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

Image_2.JPEG

<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

Image_1.JPEG

<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

Image_5.JPEG

<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

Image_4.JPEG

<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

Image_3.JPEG

<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

Synthesis and Biological Evaluation of a c(RGDyK) Peptide Conjugate of SRPIN803

In the present study, SRPIN803 and c­(RGDyK)-SRPIN803 hybrid compounds were efficiently synthesized and evaluated for their stability in human plasma and buffers of pH 7.4 and 5.2. The hybrids were mainly cytostatic against a panel of tested cancer cells, whereas one c­(RGDyK)-SRPIN803 hybrid, geo35, was the most active compound in this screen and was cytotoxic against cell lines MCF7 and MRC5 with IC50 values of 61 and 63 μM, respectively. SRPIN803 and geo35 exhibited antiangiogenic activity in zebrafish embryos, and this effect was dose-dependent. Although c­(RGDyK)-SRPIN803 hybrid compounds were found less potent compared to SRPIN803, they have shown activities interesting enough to illustrate the potential of this approach for the development of a new class of antiangiogenic compounds