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

Summary

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

Abstract

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.

Introduction

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 offspring of mice but the molecular mechanisms of the cardiac developmental toxicity of PM_2.5 is still largely unknown^4,5,6.

DNA damage can cause cell cycle arrest and induce apoptosis, which may extensively destroy the potential of progenitor cells and, consequently, impair heart development⁷⁾. It has been well documented that environmental pollutants, including PM_2.5, have the potential to attack DNA through oxidative stress mechanisms^8,9. Both human and zebrafish cardiac development are sensitive to oxidative stress^10,11,12. 8-OHdG is an oxidative DNA damage marker, and γH2AX signal is a marker of DNA double strand breaks. N-acetyl-L-cysteine (NAC), a synthetic precursor of intracellular cysteine and glutathione, is widely used as an anti-oxidative compound. In this study, we use NAC to investigate the role of oxidative stress in PM_2.5- induced DNA damage¹³.

Zebrafish as a model vertebrate has been widely used to study cardiac development and human cardiovascular diseases because mechanisms of cardiac development are highly conserved among vertebrates^14,15. The advantages of using zebrafish as a model include their small size, strong reproductive ability, and low feeding cost. Of particular interest to these studies, zebrafish embryos do not depend on the circulatory system during early development and can survive severe heart malformation¹⁴. Moreover, their transparency allows the entire body to be directly observed under a microscope. Thus, zebrafish embryos provide an outstanding opportunity for assessing the molecular mechanisms involved in the induction of cardiac developmental toxicity as a result of exposure to various environmental chemicals^5,16,17. We have previously reported that PM_2.5-induced oxidative stress leads to DNA damage and apoptosis, resulting in heart malformations in zebrafish¹⁸. In this study, we provide a detailed protocol for investigating PM_2.5-induced DNA damage in the heart of zebrafish embryos.

Protocol

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_2.5 sampler for 24 h of uninterrupted sampling.
3. Remove the filter and dry for 24 h at room temperature.
4. Quantify the filter with an analytical balance.
5. Extract organic components from the filter by Soxhlet extraction using dichloromethane as a solvent¹⁹.
6. Dry the EOM by a rotary evaporation in a 60 °C water bath and nitrogen flow. Dissolve EOM in DMSO and store at -20 °C.

2. Zebrafish embryo collection and treatment

1. Maintain the zebrafish at 28.5 ± 0.5 °C in a re-circulating aquaculture system with a 14 h light and 10 h dark photoperiod cycle.
2. Place healthy adult zebrafish into a tank at a 2:1 male to female ratio.
3. The next day, collect the embryos and wash them with system water (i.e., zebrafish breeding water).
4. Select and randomly divide zebrafish embryos demonstrating a normal development (uniform size, full grains, and no egg coagulation) into 4 groups in individual glass Petri dishes with a diameter of 7 cm (about 50 embryos per dish).
5. Treat the embryos with PM_2.5 (5 mg/L) in the presence or absence of NAC at 0.25 μM from 2 hpf until 72 hpf. Use DMSO as a vehicle control to a final concentration at 0.1% (v/v).

3. Morphological observation of zebrafish embryos and cardiac dissection

1. At 72 hpf, transfer embryos to glass slides and observe under a stereo microscope. Record heart malformations, such as pericardial edema, altered looping, and decreased size.
2. Calculate malformation rates (the percentage of embryos with heart defects out of the total living embryos) and analyze differences between groups using one-way ANOVA followed by Turkey's Multiple Comparison Test (p < 0.05 = statistically significant).
3. Anesthetize the embryos with 0.6 mg/mL MS-222 to immobilize them on glass slides.
4. Record heart beats for 30 s and quantify heart rates using ImageJ software^5,20.
5. Dissect hearts from zebrafish embryos with a disposable syringe needle under a stereo microscope. Caution: Avoid destroying the heart shape.

4. Immunofluorescence Assay

1. To use an immunofluorescence assay to detect PM_2.5 induced DNA damage in the heart of zebrafish embryos, use a hydrophobic barrier pen to draw a circle on a clean glass side.
2. Add 50 μL of 4% paraformaldehyde to 1.25 mL of Phosphate Buffered Saline (PBS) to make a fixative solution.
3. Place 3 dissected hearts into one hydrophobic barrier pen circle and incubate for 20 min at room temperature.
4. Decant the solution under the microscope and dry the samples at room temperature for at least 5 min, so that the hearts completely attach to the glass slides.
5. Wash the slides three times in PBS with 0.1% Tween 20 (PBST) for 5 min per wash.
6. Add 50 μL of bovine serum albumin (BSA) to 1000 μL of PBST to obtain a 5% BSA solution and incubate the slides in a humid chamber for 1 h to block non-specific antibody binding.
7. Decant the solution and wash the samples three times with PBST for 5 min per wash.
8. Dilute 2 μL of mouse monoclonal antibody against 8-OHdG and 2 μL of rabbit polyclonal antibody against γH2AX in 296 μL of PBST to obtain a working primary antibody cocktail solution.
9. Incubate the heart samples with 50 μL of the primary antibody cocktail solution against 8-OHdG and γH2AX in a humidified chamber for at least one hour at room temperature or overnight at 4 °C (Overnight incubation can increase signal intensity).
10. Decant the solution and wash the samples three times with PBST for 5 min per wash.
11. Dilute 1 μL of FITC-labeled goat anti-mouse secondary antibody and 1 μL of cy3 goat anti-rabbit secondary antibody in 498 μL of PBST to obtain a working secondary antibody cocktail solution and incubate the samples with the secondary antibodies (1:500 in PBST) for 1 h at room temperature in the dark.
12. Decant the solution and wash the samples three times with PBS for 5 min per wash protected from light.
13. Add 20 μL of DAPI (4',6-diamidino-2-phenylindole) to the samples for nuclear staining for 30 min at room temperature.
14. Apply a coverslip to slide and seal with nail polish to prevent drying and movement. Then image the samples under a fluorescence microscope and quantify the fluorescence signal of heart area using ImageJ software. Calculate the relative changes with the average of DMSO control samples. Determine the statistical significance of the data as in step 3.2.

Results

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

Discussion

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 ...

Materials

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