism and excretion (ADME) processes. GST superfamily, an
396 integral element of ADME, still awaits to be fully elucidated in fish species. In our previous study (Glisic et al.,
397 2015) we performed a comprehensive characterization of the GST superfamily in zebrafish, revealing a great
398 diversity of fish GSTs (in total 27 members found in zebrafish), as well as clear orthology relations with human and
399 other species Gsts. Furthermore, our tissue expression profiling, the initial functional characterization of nine
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400 cytosolic Gst enzymes, and their functional similarity to the human orthologs in respect to the xenobiotic
401 metabolism, pointed to involvement of Gst pi, Gstt1a, Gstz1, Gstr1, and Mgst3a and 3b in biotransformation of
402 xenobiotics. Gstr1 is highly expressed in toxicologically relevant tissues and, together with members of Pi class, it
403 functionally showed the most potent activities, with high affinities and turnover numbers in reactions with model
404 substrates (Glisic et al., 2015). Based on those insights in this study we aimed at detailed characterization of a
405 teleost-specific GST member, Gstr1.
406 Using the recombinant enzyme and the screening assay, we have been able to identify endo- and xenobiotic
407 compounds that interact with zebrafish Gstr1. In the next step of our study, by using inhibition enzyme assays we
408 have determined type and Ki values of observed interactions. Finally, by in silico analysis of zebrafish Gstr1
409 structure and binding sites we have confirmed the positions of identified G- and H-site forming amino acid residues
410 (Fig. 6) and substrates susceptibility to nucleophilic addition of the GSH (Fig. 7). In addition, we elucidated the
411 binding mode responsible for reversible noncompetitive inhibitor mode of action (Fig. 8).
412 There is a high degree of conservation of endocrine processes and roles of steroid hormones between zebrafish
413 and terrestrial vertebrates (Tokarz et al., 2013). As a discrepancy, it was shown that 11-ketotestosterone instead
414 of testosterone is the prominent circulating androgen in zebrafish (Waal et al., 2007). We showed that zebrafish
415 Gstr1 activity is competitively inhibited by four steroid hormones: progesterone, pregnenolone, DHEAS and
416 testosterone, with calculated Ki values in range of 14-26 μM (Tab. 1, Fig. 3, Supplementary Data Fig. S1). Molecular
417 docking of testosterone, experimentally identified as Gstr1 substrate, confirms its susceptibility to nucleophilic
418 addition of the GSH by Gstr1. These findings revealed steroid hormones as probable physiological substrates of
419 zebrafish Gstr1. It was shown that mammalian GSTs have the ability to bind hormones, and particularly sexual
420 steroids, and influence their transport, metabolism, and physiological action (Listowsky et al., 1988). A high
421 binding capacity for steroids was previously displayed by certain isoenzymes of GST (Eliasson et al., 1999; Homma
422 et al., 1986). Particularly, testosterone and progesterone have the ability to bind mammalian GSTs with moderate
423 (10-6 M < Kd < 10-4 M) or high (Kd < 10-6 M) affinity, respectively (Listowsky et al., 1988). This is in line with our
424 findings. Another study (Remoué et al., 2002) demonstrated a specific binding between testosterone and a
425 parasite Schistosoma haematobium GST enzyme, with higher affinity of binding (Kd = 5.7 x 10-7 M). Functionally,
426 cytosolic alpha class of GST enzymes has role in the GSH dependent obligatory double-bond isomerization of
427 delta(5)-androstene-3,17-dione and delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone,
428 respectively (Johansson and Mannervik, 2001; Tars et al., 2010). Demonstrated interactions of 4 steroid hormones
429 and zebrafish Gstr1 enzyme suggest possible involvement of Gstr1 in steroidogenesis, metabolism and/or
430 physiological action of these androgens. On the contrary, none of the tested estrogenic hormones showed activity
431 toward Gstr1 (Fig. 1). As we previously showed, Gstr1 expression in ovary is 5-fold lower than in testes (Glisic et
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432 al., 2015) which could be possibly explained by absence of interactions between Gstr1 enzyme and estrogenic
433 hormones.
434 GSTs have glucocorticoid-binding properties and, thereby, may influence transport, metabolism, and action of
435 steroids (Homma and Listowsky, 1985). While corticosterone is no hormone naturally occurring in teleosts (Balm
436 et al., 1989), it is almost identical to cortisol lacking only the 17α-hydroxyl group. Furthermore, it was found to
437 bind to and activate the teleostean glucocorticoid receptor (Mommsen et al., 1999). We showed that zebrafish
438 Gstr1 activity is competitively inhibited by corticosterone, with calculated Ki value of 20.5 μM (Table 1, Fig. 3). This
439 finding showed that corticosterone could be a physiological Gstr1 substrate. As previously showed, Gstr1 is highly
440 expressed in zebrafish brain of both genders (Glisic et al., 2015). Interestingy, in brain of rats a corticosterone-
441 induced decline of GST activity that may strongly promote neurodegeneration was reported (Zafir and Banu,
442 2009). Molecular docking of corticosterone, identified in this study as a substrate, confirms its susceptibility to
443 nucleophilic addition of the GSH by Gstr1. We showed that corticosterone forms alkyl-type hydrophobic
444 interaction with Val116, another H-site forming residue, but from the counterpart B monomer. In addition, H-site
445 residues from the B monomer are in close proximity of corticosterone (Fig. 7). These findings emphasize the
446 importance of using dimeric model structures of GSTs for more accurate binding studies. However, although
447 cortisol is the major stress hormone in zebrafish that acts as both glucocorticoid and mineralocorticoid (Tokarz et
448 al., 2013), it does not interact with Gstr1 (Fig. 1).
