Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Lethal and Sub-Lethal Effects and Modulation of Gene Expression Induced by T Kinase Inhibitors in Zebrafish (Danio Rerio) Embryos

Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.

Mepanipyrim and cyprodinil induce divergent temporal patterns of AhR-mediated responses in zebrafish (Danio rerio) embryos and larvae

Mepanipyrim and cyprodinil are widely used to control and/or prevent fungal diseases in fruit culture. They are widely detected in aquatic environment and numerous food commodities including fruit and fruit products. Different from TCDD, mepanipyrim and cyprodinil are more easily degraded and metabolized in the environment. However, the in vivo analysis of their metabolic dynamics is unclear and need to be further confirmed. In this study, zebrafish embryos were constantly exposed to 100 µg/L mepanipyrim or cyprodinil for 7 days. The temporal pattern of CYP1A and AhR2 expression and EROD enzyme activity at different time frames during embryonic and larval development of zebrafish were investigated. Our results showed that mepanipyrim and cyprodinil tend to accumulate in zebrafish during early embryonic developmental stages. Meanwhile, mepanipyrim and cyprodinil exposure could increase the expression level of cyp1a and ahr2 genes and EROD activity by a dynamic pattern in different developmental stages of zebrafish. Besides, their metabolites, which may accumulate in the zebrafish larvae, have strong AhR agonistic activity and showed strong AhR binding ability. Importantly, the risk of exposure to pesticides in embryo stage is huge, and should be paid more attention.

Ecotoxicological assessment of two surfactant on the emryonic development

In this study, zebrafish (Danio rerio) embryos was served as a model for marine fauna to determine if there is any potential of organ-specific toxicity (neuromuscular, hepatic, cytotoxic, and cardiac) caused by Silicone-Q-22 and Ploy-Q-47. as both surfactants are considered eco-friendly corrosion inhibitors. The calculated LC50 of Silicon-Q-22 and Poly-Q-47 was 22.36 and 8.28 mg/L, respectively. At NOEC both surfactants had resulted in teratogenic defects and cardiotoxicity, but only poly Q-47 resulted in neurotoxicity.