Synthesis, In Silico and In Vivo Toxicity Assessment of Functionalized Pyridophenanthridinones via Sequential MW-Assisted Intramolecular Friedel-Crafts Alkylation and Direct C-H Arylation.

A rapid, efficient, and original synthesis of novel pyrido[3,2,1-de]phenanthridin-6-ones is reported. First, the key cinnamamide intermediates 8a-f were easily prepared from commercial substituted anilines, cinnamic acid, and 2-bromobenzylbromide in a tandem amidation and N-alkylation protocol. Then, these N-aryl-N-(2-bromobenzyl) cinnamamides 8a-f were subjected to a TFA-mediated intramolecular Friedel-Crafts alkylation followed by a Pd-catalyzed direct C-H arylation to obtain a series of potentially bioactive 4-phenyl-4,5-dihydro-6H,8H-pyrido[3,2,1-de]phenanthridin-6-one derivatives 4a-f in good yields. Finally, the toxicological profile of the prepared final compounds, including their corresponding intermediates, was explored through in silico computational methods, while the acute toxicity toward zebrafish embryos (96 hpf-LC50, 50% lethal concentration) was also determined in the present study.

Teratogenic effects induced by paracetamol, ciprofloxacin, and their mixture on Danio rerio embryos: Oxidative stress implications.

Even though the toxic effects of paracetamol (PCM) and ciprofloxacin (CPX) have been deeply studied in the last decades, the impact of the PCM-CPX mixture may induce in aquatic organisms is poorly known. Thus, the objective of this work was to investigate the teratogenic effects and oxidative stress that PCM, CPX, and their mixture induce in Danio rerio embryos. Moreover, we aimed to determine whether the PCM-CPX mixture induces more severe effects on the embryos than the individual drugs. For this purpose, zebrafish embryos (4 hpf) were exposed to environmentally relevant concentrations of PCM, CPX, and their mixture until 96 hpf. In addition, at 72 hpf and 96 hpf, we also evaluated the oxidative stress biomarkers (superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation, and hydroperoxides and carbonyl content) in the embryos. Our results demonstrated that PCM, CPX, and their mixture reduced the survival rate of embryos by up to 75%. In addition, both drugs, induced morphological alterations in the embryos, causing their death. The most observed malformations were: scoliosis, craniofacial malformations, hypopigmentation, growth retardation, pericardial edema. Concerning oxidative stress, our integrated biomarkers response (IBR) analysis demonstrated that PCM, CPX, and their mixture induce oxidative damage on the embryos. In conclusion, PCM, CPX, and their mixture can alter zebrafish embryonic development via an oxidative stress response.

Evaluation of the environmental impact of magnetic nanostructured materials at different trophic levels.

Safety on the use of magnetic nanomaterials (MNMs) has become an active topic of research given all the recent applications of these materials in various fields. It is known that the toxicity of MNMs depends on size, shape, and surface functionalization. In this study, we evaluate the biocompatibility with different aquatic organisms of engineered MNMs-CIT with excellent aqueous dispersion and long-term colloidal stability. Primary producers (the alga Pseudokirchneriella subcapitata), primary consumers (the rotifer Lecane papuana), and predators (the fish, Danio rerio) interacted with these materials in acute and sub-chronic toxicity tests. Our results indicate that P. subcaptita was the most sensitive taxon to MNMs-CIT. Inhibition of their population growth (IC50 = 22.84 mg L-1) elicited cell malformations and increased the content of photosynthetic pigments, likely due to inhibition of cell division (as demonstrated in AFM analysis). For L. papuana, the acute exposure to MNMs shows no significant mortality. However, adverse effects such as decreased rate of population and altered swimming patterns arise after chronic interaction with MNMs. For D. rerio organisms on early life stages, their exposure to MNMs results in delayed hatching of eggs, diminished survival of larvae, altered energy resources allocation (measured as the content of total carbohydrates, lipids, and protein), and increased glucose demand. As to our knowledge, this is the first study that includes three different trophic levels to assess the effect of MNMs in aquatic organisms; furthermore, we demonstrated that these MNMs pose hazards on aquatic food webs at low concentrations (few mgL-1).

Toxicity testing of pesticides in zebrafish-a systematic review on chemicals and associated toxicological endpoints.

