Design, Synthesis, and Docking Studies of Arylhydrazone Compounds and Evaluation of Their Platelet Aggregation Inhibitory Effect and Cytotoxicity

In view of proven antiplatelet activity of hydrazone group containing compounds, two series of hydrazone derivatives were synthesized by coupling appropriate aldehydes with phenyl hydrazine and Isonicotinic acid in the presence of distilled water and a catalytic amount of glacial acetic acid. All synthesized compounds were screened for their antiplatelet activity against induced aggregation by adenosine diphosphate (ADP) and arachidonic acid (AA). The results indicate that compounds in arylhydrazone group had shown satisfactory activity. Among them, 1-(3-methoxybenzylidene)-2-phenylhydrazine (1c), 2-methoxy-4-(2-phenylhydrazono) methyl phenol (1g), and 2-((2-phenylhydrazono) methyl)-1H-pyrrole (1h) were found to be the most potent antiplatelet compounds with IC50 less than 39 μM. Furthermore, the cell toxicity assay, (MTT test) indicates their noncytotoxic in various cell lines. None of the synthesized N-isonicotinohydrazide derivatives in this study excreted sufficient antiplatelet activity.

DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 μL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0–2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA.

Single-Larva RNA Sequencing Identifies Markers of Copper Toxicity and Exposure in Early Mytilus californianus Larvae

One of the challenges facing efforts to generate molecular biomarkers for toxins is distinguishing between markers that are indicative of exposure and markers that provide evidence of the effects of toxicity. Phenotypic anchoring provides an approach to help segregate markers into these categories based on some phenotypic index of toxicity. Here we leveraged the mussel embryo-larval toxicity assay in which toxicity is estimated by the fraction of larvae that exhibit an abnormal morphology, to isolate subsets of larvae that were abnormal and thus showed evidence of copper-toxicity, versus others that while exposed to copper exhibited normal morphology. Mussel larvae reared under control conditions or in the presence of increasing levels of copper (3–15 μg/L Cu2+) were physically sorted according to whether their morphology was normal or abnormal, and then profiled using RNAseq. Supervised differential expression analysis identified sets of genes whose differential expression was specific to the pools of abnormal larvae versus normal larvae, providing putative markers of copper toxicity versus exposure. Markers of copper exposure and copper-induced abnormality were involved in many of the same pathways, including development, shell formation, cell adhesion, and oxidative stress, yet unique markers were detected in each gene set. Markers of effect appeared to be more resolving between phenotypes at the lower copper concentration, while markers of exposure were informative at both copper concentrations.

Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline

The zebrafish as an alternative animal model for developmental toxicity testing has been extensively investigated, but its assay protocol was not harmonized yet. This study has validated and optimized the zebrafish developmental toxicity assay previously reported by multiple inter-laboratory studies in the United States and Europe. In this study, using this classical protocol, of 31 ICH-positive compounds, 23 compounds (74.2%) were teratogenic in zebrafish, five had false-negative results, and three were neither teratogenic nor non-teratogenic according to the protocol standard; of 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish and two had false-positive results. After we added an additional TI value in the zebrafish treated with testing compounds at 2 dpf along with the original 5 dpf, proposed a new category as the uncategorized compounds for those TI values smaller than the cutoff both at 2 dpf and 5 dpf but inducing toxic phenotypes, refined the testing concentration ranges, and optimized the TI cut-off value from ≥ 10 to ≥ 3 for compounds with refined testing concentrations, this optimized zebrafish developmental assay reached 90.3% sensitivity (28/31 positive compounds were teratogenic in zebrafish) and 88.9% (40/45) overall predictability. Our results from this study strongly support the use of zebrafish as an alternative in vivo method for screening and assessing the teratogenicity of candidate drugs for regulatory acceptance.

Synthesis of Mono-Carbonyl Analogues of Curcumin Assisted by Microwave Irradiation

Mono-carbonyl compounds of curcumin, especially those containing hydroxy groups at the para position in the aromatic ring flanked by an electron withdrawing group (EWG) like chlorine, are known to have anti-inflammatory, antioxidant, and antibacterial activity. This study aims to synthesize mono-carbonyl compounds of curcumin with assisted microwave synthesis and determine its toxicity. The acute toxicity assay carried out on zebrafish larvae. The results showed that the synthesis of mono-carbonyl compounds of curcumin with assisted microwave synthesis gave clean products, faster reaction rates, more product yields, economical, and environmentally friendly. The optimal synthesis results obtained at 160Watt microwave radiation energy for 10 minutes. The acute toxicity assay of HGV-6, PGV-6, and GVT-6 compounds showed low toxicity to zebrafish larvae.