Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.
The zebrafish (Danio rerio) is a well-known animal model that has been widely used in research over the last 3 decades. It is a small freshwater fish that is easy to rear and breed under laboratory conditions. The zebrafish has been extensively used for various developmental and toxicological studies1,2,3,4,5,6,7,8. The zebrafish has high fecundity and short embryonic generation; the embryos are suitable for tracking different developmental stages, are visually transparent, and are amenable to varieties of genetic manipulation and high-throughput screening platforms9,10,11,12,13,14. Besides, the zebrafish provides in toto and live imaging for which its developmental process and different deformities in the presence of various toxic substances or factors can be easily studied using stereo or fluorescent microscopy7,15,16.
Radiotherapy is one of the major therapeutic modes used in treating cancer17,18,19,20,21,22,23,24. However, cancer radiotherapy demands potential radioprotectors to protect normal healthy cells from dying while killing malignant cells or safeguard human health during therapy involving high energy radiations25,26,27,28,29. Conversely, potent radiosensitizers are also being investigated to increase the efficiency of radiation to kill malignant cells, especially in targeted and precision therapies30,31,32,33. Therefore, to validate potent radioprotectors and sensitizers, a model suitable for semi-high-throughput drug screening and measurably exhibiting radiation effects is highly solicited. Several available models are used in radiation studies and involved in drug screening experiments. However, higher vertebrates and even the most commonly used in vivo model, mice, are unsuitable for large-scale drug screening because it is time-consuming, costly, and challenging to design such screening experiments with these models. Similarly, cell culture models are ideal for varieties of high-throughput drug screening experiments34,35. However, experiments involving cell culture are not always pragmatic, highly reproducible, or reliable as cells in culture may markedly change their behavior according to the growth conditions and kinetics. Also, varieties of cell types show differential radiation sensitization. Notably, 2D and 3D cell culture systems do not represent the whole organism scenario, and, thus, the results obtained may not recapitulate the actual level of radiotoxicity36,37. In this regard, the zebrafish provides several advantages in screening for novel radiosensitizers and radioprotectors. The ease of handling, large clutch size, short life span, rapid embryonic development, embryo transparency, and small body size make the zebrafish a suitable model for large-scale drug screening. Due to the above advantages, experiments can be readily repeated in a short time, and the effect can be observed easily under a dissecting microscope in multi-well plates. Hence, the zebrafish is gaining popularity in drug screening research involving radiation studies38,39.
The potential of zebrafish as a bonafide model to screen radiation modifiers has been demonstrated in various studies40,41,42,43,44,45. The radioprotective effect of potential radio modifiers, such as nanoparticle DF1, amifostine (WR-2721), DNA repair proteins KU80 and ATM, and transplanted hematopoietic stem cells, and the effects of radiosensitizers, such as flavopiridol and AG1478, in the zebrafish model have been reported19,41,42,43,44,45,46. Using the same system, the radioprotective effect of DF-1 (fullerene nanoparticle) was assessed both at systemic and organ-specific levels, and also the use of zebrafish embryos for radioprotector screening was further explored47. Recently, the Kelulut honey was reported as a radioprotector in zebrafish embryos and was found to increase embryo survival and prevent organ-specific damage, cellular DNA damage, and apoptosis48.
Similarly, the radioprotective effects of polymers generated via Hantzsch's reaction were checked on zebrafish embryos in a high-throughput screening, and the protection was mainly conferred by protecting cells from DNA damage49. In one of the previous studies, the lipophilic statin fluvastatin was found as a potential radiosensitizer using the zebrafish model with this approach50. Similarly, gold nanoparticles are considered to be an ideal radiosensitizer and have been used in many studies51,52.
The embryonic development in zebrafish involves cleavage in the initial 3 h in which a single-celled zygote divides to form 2 cells, 4 cells, 8 cells, 16 cells, 32 cells, and 64 cells that are easily identified with a stereomicroscope. Then, it attains the blastula stage with 128 cells (2.25 h post-fertilization, hpf), where the cells double every 15 min and proceed through these following stages: 256 cells (2.5 hpf), 512 cells (2.75 hpf), and reaching 1,000+ cells in just 3 h (Figure 1). At 4 h, the egg attains the sphere stage, followed by the formation of a dome shape in the embryonic mass7,53,54. The gastrulation in zebrafish starts from 5.25 hpf54, where it reaches the shield stage. The shield clearly indicates the rapid convergence movement of the cells to one side of the germ ring (Figure 1) and is a prominent and distinct phase of gastrulating embryos that can be easily identified53,54. Although radiation exposure to embryos could be done at any stage of their development, radiation exposure during gastrulation might have more distinct morphological changes facilitating better readouts of radiation-induced toxicities55; similarly, administration of drugs to embryos can be started as early as 2 hpf54.