The use of zebrafish (Danio rerio) has arisen as a promising biological platform for toxicity testing of pesticides such as herbicides, insecticides, and fungicides. Therefore, it is relevant to assess the use of zebrafish in models of exposure to investigate the diversity of pesticide-associated toxicity endpoints which have been reported. Thus, this review aimed to assess the recent literature on the use of zebrafish in pesticide toxicity studies to capture data on the types of pesticide used, classes of pesticides, and zebrafish life stages associated with toxicity endpoints and phenotypic observations. A total of 352 articles published between September 2012 and May 2019 were curated. The results show an increased trend in the use of zebrafish for testing the toxicity of pesticides, with a great diversity of pesticides (203) and chemical classes (58) with different applications (41) being used. Furthermore, experimental outcomes could be clustered in 13 toxicity endpoints, mainly developmental toxicity, oxidative stress, and neurotoxicity. Organophosphorus, pyrethroid, azole, and triazine were the most studied classes of pesticides and associated with various toxicity endpoints. Studies frequently opted for early life stages (embryos and larvae). Although there is an evident lack of standardization of nomenclatures and phenotypic alterations, the information gathered here highlights associations between (classes of) pesticides and endpoints, which can be used to relate mechanisms of action specific to certain classes of chemicals.

Exposure to the azo dye Direct blue 15 produces toxic effects on microalgae, cladocerans, and zebrafish embryos.

Aquatic pollution caused by dyes has increased together with the growth of activities using colorants such as the textile, leather, food, and agrochemicals industries. Because most popular azo dyes are synthesized from benzidine, a carcinogenic compound, a threat to aquatic biota could be expected. The use of single species for toxicity assessment provides limited data, so a battery of test organisms, including representatives of different trophic levels such as algae, zooplankters, and fish, could undoubtedly provide more information. Therefore, our study was aimed at evaluating the toxic effect of the azo dye Direct blue 15 (DB15) on a battery of bioassays using a primary producer (Pseudokirchneriella subcapitata), a primary consumer (Ceriodaphnia dubia), and a secondary consumer (Danio rerio). P. subcapitata was more sensitive to DB15 (IC50 = 15.99 mg L-1) than C. dubia (LC50: 450 mg L-1). In the algae exposed to DB15, chlorophyll-a and -b were significantly increased, and carotenoids were reduced. The concentrations of protein, carbohydrates, and lipids per cell in P. subcapitata exposed to all DB15 concentrations were significantly higher than that measured in control. At 25 mg L-1 of DB15, survival, total progeny, and the number of released clutches were significantly decreased, and the start of reproduction was delayed in C. dubia. DB15 did not induce lethal or sublethal effects in D. rerio embryos at any of the tested concentrations from 24 to 72 h post-fertilization (hpf), but from 96 to 144 hpf, the larvae exposed to 100 and 500 mg L-1 developed yolk sac edema, curved tail, and skeletal deformations. After 144 hpf, DB15 produced a significant increase in embryos without a heartbeat, as the concentration of dye raised. The textile-used, azo dye DB15, caused toxic effects of different magnitude on microalgae, cladocerans, and zebrafish embryos; for this reason, the discharge of this colorant into waterbodies should be regulated to prevent environmental impacts.

Production of White, Red and Black Quinoa (Chenopodium quinoa Willd Var. Real) Protein Isolates and Its Hydrolysates in Germinated and Non-Germinated Quinoa Samples and Antioxidant Activity Evaluation.

Red, black and white seeds quinoa were germinated at 28 °C during 24 (G1), 48 and 72 h (G3). Red quinoa presented a higher percentage of germination with a value of 46% of germination at 72 h. Quinoa protein isolate (QPI) was obtained by alkaline extraction (pH 8.0) followed by an isoelectric precipitation (pH 4.5) from white, red and black quinoa seeds, germinated QPI-G1 or QPI-G3 and non-germinated QPI-NG, Chenopodium quinoa Willd var. Real. QPI-G1, QPI-G3 and QPI-NG were subject to a simulated gastric digestion (DG) and in vitro duodenal digestion (DD). The antioxidant activity was evaluated using the 1, 1-diphenyl-2-picryl hydrazyl (DPPH), azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) methods. Gastric and duodenal digest of QPI-NG and QPI-G1 and QPI-G3 from white, red and black quinoa presented antioxidant activity. QPI-G1-DD of white quinoa presented the highest antioxidant activity with a DPPH value of 167.98 µmoL TE/g of digest, QPI-G1-DD of red quinoa with an ABTS value of 204.86 µmoL TE/g of digest and QPI-G1-DD of black quinoa with an ORAC value of 401.42 µmoL TE/g of digest. QPI-G3-DD of white quinoa presented higher antioxidant activity with a DPPH value of 186.28 µmoL TE/g of sample, QPI-G3-DD of red quinoa with an ABTS value of 144.06 µmoL TE/g of digest and QPI-G3-DD of black quinoa with an ORAC value of 395.14 µmoL TE/g of digest. The inhibition of reactive oxygen species (ROS) production in the zebrafish embryo model (Danio rerio) was evaluated. Protein profiles of QPI from white, red and black from germinated quinoa and non-germinated quinoa were similar with proteins between 10 kDa to 100 kDa with the presence of globulins 11S and 7S and 2S albumins.

Congo red dye diversely affects organisms of different trophic levels: a comparative study with microalgae, cladocerans, and zebrafish embryos.

