The research work in the corresponding author&#8217;s laboratory is funded by the National Nature Science Foundation of China (Grant numbers: 81972999, 81870239, and 82171689) and The Priority Academic Program Development of Jiangsu Higher Education Institutions.

<ol>
	<li>Bouma, B. J., Mulder, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+landscape+of+congenital+heart+disease.">Changing landscape of congenital heart disease.</a> Circulation Research. 120 (6), 908-922 (2017).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+immunofluorescence+to+detect+PM2.5-induced+DNA+damage+in+zebrafish+embryo+hearts.">Using immunofluorescence to detect PM2.5-induced DNA damage in zebrafish embryo hearts.</a> Journal of Visualized Experiments. (168), e62021 (2021).</li><li>Jiang, Q. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+chicken+embryo+as+a+powerful+tool+in+assessment+of+developmental+cardiotoxicities.">Using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities.</a> Journal of Visualized Experiments. (169), e62189 (2021).</li><li>Ding, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Alizarin+Red+staining+to+detect+chemically+induced+bone+loss+in+zebrafish+larvae.">Using Alizarin Red staining to detect chemically induced bone loss in zebrafish larvae.</a> Journal of Visualized Experiments. (178), e63251 (2021).</li><li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+effect+of+pesticides+on+the+larvae+of+the+solitary+bees.">Evaluating the effect of pesticides on the larvae of the solitary bees.</a> Journal of Visualized Experiments. (176), e62946 (2021).</li><li>Ankley, G. T., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adverse+outcome+pathway:+A+multifaceted+framework+supporting+21(st)+century+toxicology.">The adverse outcome pathway: A multifaceted framework supporting 21(st) century toxicology.</a> Current Opinion in Toxicology. 9, 1-7 (2018).</li><li>Leist, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+outcome+pathways:+Opportunities,+limitations+and+open+questions.">Adverse outcome pathways: Opportunities, limitations and open questions.</a> Archives of Toxicology. 91 (11), 3477-3505 (2017).</li></ol>

The commercial application of the author&#8217;s research is covered by a patent application (CN111024935A).

At the developmental stage, animals are highly susceptible to the effects of environmental pollution. Chemical exposure during critical gestation stages have been shown to lead to congenital malformations and spontaneous abortions. Therefore, understanding the mode of action of potential toxicants would be beneficial in the prevention or treatment of spontaneous abortions or congenital diseases caused by environmental pollutants. However, it is challenging to study the mechanisms underpinning the developmental toxicity of environmental chemicals using traditional animal models due to the complexity, tediousness, and excessive costs associated with these experiments. In this methods collection, authors from different areas of research have employed novel techniques and alternative models to study the developmental toxicity of environmental chemicals.
It is known that the heart is one of the earliest organs to be formed during embryogenesis, and this organ is particularly prone to environmental insults. Research estimates show that congenital heart diseases account for approximately one-third of all known birth defects, in addition to being the primary cause of infant mortality1. Unfortunately, traditional animal models are either too laborious or insensitive to detect cardiac developmental toxic effects. Two papers from this methods collection investigate the adverse effects of ambient fine particulate matter (PM2.5), per- and polyfluoroalkyl substances (PFAS), diesel exhaust (DE), and nanomaterials on heart development using zebrafish and chicken embryos2,3. By utilizing the principles of the immunofluorescence assay technique, 8-hydroxy-2&#39;deoxygenase (8-OHdG), a marker of oxidative DNA damage, and H2AX, a sensitive marker of DNA double-strand breaks, are consistently detected in the heart of zebrafish embryos after exposure to extractable organic matter (EOM) from PM2.52. Similarly, cardiomyocyte DNA damage and right ventricular wall thickness are also clearly demonstrated in isolated heart tissue using the same immunofluorescence and histochemistry techniques3.
The zebrafish model can also be used to study skeletal morphogenesis. It has been widely reported that lead exposure causes this chemical to accumulate in the skeleton throughout developmental stages, leading to bone damage. Ding et al. used Alizarin Red to label the bone structures and demonstrated that lead exposure significantly decreased bone mineralization in zebrafish larvae, indicating that lead exposure is a risk factor for bone loss during skeletal development4.
Pesticides are well-known for their high persistence in the environment. The ubiquitous use of pesticides is a risk factor for the abundance and diversity of pollinators, which play an essential role in the ecosystem services of modern global agriculture. Song et al. presented a novel immersion method to investigate the toxic effects of chlorpyrifos, a very popular pesticide, on the larvae of the solitary bee, Osmia excavata5. They identified the LD50 value for chlorpyrifos using the larvae of O. excavata and found that chlorpyrifos exposure significantly reduced the eclosion rate in a dose-dependent manner.
The adverse outcome pathway (AOP) framework, which emphasizes molecular causes and makes alternative testing models easier to utilize6, is a crucial component of Toxicology in the 21st Century (Tox21)6. According to Leist et al., AOP establishes a connection between a molecular initiating event (MIE) and an undesirable adverse outcome (AO) through key events (KE), as defined by key event relationships (KER)7. Thus, the AOP framework can facilitate the translation of molecular and mechanistic data from animal studies into endpoints for use in safety assessments6. The articles in this methods collection provide a powerful set of techniques and models for investigating the molecular mechanisms through which environmental pollutants cause various developmental toxicities. We hope that this collection will be of great benefit in the risk assessment of environmental pollutants.

current techniques and models for assessing the developmental toxicity of environmental pollutants

ARTICLES DISCUSSED:
Ding, J. et al. Using Alizarin Red staining to detect chemically induced bone loss in zebrafish larvae. Journal of Visualized Experiments. (178), e63251 (2021).
Huang, Y. et al. Using immunofluorescence to detect PM2.5-induced DNA damage in zebrafish embryo hearts. Journal of Visualized Experiments. (168), e62021 (2021).
Jiang, Q. X., Xu, X. H., DeWitt, J. C., Zheng, Y. X. Using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities. Journal of Visualized Experiments. (169), e62189 (2021).
Song, Y., Li, R., Li, L., Ouyang, F., Men, X. Evaluating the effect of pesticides on the larvae of the solitary bees. Journal of Visualized Experiments. (176), e62946 (2021).

Read this Scientific Article Protocol about Current Techniques and Models for Assessing the Developmental Toxicity of Environmental Pollutants at JoVE.com