449 One of the two major primary bile salts in many ray-finned fish is chenodeoxycholyltaurine
450 (taurochenodeoxycholic acid), while zebrafish is the only teleost model species that synthesizes mainly 5α (trans
451 A/B ring) bile salts (Hagey et al., 2010). Herewith we show that taurochenodeoxycholate (TCDC) is a competitive
452 inhibitor, i.e. probable physiological substrate of zebrafish Gstr1 (Table 1, Fig. 3) with calculated Ki value of 33 μM.
453 Therefore, we suggest that Gstr1 activity could influence physiological actions of TCDC in zebrafish.
454 Among tested industrial chemicals (Fig. 2) only tributyltin chloride, an organometal used as an antifouling biocide,
455 demonstrated a strong noncompetitive inhibition of Gstr1 with calculated Ki value of 0.26 μM (Table 1, Fig. 4).
456 These findings, together with molecular docking results, revealed reversible inhibitor character of tributyltin
457 chloride toward Gstr1 enzyme. Our results are similar to some previous observations for inhibitory effect of
458 tributyltin on GST activity in fish. A very high potency of organotin compounds (tributyltin, triphenyltin) for
459 inhibition of plaice cytosolic GST activity in vitro was shown by George and Buchanan (1990). Also, organotins
460 including tributyltin are reported to inhibit GST activity in fish (Al-Ghais and Ali, 1999; Padrós et al., 2003; WU et
461 al., 2007), and it was speculated that organotin-GSH complexes bind to the first and the second catalytic sites of
462 GST. On the contrary, it was shown that long-term exposure to sub-lethal concentrations of tributyltin, in a range
463 of Ki value we calculated, caused ROS stress in the liver of common carp and significantly induced GST activity (Li
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464 et al., 2015). Also, Wang et al. (2006) showed that GST activity increased after exposure to lower dose of tributyltin
465 in liver of Sebastiscus marmoratus, and opposite effect was noted with higher dose. Studies with rats confirmed
466 that tributyltin induces oxidative stress by inhibiting GST which results in apoptosis (Ishihara et al., 2012; Mitra et
467 al., 2013). GST activity inhibition leads to increase lipid peroxidation as its substrates inside the cells are organic
468 hydroperoxides and 4-hydroxyalkenals. Generally, inhibition of the GST activity by tributyltin reduced the capacity
469 of cells/organs to detoxify other chemicals and increase the vulnerability to oxidative stress.
470 Of all tested pesticides, group of organophosphate insecticides, chlorpyrifos-methyl (CPF-methyl), diazinon and
471 malathion caused inhibition of Gstr1 activity in the range of 49-66% in comparison to control (Fig. 2). The absence
472 of interactions of Gstr1 enzyme with other tested pesticides is in line with data published so far. Actually, Trute et
473 al. (2007) showed that major hepatic coho salmon (Oncorhynchus kisutch) GST isoforms, belonging to Pi and a
474 Rho-class, have no activity towards the pesticides, including atrazine. However, Booth and O’Halloran (2001)
475 revealed that GST catalyses the conjugation of glutathione with xenobiotics, including organophosphorus
476 pesticides. Our results revealed that diazinon competitively inhibits Gstr1 activity with calculated Ki value of 27
477 μM (Table 1, Fig. 4). Gstr1 activity changes caused by different concentration of CPF-methyl and malathion did not
478 reveal clear and consistent inhibition pattern and it was not possible to calculate Ki values (Table 1, Supplementary
479 Data Fig. S2). Because of that, the nature of interactions of these pesticides with Gstr1 remains unclear. However,
480 their environmental loads (Murray et al., 2010) are not sufficient to reach effective concentrations required to
481 modulate zebrafish Gstr1 activity (Fig. 2). Based on presented results, we propose that diazinon is possible Gstr1
482 substrate, which could qualify zebrafish Gstr1 as a diazinon-metabolizing enzyme. Diazinon-metabolizing GST
483 enzyme was revealed for silkworm, suggesting that it may detoxify diazinon (Yamamoto and Yamada, 2016).
484 Another study revealed that diazinon exposure to common carp (Cyprinus carpio) induces GST enzyme activities
485 in liver, which were assumed to have resulted from the defense against the toxicity of diazinon (Oruc, 2011).