Global consumption of synthetic dyes is roughly 7 × 105 tons per year, of which the textile industry expends about two-thirds. Consumption of synthetic dyes produces large volumes of wastewater discharged into aquatic ecosystems. Colored effluents produce toxic effects in the hydrobionts, reduce light penetration, and alter the photosynthetic activity, causing oxygen depletion, among other effects. Some dyes, such as Congo red (CR), are elaborated with benzidine, a known carcinogenic compound. Information regarding dye toxicity in aquatic ecosystems is scarce; therefore, our study was aimed at evaluating the toxicity of CR on a battery of bioassays: the microalga Pseudokirchneriella subcapitata, the cladocerans Daphnia magna and Ceriodaphnia rigaudi, and the zebrafish Danio rerio. P. subcapitata was the most sensitive species to CR (IC50, 3.11 mg L-1); in exposed individuals, population growth was inhibited, but photosynthetic pigments and macromolecule concentrations were stimulated. D. magna was tolerant to high dye concentrations, the determined LC50 (322.9 mg L-1) is not an environmentally relevant value, but for C. rigaudi, LC50 was significantly lower (62.92 mg L-1). In zebrafish embryos, exposure to CR produced yolk sac edema, skeletal deformities, and stopped larvae hatching; lack of heart beating was the only observed lethal effect. CR affected organisms of different trophic levels diversely. Particularly, the effects observed in microalgae confirm the vulnerability of primary producers to dye-polluted wastewaters, because dyes produced toxic effects and interfered with photosynthesis. Different cladoceran species displayed different acute effects; thus, species sensitivity must also be considered when toxicity of dyes is assessed. Inhibition of fish larvae hatching is a significant effect not previously reported that warns about the toxicity of dyes in fish population dynamics. Synthetic azo colorants should be considered as emerging pollutants because they are discharged into the aquatic environment and are not currently included in the environmental regulation of several countries.

Biological impacts of organophosphates chlorpyrifos and diazinon on development, mitochondrial bioenergetics, and locomotor activity in zebrafish (Danio rerio).

The objectives of this study were to compare the biological responses in developing zebrafish to two organophosphate insecticides, chlorpyrifos (CPF) and diazinon (DZN). Zebrafish embryos were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 1.0, 10.0 and 25.0 µM CPF, as well as one dose of 0.1, 1.0, 10.0 and 100.0 µM DZN for 96 h. CPF at 10.0 and 25.0 µM caused 70-80% and 100% mortality in embryos after 96 h exposure, whereas embryos treated with 10.0 and 100.0 µM DZN showed 30-40% and 70-80% lethality. CPF at 10.0 µM significantly decreased cumulative hatching rate, whereas hatching rate was significantly reduced in embryos treated with 100.0 µM DZN. Spinal lordosis was primarily observed in larvae exposed to 1.0 and 10.0 µM CPF, whereas pericardial edema was mainly detected with 10.0 and 100.0 µM DZN exposure. Embryo exposed to 1.0, 10.0 and 25.0 µM CPF exhibited no mitochondrial dysfunction; exposure to 100.0 µM DZN significantly inhibited mitochondrial bioenergetics. To determine if CPF and DZN affected larval activity, dark photokinesis response was assessed in larvae following 7 days exposure to 0.1 and 1.0 µM CPF, as well as to 0.1 1.0, and 10.0 µM DZN. Larvae exposed to 1.0 µM CPF showed hypoactivity, whereas the activity in the dark was not overtly changed in larvae exposed to DZN. In summary, CPF showed higher developmental toxicity compared to DZN. Moreover, based on the types of morphological deformities noted, as well as differences in locomotor activity, we conclude that OPs have unique chemical-specific modes of action that can result in varied biological responses during early development.

Inhibition of Lipid Peroxidation of Kiwicha (Amaranthus caudatus) Hydrolyzed Protein Using Zebrafish Larvae and Embryos.

Amaranth protein concentrate (APC) was hydrolyzed under in vitro gastrointestinal conditions. APC proteins were partially degraded by pepsin at pHs 1.2, 2.0, and 3.2. During the intestinal phase (pepsin/pancreatin enzymes at pH 7.0), no polypeptide bands were observed in the gel, suggesting the susceptibility of amaranth proteins to the action of digestive enzymes. The potent in vitro inhibition of lipid peroxidation, shown by the gastric and intestinal digests, was confirmed in the zebrafish larvae, with a 72.86% reduction in oxidation of lipids in the presence of the gastric hydrolysate at pH 2.0, compared to a 95.72% reduction in the presence of the gastrointestinal digest. APC digests were capable of reducing reactive oxygen species (ROS) production in the zebrafish embryo model with a value of fluorescence of 52.5% for the gastric hydrolysate, and 48.4% for the intestinal hydrolysate.