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The present study was conducted with prior approval from and following the guidelines of the Institutional Animal Ethical Committee, Institute of Life Sciences, Bhubaneswar. All zebrafish maintenance and breeding were conducted at an ambient fish culture facility at 28.5 °C, and the embryos were maintained in a biological oxygen demand (BOD) incubator at a temperature of 28.5 °C. Here, the zebrafish AB strain was used, and the staging was carried out according to Kimmel et al.54. X-ray radiation was given at 6 hpf (shield stage), and different phenotypes were observed until 120 hpf.
1. Breeding setup and embryo collection
- Set the breeding tanks (made up of polycarbonate, capacity 1 L, see Table of Materials). Pour system water (pH, 6.8-7.5; conductivity, 500 µS; and temperature, 28.5 °C) into the breeding tanks covering nearly 40% of its volume. Place the divider in the tank to create two chambers, one for females and the other for males.
- From the parent tanks, carefully collect two healthy females and one healthy male with the help of a net, put them in their respective halves, and keep them in the dark overnight (minimum 10 h) at 28.5 °C.
- The next morning, remove the divider and allow the fishes to mate without disturbing the breeding tanks.
NOTE: The females will start spawning, and the eggs will be seen lying on the bottom of the tank within 10-15 min after the fishes are allowed to mate56,57,58.
- Return the fishes to their tanks after spawning, collect the embryos from the breeding tank using a strainer, wash them properly with the system water, and keep the collected eggs in a Petri plate with E-3 media (4.94 mM of NaCl, 0.17 mM of KCl, 0.43 mM CaCl2, 0.85 mM of MgCl2 salts, 1% w/v of methylene blue, see Table of Materials).
- Observe the eggs under a dissecting microscope, remove the unfertilized or dead embryos using a Pasteur pipette, and keep the Petri plates containing fertilized eggs in the E-3 medium at 28.5 °C in an incubator for their proper growth and maintenance.
NOTE: Unfertilized eggs can be identified with a milky white appearance with a coagulated chorion or with ruptured cells inside the chorion. Along with unfertilized eggs, eggs not undergoing cleavage and eggs with deformities like irregularities during cleavage, e.g., asymmetry, vesicle formation, or injuries of the chorion, or not actively developing, must be discarded to keep the collected embryos healthy and to keep the media clean7,56.
2. Monitoring embryos and selection for radiation experiments
- Monitor the growing embryos under the dissecting microscope, identify the proper stage7,54, and remove any dead or unhealthy embryos. Ensure adequate embryo staging as the radiation and drug doses will be given at a particular gastrulation stage.
NOTE: Every day, check for the level and quality of media in the culture dishes. Change the media every 24 h, along with removing dead embryos. Pasteur pipettes are preferred to be used for picking embryos or changing media.
- Before starting the experiment, carefully distribute the healthy embryos in the experimental plates with the help of a Pasteur pipette. For each experimental group, take 15-20 embryos.
NOTE: Place only healthy embryos of the desired developmental stages in the experimental plate. Suppose the drug treatment has to be done with embryos at 6 hpf, then start seeding them in experimental plates at least 30-60 min earlier.
3. Drug treatment
- Add drugs of desired concentration to the zebrafish embryos. Prepare the drug-containing E-3 media well in advance. Ensure the stock solution of the drug has no undissolved drug before preparing the working media for treating zebrafish embryos.
- Before adding any drug to a medium for radiation screening, check the cytotoxic effect of the drug with the grades of concentrations of the drug. Follow the OECD guidelines to evaluate the LC 50 of the drugs under evaluation59,60,61.
NOTE: Be careful while moving the plates and dishes during the irradiation or observation time. There are many chances that the plates will be disturbed during this handling, causing the media to leak out of the wells or the embryos to spill out of their respective wells, potentially contaminating nearby wells and ruining the experiment.
4. X-ray irradiation
- While setting up a radiation experiment, include a control/non-irradiated and a radiation-only group. Similarly, while performing a drug screening, include another group where the drugs will be given with the same concentration as those administered in the screening experiment along with radiation.
NOTE: Label both the lid and base of well plates or culture dishes so that the lids do not get misplaced.
- Distribute the embryos in a well plate if the radiation shields can cover and protect the extra wells from radiation while the other wells are exposed to a particular radiation dose; otherwise, use individual plates or discs to seed the embryos per radiation dose.
- Turn on the X-ray irradiator machine (see Table of Materials), and initiate the machine initialization and warm-up.
NOTE: The source to subject distance (SSD) value must be 50 cm; one can use different SSDs yet again, which requires standardization.
- Place the experimental plate under the irradiator inside the machine in the center, ensuring that the plate is directly below the X-ray source, and then set the dose (e.g., 5 GY) and start the X-ray.
NOTE: Seal the plates with paraffin film to avoid any unwanted spillage or contamination during the transportation of the plates from the incubator to the irradiator and back.
