Optimising the use of zebrafish embryos and in vitro models as alternatives to rodent testing for assessing neutrophil responses to nanomaterials

Optimising the use of zebrafish embryos and in vitro models as alternatives to rodent testing for assessing neutrophil responses to nanomaterials
Nanomaterials (NMs), defined as materials with at least one dimension measuring 100 nm or less, exhibit unique properties making them highly attractive for use in a variety of consumer products such as cosmetics, clothing, food packaging, electronics, and medicines. There are uncertainties regarding the potential adverse effects of engineered NMs on human health. The safety of engineered NMs must therefore be thoroughly assessed to ensure the responsible exploitation of nanotechnology. Assessment of NM safety currently relies on the use of rodents. In rodent studies, neutrophil accumulation (e.g. in the lung) is commonly used as a marker of NM toxicity. However, there is an urgent need to better align nanotoxicology with the principles of the 3Rs: the replacement, reduction, and refinement of the use of animals in research. To achieve this, it is proposed that zebrafish (Danio rerio) embryos and in vitro models should be utilised more widely when screening NM toxicity.
In this project, transgenic zebrafish larvae with fluorescently-labelled neutrophils (Tg (mpx:EGFP)) were exploited to assess inflammatory responses to NMs using two exposure methods: aqueous exposure of injured fish (the tail fin injury model), and microinjection into the otic vesicle. Larvae were exposed to NMs at 3 dpf and inflammatory responses monitored for 48 hours. In the tail fin injury model it was shown that post-injury aqueous treatment with silver (Ag) and zinc oxide (ZnO) NMs (0.02, 0.375, 0.75, 1.5 Ag µg/mL, and 0.5, 1, 5, 7.5 Zn µg/mL) stimulated an enhanced and sustained neutrophilic inflammatory response. Following microinjection, Ag NMs (1.95, 3.9, and 7.8 Ag µg/mL) stimulated a neutrophilic inflammatory response at the otic vesicle. These results show that the zebrafish larvae can be used to quickly determine the pro-inflammatory effects of NMs.
An in vitro investigation of neutrophil responses was performed to investigate the migration of primary human neutrophils and a neutrophil-like cell line (HL-60) towards conditioned medium (CM) generated by lung epithelial cells (A549 cells) exposed to Ag NMs. Moreover, the apoptosis of neutrophils was assessed following direct exposure to Ag NMs. It was found that the migration of neutrophils was not observed in response to CM. However, apoptosis in response to Ag NM treatment was observed, in a time and dose dependent manner. These results highlight that further optimisation is required to improve upon existing neutrophil in vitro models, in order to fully assess the pro-inflammatory effects of NMs in vitro.
Overall, the results demonstrate that zebrafish larvae are a suitable alternative model for assessing NM toxicity, and the tail fin injury model should be prioritised for use by the wider scientific community for assessing chemical safety (e.g. NMs, pathogens, pharmaceuticals). In addition, the responses of neutrophils in vitro can be studied to quickly assess NM toxicity. The more widespread use of such alternative, non-rodent models will make nanotoxicology testing quicker, cheaper, and more ethical.