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Developmental Biology

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Published: February 15th, 2021

DOI:

10.3791/62021

1School of Public Health, Soochow University, 2Toxicology, Risk Assessment and Research Division, Texas Commission on Environmental Quality
* These authors contributed equally

This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 μg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 μM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Air pollution is now a serious environmental problem facing the world. Ambient fine particulate matter (PM2.5), which is one of the most important indicators of air quality, can carry a large number of harmful substances and enter the blood circulatory system, causing serious harm to human health1. Epidemiology studies have demonstrated that PM2.5 exposure can lead to an increased risk of congenital heart defects (CHDs)2,3. Evidence from animal experiments also showed that PM2.5 can cause abnormal cardiac development in zebrafish embryos and the offsprin....

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Wild type zebrafish (AB) used in this study were obtained from the National Zebrafish Resource Center in Wuhan, China. All animal procedures outlined here have been reviewed and approved by the Animal Care Institution of The Ethics Committee of Soochow University.

1. PM2.5 sampling and organic compound extraction

NOTE: PM2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously5.

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This immunofluorescence assay is a sensitive and specific method for measuring protein expression changes in the hearts of zebrafish embryos exposed to environmental chemicals.

In this representative analysis, embryos exposed to PM2.5 in the absence or presence of the antioxidant NAC were evaluated for the presence the presence of heart malformations (Figure 1). As observed, EOM from PM

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Although zebrafish is an excellent vertebrate model for studying the cardiac developmental toxicity of environmental chemicals, due to the small size of the embryo heart, it is difficult to obtain enough protein for western blot analysis. Therefore, we present a sensitive immunofluorescence method for quantifying the protein expression levels of DNA damage biomarkers in the hearts of zebrafish embryos exposed to PM2.5.

During dissection, it is important to keep the integrity of the .......

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This work was supported by the National Nature Sciences Foundation of China (Grant number: 81870239, 81741005, 81972999) and The Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Name Company Catalog Number Comments
8-OHdG Antibody Santa Cruz Biotechnology, USA sc-66036 Primary antibody
Analytical balance Sartorius,China BSA124S
BSA Solarbio,Beijing,China SW3015 For blocking
DAPI Abcam, USA ab104139 For nuclear counterstain.
DMSO Solarbio,Beijing,China D8371
Fluorescence microscope Olympus, Japan IX73 For imaging fluorescence signals/
Goat Anti-Rabbit IgG Cy3 Carlsbad,USA CW0159 Secondary antibody
Goat Anti-Rabbit IgG FITC Carlsbad,USA RS0003 Secondary antibody
N-Acetyl-L-cysteine(NAC) Adamas-Beta, Shanghai, China 616-91-1
Orbital shaker QILINBEIER,China TS-1
Paraformaldehyde Sigma,China P6148 Make 4% paraformaldehyde for fixation.
Phosphate Buffered Saline HyClone,USA SH30256.01 Prepare 0.1% Tween in PBS for washing.
PM2.5 sampler TianHong,Wuhan, China TH-150C For 24-hr uninterrupted PM2.5 sampling.
Re-circulating aquaculture system HaiSheng,Shanghai,China The zebrafish was maintained in it.
Soxhlet extractor ZhengQiao,Shanghai, China BSXT-02 For organic components extraction.
Stereomicroscope Nikon,Canada SMZ645 For heart dissection from zebrafish embryos.
Tricaine methanesulfonate (MS222) Sigma,China E10521 To anesthetize zebrafish embryos
Tween 20 Sigma,China P1379
γH2AX Antibody Abcam, USA ab26350 Primary antibody

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