486 Previous studies on effect of chlorpyrifos on GST activity showed statistically significant decrease in the GSTs
487 activity in the zebrafish larvae, common carp and rats (Jin et al., 2015; Xing et al., 2012; Mansour and Mossa,
488 2009). Interestingly, in fish species Labeo rohita and Carassius auratus gibelio, malathion caused modulation of
489 GST activity in time-dependent and tissue-specific manner (Thenmozhi et al., 2011; Huculeci et al., 2008). Similarly,
490 increase and decrease of GST activity across different organs were observed after exposure of neonatal rats to
491 malathion (Timur et al., 2003). Overall, according to our results and previously published data, Gstr1 probably
492 belongs to group of enzymes capable of metabolizing organophosphate insecticides, thus protecting fish against
493 those deleterious environmental contaminants.
494 Screening assay performed in this study revealed that from all tested pharmaceuticals and personal care products
495 (PPCPs), only erythromycin, methotrexate, rifampicin and tetracycline interact with zebrafish Gstr1 (Fig. 2).
11211122112311241125112611271128112911301131113211331134113511361137113811391140114111421143114411451146114711481149115011511152115311541155115611571158115911601161116211631164116511661167116811691170117111721173117411751176
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496 However, these pharmaceuticals interact with Gstr1 differently. E.g., macrocyclic antibiotic rifampicin is a
497 noncompetitive reversible inhibitor of Gstr1 activity with calculated Ki value of 65 μM (Table 1, Fig. 4). Molecular
498 docking confirmed that rifampicin is reversible noncompetitive inhibitor of zebrafish Gstr1 enzyme and elucidated
499 the binding mode responsible for its inhibitory action. Binding studies suggest that rifampicin occupies G-site of
500 Gstr1, thus blocking the approach of GSH. Actually, rifampicin interacts with neighboring residues via different
501 types of non-covalent interactions (Fig. 8). Although several previous studies have shown induction of expression
502 of GST transcripts by rifampicin in fish and human hepatocytes (Corcoran et al., 2012; Rae et al., 2001), and
503 induction of GST activity in rats (Adachi et al., 1985), there are no reports describing rifampicin-dependent
504 modulation of GSTs enzyme activity in fish. To the best of our knowledge, this is the first report describing
505 interaction of rifampicin to GST enzyme. On the other hand, pharmaceuticals erythromycin, methotrexate and
506 tetracycline competitively inhibit Gstr1 activity with calculated Ki values in a range of 17.5-36.5 μM (Table 1, Fig.
507 4, Supplementary Data Fig. S3). Competitive type of inhibition categorizes these drugs as possible substrates which
508 are metabolized by zebrafish Gstr1 enzyme. There is evidence that erythromycin, a macrolide antibiotic, inhibits
509 GST activity in liver of crucian carp (Carassius auratus), where the inhibition rate decreased with exposure time
510 and concentration. Among all tested xenobiotics, tetracycline expressed the most potent competitive inhibition
511 (Ki = 17.5 μM) of Gstr1 enzyme activity (Table 1, Fig. 4). This polyketide antibiotic inhibits human recombinant
512 GSTP1‐1 and GSTM3-3 enzymes with IC50 values of 13 μM and 47 μM, respectively (Mukanganyama et al., 2002).
513
514 5. Conclusion
515 Our study provides the first functional characterization of a teleost-specific GST Rho member protein in zebrafish,
516 Gstr1. Combining experimental data obtained using the described in vitro inhibition assays, insights based on the
517 obtained 3D structure of zebrafish Gstr1, and finally molecular docking studies, we have shown that
518 pregnenolone, progesterone, testosterone, DHEAS and corticosterone are probable physiological substrates of
519 zebrafish Gstr1. Based on the obtained data we hypothesize that Gstr1 probably has an important role in
520 steroidogenesis, metabolism and/or physiological actions of androgens, but not estrogens in fish. In addition,
521 reversible inhibitors of fish Gstr1 were identified among environmental contaminants, and our results imply the
522 role of Gstr1 in metabolism of xenobiotics and protection of fish against deleterious environmental contaminants
523 such as organophosphate insecticides and pharmaceuticals.
524
525 Acknowledgments
526 This work was supported by the Croatian National Science Foundation (Project No. 4806), the SCOPES programme
527 joint research project granted by the Swiss National Science Foundation (SNSF) (Grant No. SCOPES -
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528 IZ73ZO_152274/1), Serbian Ministry of Education, Sciences and Technological Development (No. 173037), and by
529 STIM - REI, a project funded by European Union from European Structural and Investment Funds 2014 - 2020,
530 Contract Number: KK.01.1.1.01.0003. The Biovia Discovery Studio Client v17.2 (Accelrys, San Diego, CA, USA)
531 software, used for homology modeling and molecular docking studies, was provided through Croatian Science
532 Foundation project (project CHOLINESTERASE, grant number IP-11-2013-4307).
533
534 References
535 Adachi, Y., Nanno, T., Yamashita, M., Ueshima, S., Yamamoto, T., 1985. Induction of rat liver bilirubin-
536 conjugating enzymes and glutathione s-transferase by rifampicin. Gastroenterol. Jpn. 20, 104–110.
