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

Podsumowanie

This protocol uses an immunofluorescence assay to detect PM_2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Streszczenie

Ambient fine particulate matter (PM_2.5) 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 PM_2.5-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 PM_2.5 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 PM_2.5 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 PM_2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Wprowadzenie

Air pollution is now a serious environmental problem facing the world. Ambient fine particulate matter (PM_2.5), 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 PM_2.5 exposure can lead to an increased risk of congenital heart defects (CHDs)^2,3. Evidence from animal experiments also showed that PM_2.5 can cause abnormal cardiac development in zebrafish embryos and the offsprin....

Protokół

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. PM_2.5 sampling and organic compound extraction

NOTE: PM_2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously⁵.

1. Bake 47 mm quartz membrane filters in a 500 °C muffle furnace for 2 h to remove the organic components.
2. Place a filter in a PM<....

Wyniki

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 PM_2.5 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

Dyskusje

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 PM_2.5.

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

Materiały

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