Assessment of the Acute Toxicity, Uptake and Biotransformation Potential of Benzotriazoles in Zebrafish (<i>Danio rerio</i>) Larvae Combining HILIC- with RPLC-HRMS for High-Throughput Identification

The current study reports on the toxicity, uptake, and biotransformation potential of zebrafish (embryos and larvae) exposed to benzotriazoles (BTs). Acute toxicity assays were conducted. Cardiac function abnormalities (pericardial edema and poor blood circulation) were observed from the phenotypic analysis of early life zebrafish embryos after BTs exposure. For the uptake and biotransformation experiment, extracts of whole body larvae were analyzed using liquid chromatography–high-resolution tandem mass spectrometry (UPLC-Q-TOF-HRMS/MS). The utility of hydrophilic interaction liquid chromatography (HILIC) as complementary technique to reversed phase liquid chromatography (RPLC) in the identification process was investigated. Through HILIC analyses, additional biotransformation products (bio-TPs) were detected, because of the enhanced sensitivity and better separation efficiency of isomers. Therefore, reduction of false negative results was accomplished. Both oxidative (hydroxylation) and conjugative (glucuronidation, sulfation) metabolic reactions were observed, while direct sulfation proved the dominant biotransformation pathway. Overall, 26 bio-TPs were identified through suspect and nontarget screening workflows, 22 of them reported for the first time. 4-Methyl-1-<i>H</i>-benzotriazole (4-MeBT) demonstrated the highest toxicity potential and was more extensively biotransformed, compared to 1-<i>H</i>-benzotriazole (BT) and 5-methyl-1-<i>H</i>-benzotriazole (5-MeBT). The extent of biotransformation proved particularly informative in the current study, to explain and better understand the different toxicity potentials of BTs

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.

<p>List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.</p

Flow cytometric analysis of expression of Myc-tagged OR2 on the surface of Bm5 cells.

<p>(<b>A</b>) N- or (<b>B</b>) C-terminally Myc-tagged OR2 were stably expressed in Bm5 cells in the presence of flagOR7 and analyzed by FACS for the extracellular localization of the Myc tag. For each panel, the green tracing and number represent fluorescence values obtained from the staining of the cells with only the FITC labelled secondary antibodies, while the red tracing and number represent values obtained from cells incubated with both the primary anti-Myc and the FITC labelled secondary antibodies. Increased fluorescence intensity (2.59-fold over the background value) was observed for the C-terminally Myc-tagged receptor in comparison to the receptor that was Myc-tagged at the N-terminal end (1.43-fold over the background value). (<b>C</b>) Values (increases over background) indicated in this panel represent the mean ± S.E.M. of three independent experiments. (<b>D</b>) Western blot analyses of whole cell lysates from the stable cell lines used for FACS analysis and control, mock-transformed cells, probed with anti-Myc (upper) and anti- tubulin (lower) antibodies. (<b>E</b>) FACS analysis of cells expressing the N-terminally Myc-tagged µ-opioid receptor used as a positive control for the extracellular localization of the Myc tag. Green and red tracing/numbers are as in panels A and B. Inset shows the detection of µOR in these cells by western blotting with the anti-myc antibody. The arrowhead and the arrow point to major bands detected (putative monomer and dimer, respectively), while the positions of 50, 90 and 118-kDa molecular mass markers are indicated at left.</p

Expression of <i>A. gambiae</i> OR1, OR2 and OR7 in insect cells.

<p>(<b>A</b>) Schematic representation of the basic backbone vector (pEIA) used for the heterologous expression of various forms (tagged and untagged) of ORs in lepidopteran cells. <i>hr3</i> enhancer, baculoviral (BmNPV) homologous region 3 enhancer sequence; pActin, <i>Bombyx mori</i> A3 cytoplasmic actin promoter; MCS, multiple cloning site; actin pA, 3′untranslated region of <i>B. mori</i> actin gene containing polyadenylation signals; IE1 cassette, baculoviral (BmNPV) DNA fragment containing the <i>ie-1</i> transactivator gene under the control of its native viral promoter; OR; <i>A. gambiae</i> odorant receptor ORF; Myc, Flag and MycHis, epitope tags. (<b>B</b>) Detection of heterologous expression of C-terminally MycHis-tagged ORs in transfected Hi5 cells using Myc monoclonal antibody. (<b>C</b>) Detailed western blot analysis of OR2. Hi5 cells were transfected with plasmids expressing different versions of OR2, and lysates were analyzed using a specific polyclonal antibody against OR2 (left panel, lanes 1–5) or monoclonal antibodies against the Myc (middle panel, lanes 6–9) or the Flag epitope (right panel, lanes 10–11). Arrowheads and arrows indicate major bands corresponding to monomers and putative dimers, respectively. (<b>D</b>) Detailed western blot analysis of OR1. Hi5 cells were transfected with plasmids expressing different versions of OR1, and lysates were analyzed using monoclonal antibodies against the Myc (middle panel, lanes 1–4) or the Flag epitope (right panel, lanes 5–6). In the left panel immunoreactivity of the specific polyclonal antibody against OR1 is shown, with lysates from cells expressing mycOR1 after treatment with the proteasome inhibitor MG132. Molecular weight markers are shown on the left. (<b>E</b>) and (<b>F</b>) Effect of coexpression of OR7 on the expression levels of OR1 and OR2. Hi5 cells were transfected with constant amounts (45% of total DNA) of Myc-tagged OR1 or OR2, along with equal amounts of Flag-tagged OR7 or empty vector (pEIA), and pEIA-GFP (10% of total DNA, for evaluation of the efficiency of transfection). Whole cell lysates (<b>E</b>) and membrane fractions (<b>F</b>) were analysed by SDS-PAGE and western blot. Detection of OR1, OR2 and OR7 was done using the anti-Myc and anti-Flag antibodies either consecutively (in <b>E</b>) or simultaneously (in <b>F</b>). To control for loading, the whole lysate fractions were also probed with an anti-tubulin antibody.</p

Topology assays for the mosquito OR1 and OR2.

<p>Chimeric receptor proteins fused at either their N- or the C- terminus to the TEV cleavage sequence (THE) and the HR3 transcription factor are co-expressed in Hi5 cells with a GFP reporter construct together with or without TEV protease. For both OR1 (<b>A</b>) and OR2 (<b>B</b>) N-terminal fusions (HR3-THE-OR1 and HR3-THE-OR2), expression of the TEV protease resulted in increase of fluorescence of the cells. No significant increase of fluorescence was detected when the C-terminal fusions of ORs were used (OR1-THE-HR3 and OR2-THE-HR3). Similar constructs of the human opioid receptor δ (δOR) that was used as control (<b>C</b>) give opposite results with an increased fluorescence for the C-terminal fusion (δOR-THE-HR3). For each chimeric receptor protein, both representative images (left) and quantitative results (right, with values representing the mean ± S.E.M. of four experiments) from the fluorometric analysis are shown.</p