537 Al-Ghais, S.M., Ali, B., 1999. Inhibition of glutathione S-transferase catalyzed xenobiotic detoxication by
538 organotin compounds in tropical marine fish tissues. Bull. Environ. Contam. Toxicol. 62, 207–213.
539 https://doi.org/10.1007/s001289900861
540 Allocati, N., 2018. Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and
541 neurodegenerative diseases. Oncogenesis. https://doi.org/10.1038/s41389-017-0025-3
542 Armstrong, R.N., 1997. Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases. Chem.
543 Res. Toxicol. 10, 2–18.
544 Balm, P.H.M., Lambert, J.D.G., Wendelaar Bonga, S.E., 1989. Corticosteroid biosynthesis in the interrenal cells of
545 the teleost fish, Oreochromis mossambicus. Gen. Comp. Endocrinol. 76, 53–62.
546 https://doi.org/10.1016/0016-6480(89)90032-4
547 Booth, L.H., O’Halloran, K., 2001. A comparison of biomarker responses in the earthworm Aporrectodea
548 caliginosa to the organophosphorus insecticides diazinon and chlorpyrifos. Environ. Toxicol. Chem. 20,
549 2494–2502. https://doi.org/10.1002/etc.5620201115
550 Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., Karplus, M., 1983. CHARMM: A
551 program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–
552 217. https://doi.org/10.1002/jcc.540040211
553 Copeland, R.A., 2005. Evaluation of Enzyme Inhibitors in Drug Discovery, Journal of Chemical Information and
554 Modeling. A John Wiley & Sons, Inc., Hoboken, New Jersey.
555 https://doi.org/10.1017/CBO9781107415324.004
556 Corcoran, J., Lange, A., Winter, M.J., Tyler, C.R., 2012. E ff ects of Pharmaceuticals on the Expression of Genes
557 Involved in Detoxi fi cation in a Carp Primary Hepatocyte Model. Environ. Sci. Technol. 46, 6306–6314.
558 De Luca, A. De, Hartinger, C.G., Dyson, P.J., Lo, M., Casini, A., 2013. A new target for gold ( I ) compounds :
559 Glutathione-S-transferase inhibition by aurano fi n. J. Inorg. Biochem. 119, 38–42.
12331234123512361237123812391240124112421243124412451246124712481249125012511252125312541255125612571258125912601261126212631264126512661267126812691270127112721273127412751276127712781279128012811282128312841285128612871288
24
560 https://doi.org/10.1016/j.jinorgbio.2012.08.006
561 Dohnal, V., Wu, Q., Kuc, K., 2014. Metabolism of aflatoxins : key enzymes and interindividual as well as
562 interspecies differences. https://doi.org/10.1007/s00204-014-1312-9
563 Eaton, D.L., Bammler, T.K., 1999. Concise review of the glutathione S-transferases and their significance to
564 toxicology. Toxicol. Sci. 49, 156–64. https://doi.org/10.1093/toxsci/49.2.156
565 Eliasson, M., Stark, T., Depierre, J.W., 1999. Expression of glutathione transferase isoenzymes in the porcine
566 ovary in relationship to follicular maturation and luteinization 117, 35–48.
567 Espinoza, H.M., Shireman, L.M., McClain, V., Atkins, W., Gallagher, E.P., 2013. Cloning, expression and analysis of
568 the olfactory glutathione S-transferases in coho salmon. Biochem. Pharmacol. 85, 839–848.
569 https://doi.org/10.1016/j.bcp.2012.11.018
570 Gehlhaar, D.K., Verkhivker, G.M., Rejto, P.A., Sherman, C.J., Fogel, D.B., Fogel, L.J., Freer, S.T., 1995. Molecular
571 recognition of the inhibitor AG-1243 by HIV-1 protease: conformationally exible docking by evolutionary
572 programming. Chem. Biol. 2, 317–324.
573 Glisic, B., Hrubik, J., Fa, S., Dopudj, N., Kovacevic, R., Andric, N., 2016. Transcriptional profiles of glutathione-S-
574 Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos. Environ. Toxicol. 31.
575 https://doi.org/10.1002/tox.22038
576 Glisic, B., Mihaljevic, I., Popovic, M., Zaja, R., Loncar, J., Fent, K., Kovacevic, R., Smital, T., 2015. Characterization
577 of glutathione-S-transferases in zebrafish (Danio rerio). Aquat. Toxicol. 158, 50–62.
578 https://doi.org/10.1016/j.aquatox.2014.10.013
579 Hagey, L.R., Møller, P.R., Hofmann, A.F., Krasowski, M.D., 2010. Diversity of Bile Salts in Fish and Amphibians:
580 Evolution of a Complex Biochemical Pathway. Physiol. Biochem. Zool. 83, 308–321.
581 https://doi.org/10.1086/649966
582 Harshbarger, W., Gondi, S., Ficarro, S.B., Hunter, J., Udayakumar, D., Gurbani, D., Singer, W.D., Liu, Y., Li, L.,
583 Marto, J.A., Westover, K.D., 2017. Structural and Biochemical Analyses Reveal the Mechanism of
584 Glutathione S -Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine * Edited by Norma
585 Allewell 292, 112–120. https://doi.org/10.1074/jbc.M116.750299
586 Hayes, J.D., Flanagan, J.U., Jowsey, I.R., 2005. GLUTATHIONE TRANSFERASES. Annu. Rev. Pharmacol. Toxicol. 45,
587 51–88. https://doi.org/10.1146/annurev.pharmtox.45.120403.095857
588 Hayes, J.D., Pulford, D.J., 1995. The glutathione S-transferase supergene family: regulation of GST and the
589 contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol.
590 Biol. 30, 445–600. https://doi.org/10.3109/10409239509083491
591 Higgins, L.G., Hayes, J.D., 2011. Mechanisms of induction of cytosolic and microsomal glutathione transferase
12891290129112921293129412951296129712981299130013011302130313041305130613071308130913101311131213131314131513161317131813191320132113221323132413251326132713281329133013311332133313341335133613371338133913401341134213431344
25
592 (GST) genes by xenobiotics and pro-inflammatory agents. Drug Metab. Rev. 43, 92–137.
593 https://doi.org/10.3109/03602532.2011.567391
594 Hodgson, E., 2010. Introduction to Biotransformation (Metabolism), in: Krieger, R. (Ed.), Hayes’ Handbook of
595 Pesticide Toxicology. Elsevier Inc., pp. 865–875. https://doi.org/10.1016/B978-0-12-374367-1.00036-7
596 Homma, H., Listowsky, I., 1985. Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled
597 with dexamethasone 21-methanesulfonate. Proc. Natl. Acad. Sci. U. S. A. 82, 7165–9.
598 https://doi.org/10.1073/PNAS.82.21.7165
599 Homma, H., Maruyama, H., Niitsu, Y., Listowsky, I., 1986. A subclass of glutathione S-transferases as intracellular
600 high-capacity and high-affinity steroid-binding proteins 235, 763–768.
601 Hu, X., O’Donnell, R., Srivastava, S.K., Xia, H., Zimniak, P., Nanduri, B., Bleicher, R.J., Awasthi, S., Awasthi, Y.C., Ji,
602 X., Singh, S. V., 1997. Active site architecture of polymorphic forms of human glutathione S-transferase P1-
603 1 accounts for their enantioselectivity and disparate activity in the glutathione conjugation of 7β,8α-
604 dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Biochem. Biophys. Res. Commun. 235, 424–
605 428. https://doi.org/10.1006/bbrc.1997.6777
606 Huculeci, R., Dinu, D., Staicu, C., Munteanu, M.C., Costache, M., Dinischiotu, A., 2008. Malathion-Induced
607 Alteration of the Antioxidant Defence System in Kidney, Gill, and Intestine of Carassius auratus gibelio.
608 Environ. Toxicol. 523–530. https://doi.org/10.1002/tox.20454
609 Ishihara, Y., Kawami, T., Ishida, A., Yamazaki, T., 2012. Tributyltin induces oxidative stress and neuronal injury by
610 inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures. Neurochem. Int. 60, 782–
611 790. https://doi.org/10.1016/j.neuint.2012.03.004
612 Jain, A.N., 1996. Scoring noncovalent protein-ligand interactions: A continuous differentiable function tuned to
613 compute binding affinities. J. Comput. Aided. Mol. Des. 10, 427–440. https://doi.org/10.1007/BF00124474
614 Jin, Y., Liu, Z., Peng, T., Fu, Z., 2015. The toxicity of chlorpyrifos on the early life stage of zebrafish: A survey on
615 the endpoints at development, locomotor behavior, oxidative stress and immunotoxicity. Fish Shellfish
616 Immunol. 43, 405–414. https://doi.org/10.1016/j.fsi.2015.01.010
617 Johansson, A.S., Mannervik, B., 2001. Human glutathione transferase A3-3, a highly efficient catalyst of double-
618 bond isomerization in the biosynthetic pathway of steroid hormones. J. Biol. Chem. 276, 33061–5.
619 https://doi.org/10.1074/jbc.M104539200
620 Johnstone, T.C., Suntharalingam, K., Lippard, S.J., 2016. The Next Generation of Platinum Drugs : Targeted Pt ( II )
621 Agents , Nanoparticle Delivery , and Pt ( IV ) Prodrugs. https://doi.org/10.1021/acs.chemrev.5b00597
622 Krammer, A., Kirchhoff, P.D., Jiang, X., Venkatachalam, C.M., Waldman, M., 2005. LigScore: A novel scoring
623 function for predicting binding affinities. J. Mol. Graph. Model. 23, 395–407.
13451346134713481349135013511352135313541355135613571358135913601361136213631364136513661367136813691370137113721373137413751376137713781379138013811382138313841385138613871388138913901391139213931394139513961397139813991400
26
624 https://doi.org/10.1016/j.jmgm.2004.11.007
625 Li, Z.H., Li, P., Shi, Z.C., 2015. Responses of the hepatic glutathione antioxidant defense system and related gene
626 expression in juvenile common carp after chronic treatment with tributyltin. Ecotoxicology 24, 700–705.
627 https://doi.org/10.1007/s10646-014-1416-2
628 Listowsky, I., Abramovitz, M., Homma, H., Niitsu, Y., 1988. Intracellular Binding and Transport of Hormones and
629 Xenobiotics by Glutathiones-Transferases. Drug Metab. Rev. 19, 305–318.
630 https://doi.org/10.3109/03602538808994138
631 Mansour, S.A., Mossa, A.-T.H., 2009. Lipid peroxidation and oxidative stress in rat erythrocytes induced by
632 chlorpyrifos and the protective effect of zinc. Pestic. Biochem. Physiol. 93, 34–39.
633 https://doi.org/10.1016/j.pestbp.2008.09.004
634 Maraković, N., Knežević, A., Vinković, V., Kovarik, Z., Šinko, G., 2016. Design and synthesis of N-substituted-2-
635 hydroxyiminoacetamides and interactions with cholinesterases. Chem. Biol. Interact. 259, 122–132.
636 https://doi.org/10.1016/j.cbi.2016.05.035
637 Mihaljević, I., Popović, M., Žaja, R., Maraković, N., Šinko, G., Smital, T., 2017. Interaction between the zebrafish (
638 Danio rerio ) organic cation transporter 1 (Oct1) and endo- and xenobiotics. Aquat. Toxicol. 187, 18–28.
639 https://doi.org/10.1016/j.aquatox.2017.03.012
640 Mitra, S., Srivastava, A., Khandelwal, S., 2013. Tributyltin chloride induced testicular toxicity by JNK and p38
641 activation, redox imbalance and cell death in sertoli-germ cell co-culture. Toxicology 314, 39–50.
642 https://doi.org/10.1016/j.tox.2013.09.003
643 Momany, F.A., Rone, R., 1992. Validation of the general purpose QUANTA ®3.2/CHARMm® force field. J.
644 Comput. Chem. 13, 888–900. https://doi.org/10.1002/jcc.540130714
645 Mommsen, T.P., Vijayan, M.M., Moon, T.W., 1999. Cortisol in teleosts: dynamics, mechanisms of action, and
646 metabolic regulation. Rev. Fish Biol. Fish. 9, 211–268. https://doi.org/10.1023/A:1008924418720
647 Muegge, I., 2006. PMF scoring revisited. J. Med. Chem. 49, 5895–5902. https://doi.org/10.1021/jm050038s
648 Muegge, I., Martin, Y.C., 1999. A general and fast scoring function for protein-ligand interactions: A simplified
649 potential approach. J. Med. Chem. 42, 791–804. https://doi.org/10.1021/jm980536j
650 Mukanganyama, S., Widersten, M., Naik, Y., Mannervik, B., Hasler, J., 2002. IN H IB ITIO N O F G LU TA TH IO N E
651 S-TR A N SFER A SES B Y A N TIM A LA R IA L D R U G S PO SSIB LE IM PLIC A TIO N S FO R C IR C U M V EN
652 TIN G A N TIC A N C ER D R U G R ESISTA N C E. Int. J. Cancer 97, 700–705.
653 Murray, K.E., Thomas, S.M., Bodour, A.A., 2010. Prioritizing research for trace pollutants and emerging
654 contaminants in the freshwater environment. Environ. Pollut. 158, 3462–3471.
655 https://doi.org/10.1016/j.envpol.2010.08.009
14011402140314041405140614071408140914101411141214131414141514161417141814191420142114221423142414251426142714281429143014311432143314341435143614371438143914401441144214431444144514461447144814491450145114521453145414551456
27
656 Oakley, A., 2011. Glutathione transferases: a structural perspective. Drug Metab. Rev. 43, 138–51.
657 https://doi.org/10.3109/03602532.2011.558093
658 Oruc, E., 2011. Effects of diazinon on antioxidant defense system and lipid peroxidation in the liver of Cyprinus
659 carpio (L.). Environ. Toxicol. 26, 571–578. https://doi.org/10.1002/tox.20573
660 Padrós, J., Pelletier, É., Ribeiro, C.O., 2003. Metabolic interactions between low doses of benzo[a]pyrene and
661 tributyltin in arctic charr (Salvelinus alpinus): A long-term in vivo study. Toxicol. Appl. Pharmacol. 192, 45–
662 55. https://doi.org/10.1016/S0041-008X(02)00042-X
663 Park, A.K., Moon, J.H., Jang, E.H., Park, H., Ahn, I.Y., Lee, K.S., Chi, Y.M., 2013. The structure of a shellfish specific
664 GST class glutathione S -transferase from antarctic bivalve Laternula elliptica reveals novel active site
665 architecture. Proteins Struct. Funct. Bioinforma. 81, 531–537. https://doi.org/10.1002/prot.24208
666 Parrill, A.L., Reddy, R.M., 1999. Rational Drug Design, in: Novel Methodology and Practical Applications.
667 American Chemical Society.
668 Rae, J.M., Johnson, M.D., Lippman, M.E., Flockhart, D.A., 2001. Rifampin is a selective, pleiotropic inducer of
669 drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays. J.
670 Pharmacol. Exp. Ther. 299, 849–857.
671 Remoué, F., Mani, J., Pugnière, M., Schacht, A., Capron, A., Riveau, G., Remoue, F., 2002. Functional Specific
672 Binding of Testosterone to Schistosoma haematobium Functional Specific Binding of Testosterone to
673 Schistosoma haematobium 28-Kilodalton Glutathione S -Transferase †. Infect. Immun. 70, 601–605.
674 https://doi.org/10.1128/IAI.70.2.601
675 Sali, A., Blundell, T.L., 1993. Comparative Protein Modelling by Satisfaction of Spatial Restraints. J. Molecuilar
676 Biol. 234, 779–815.
677 Sau, A., Pellizzari Tregno, F., Valentino, F., Federici, G., Caccuri, A.M., 2010. Glutathione transferases and
678 development of new principles to overcome drug resistance. Arch. Biochem. Biophys. 500, 116–22.
679 https://doi.org/10.1016/j.abb.2010.05.012
680 Seidegard, J., Pero, R., Stille, B., 1989. Identification of the trans-stilbene oxide-active glutathione transferase in
681 human mononuclear leukocytes and in liver as GST1. Biochem. Genet. 27, 253–261.
682 Sheehan, D., Meade, G., Foley, V.M., Dowd, C.A., 2001. Structure, function and evolution of glutathione
683 transferases: implications for classification of an ancient enzyme superfamily. Biochem. J. 360, 1–16.
684 https://doi.org/10.1042/0264-6021:3600001
685 Singhal, S.S., Singh, S.P., Singhal, P., Horne, D., Singhal, J., Awasthi, S., 2015. Antioxidant role of glutathione S-
686 transferases : 4-Hydroxynonenal , a key molecule in stress-mediated signaling. Toxicol. Appl. Pharmacol.
687 289, 361–370. https://doi.org/10.1016/j.taap.2015.10.006
14571458145914601461146214631464146514661467146814691470147114721473147414751476147714781479148014811482148314841485148614871488148914901491149214931494149514961497149814991500150115021503150415051506150715081509151015111512
28
688 Stegeman, J.J., Goldstone, J. V., Hahn, M.E., 2010. Perspectives on zebrafish as a model in environmental
689 toxicology. Fish Physiol. 29, 367–439. https://doi.org/10.1016/S1546-5098(10)02910-9
690 Tars, K., Olin, B., Mannervik, B., 2010. Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class
691 Glutathione Transferases. J. Mol. Biol. 397, 332–340. https://doi.org/10.1016/j.jmb.2010.01.023
692 Tentori, L., Susanna, A., Mazzon, E., Muzi, A., Sau, A., Cuzzocrea, S., Vernole, P., Federici, G., Maria, A., Graziani,
693 G., 2011. benzoxadiazol-4-ylthio ) hexanol ( NBDHEX ) increases temozolomide efficacy against malignant
694 melanoma. Eur. J. Cancer 47, 1219–1230. https://doi.org/10.1016/j.ejca.2010.12.008
695 Thenmozhi, C., Vignesh, V., Thirumurugan, R., Arun, S., 2011. Impacts of Malathion on mortality and biochemical
696 changes of freshwater fish Labeo rohita. Iran J. Environ. Heal. Sci. Eng. 8, 387–394.
697 Tierbach, A., Groh, K.J., Schönenberger, R., Schirmer, K., Suter, M.J.F., 2018. Glutathione S-Transferase protein
698 expression in different life stages of zebrafish (Danio rerio). Toxicol. Sci. 162, 702–712.
699 https://doi.org/10.1093/toxsci/kfx293
700 Timur, S., Önal, S., Karabay, N.Ü., Sayim, F., Zihnioǧlu, F., 2003. In vivo effects of Malathion on Glutathione-S-
701 transferase and acetylcholinesterase activities in various tissues of neonatal rats. Turkish J. Zool. 27, 247–
702 252.
703 Tokarz, J., Möller, G., Hrabˇ, M., 2013. Journal of Steroid Biochemistry and Molecular Biology Zebrafish and
704 steroids : What do we know and what do we need to know ? ଝ 137, 165–173.
705 https://doi.org/10.1016/j.jsbmb.2013.01.003
706 Trute, M., Gallis, B., Doneanu, C., Shaffer, S., Goodlett, D., Gallagher, E., 2007. Characterization of hepatic
707 glutathione S-transferases in coho salmon (Oncorhynchus kisutch). Aquat. Toxicol. 81, 126–36.
708 https://doi.org/10.1016/j.aquatox.2006.11.009
709 Waal, P.P. De, Wang, D.S., Nijenhuis, W.A., Schulz, R.W., Bogerd, J., 2007. Functional characterization and
710 expression analysis of the androgen receptor in zebrafish ( Danio rerio ) testis.
711 https://doi.org/10.1530/REP-08-0055
712 Wang, C., Zhao, Y., Zheng, R., Ding, X., Wei, W., Zuo, Z., Chen, Y., 2006. Effects of tributyltin, benzo[a]pyrene, and
713 their mixture on antioxidant defense systems in Sebastiscus marmoratus. Ecotoxicol. Environ. Saf. 65, 381–
714 387. https://doi.org/10.1016/j.ecoenv.2005.08.003
715 WU, Y. qiong, WANG, C. gang, WANG, Y., ZHAO, Y., CHEN, Y. xin, ZUO, Z. hong, 2007. Antioxidant responses to
716 benzo[a]pyrene, tributyltin and their mixture in the spleen of Sebasticus marmoratus. J. Environ. Sci. 19,
717 1129–1135. https://doi.org/10.1016/S1001-0742(07)60184-3
718 Xing, H., Wang, X., Sun, G., Gao, X., Xu, S., Wang, X., 2012. Effects of atrazine and chlorpyrifos on activity and
719 transcription of glutathione S-transferase in common carp (Cyprinus carpio L.). Environ. Toxicol. Pharmacol.
15131514151515161517151815191520152115221523152415251526152715281529153015311532153315341535153615371538153915401541154215431544154515461547154815491550155115521553155415551556155715581559156015611562156315641565156615671568
29
720 33, 233–244. https://doi.org/10.1016/j.etap.2011.12.014
721 Yamamoto, K., Yamada, N., 2016. Identification of a diazinon- metabolizing glutathione S - transferase in the
722 silkworm , Bombyx mori. Nat. Publ. Gr. 6, 1–9. https://doi.org/10.1038/srep30073
723 Zafir, A., Banu, N., 2009. Modulation of in vivo oxidative status by exogenous corticosterone and restraint stress
724 in rats. Stress 12, 167–177. https://doi.org/10.1080/10253890802234168
15691570157115721573157415751576157715781579158015811582158315841585158615871588158915901591159215931594159515961597159815991600160116021603160416051606160716081609161016111612161316141615161616171618161916201621162216231624
Supplementary Data
Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class
Authors:
Branka Bašicaa, Ivan Mihaljevićb, Nikola Marakovićc, Radmila Kovačevića, Tvrtko Smitalb,*
Affiliations:a University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovica
2, 21000 Novi Sad, Serbiab Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer
Bošković Institute, Bijenicka 54, 10000 Zagreb, Croatiac Instiute for Medical Research and Occupational Medicine, Ksaverska cesta 2, 10000 Zagreb, Croatia
These authors contributed equally to this work
*Corresponding author
Tvrtko Smital, PhD
Laboratory for Molecular Ecotoxicology
Division for Marine and Environmental Research
Ruđer Bošković Institute
Bijenicka 54
10 000 Zagreb, CROATIA
Tel.: **385 1 45 61 039
E-mail: smital@irb.hr
Figure S1. Inhibition of zebrafish Gstr1 activity by endogenous compounds (progesterone and DHEAS).
Concentration dependence of Gstr1 mediated monochlorobimane (MCB) transformation is expressed in
fluorescence units normalized to time and protein concentration (FU/min/mg proteins) over MCB
concentration (μM) at 25 °C in presence of 1 mM GSH co-substrate. Inserted figures: Lineweaver-Burk
plots that show the type of inhibition. Data were fitted in the GraphPad Prism 6. Mean, SEM and
confidence interval (c.i.) were calculated from 4 replicates of two independent experiments.
Fig. S2. Inhibition of zebrafish Gstr1 activity by organophosphate insecticides (chlorpyrifos-methyl and
malathion). Concentration dependence of Gstr1 mediated monochlorobimane (MCB) transformation is
expressed in fluorescence units normalized to time and protein concentration (FU/min/mg proteins) over
MCB concentration (μM) at 25 °C in presence of 1 mM GSH co-substrate. Inserted figures: Lineweaver-
Burk plots. Data were fitted in the GraphPad Prism 6. Mean, SEM and confidence interval (c.i.) were
calculated from 4 replicates of two independent experiments.
Fig. S3. Inhibition of zebrafish Gstr1 activity by pharmaceuticals (erythromycin and methotrexate).
Concentration dependence of Gstr1 mediated monochlorobimane (MCB) transformation is expressed in
fluorescence units normalized to time and protein concentration (FU/min/mg proteins) over MCB
concentration (μM) at 25 °C in presence of 1 mM GSH co-substrate. Inserted figures: Lineweaver-Burk
plots that show the type of inhibition. Data were fitted in the GraphPad Prism 6. Mean, SEM and
confidence interval (c.i.) were calculated from 4 replicates of two independent experiments.
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