- After the completion of irradiation, take out the plates, shut down the machine program, switch off the machine, and check the plates under the microscope immediately after radiation. Remove the dead embryos and return the plates to the incubator at 28.5 °C. Record the number of dead embryos after evaluating them under the dissecting microscope.
NOTE: Irradiate the different groups of embryos with designated radiation doses without much delay between individual groups as the effect of radiation may be significantly affected by the difference in the developmental stage.
CAUTION: While operating the X-ray machine, take proper protective measures.
5. Data collection, imaging, and analysis
- Collect data at predetermined time intervals, such as every 24 h after the radiation is given. Record all possible observations such as survival, hatching efficiency, stage of development, heartbeat count, body and tail curvature, pericardial edema, the extension of the yolk sac, microcephaly, swim bladder development, general motility or activity, etc.62,63,64.
- To capture images, choose representative embryos on a clean slide, check the embryos under the microscope, orient them in a particular direction, and click on images. Rename the image files according to the group and time.
NOTE: The same magnification and illumination must be used while capturing pictures at different time intervals.
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The overall layout of the protocol is depicted in Figure 2. The effect of radiation and the characterization in a dose-dependent manner was evaluated with the following analyses.
Assessment of X-ray-induced toxicities
Using a stereomicroscope, the following abnormalities were assessed and characterized after the drug treatment and/or radiation. As per the OECD guidelines61, for toxicity evaluation in fishes, four major apical endpoints, including the coagulation of embryos, deformities in somite formation, non-detachment of the tail from the yolk sac, and reduction or absence of heartbeat, were included to analyze the overall toxicity61. The acute toxicity was determined based on a positive outcome in any of the above abnormalities. In addition to these four major endpoints, morphological observation for spine or tail bending, head malformation and microcephaly, defects in development, pericardial edema, yolk sac deformities, swim bladder deformities, and changes in eye structure were also carried out (Figure 3C and Figure 4). The scoring for the radiotoxicity could be based on the survival percentage and/or scoring of different morphological abnormalities.
Survival percentage and survival curve
The survival percentage was calculated by dividing the total live embryos by the total number of embryos initially taken in a group and multiplying the result by 10038,50,65. Then, the values corresponding to different time points and different experimental groups were plotted to obtain the survival curve. This study provides the survival curve for embryos irradiated at 6 hpf (Figure 3A).
Major abnormalities associated with radiation-induced toxicity (Figure 3 and Figure 4)
Body curvature and tail bending
This is one of the most common parameters to assess any toxicity-induced deformities in zebrafish embryos50,65,66. Body curvature deformities can be seen in different patterns ranging from low, to moderate, to severe, with bending in the post-hepatic tail region or in the main body axis or even with a completely semicircular spine or more than one bending in the body axis and in the tail. At lower radiation doses, the bending may not appear in all the embryos but can develop in most of the embryos. With an increase in dose, the severity of bending also increases and affects all individuals. In this study, these deformities were observed in the embryos treated with a 10 GY dose of radiation.
Pericardial and cardiac edema
Embryos treated with toxic exposures like radiation and drugs beyond tolerable ranges or in toxic doses also develop pericardial edema65,66. Embryos exposed to X-ray radiation show pericardial and cardiac edema, in which fluid accumulates in the pericardial cavity and heart, resulting in a swollen pericardium and heart.
Yolk sac edema, thickening of the yolk, and yolk sac constriction
After the X-ray exposure, the yolk sac in some fishes is seen to be thickened or retained, implying the toxicity of X-ray radiation. In some cases, overall yolk sac constriction, where the yolk extension is short, or edema development in the yolk region can also be seen.
Reduction in head size (microcephaly)
One expected outcome of heavy radiation is the reduction in the size of the head, or microcephaly, which can be identified when treated embryos are compared with the embryos in the control group.
Swim bladder deformities
Post irradiation, the swim bladder is seen to be reduced or compromised in a few embryos, and the swim bladder deformity is greater in the case of embryos subjected to higher radiation doses, which may contribute to the low locomotion or reduced swimming abilities in embryos exposed to high X-ray doses.
Change in eye structure
Radiation can cause enormous DNA damage and protein alterations, which eventually cause cell death and a reduction in cell numbers or the death of specific cell types65. The eye can be affected by intense radiation doses, and small eye size and a decrease in its cell layers have been observed55.
Heartbeats per minute (bpm)
The heartbeats per minute were counted by observing the embryos under the stereomicroscope. As the radiation dose increases, the bpm tends to decrease (Figure 3B). Five larvae were considered to calculate bpm at each time point per group. A decrease in heartbeat count could indicate cardiac dysfunction66.
Using this protocol, the X-ray radiation dose of 10 GY was visibly toxic in zebrafish embryos irradiated at 6 hpf. In the control group and embryos exposed to 2 GY and 5 GY, there was no significant death in embryos (Figure 3A). Similarly, the heart beats per minute count suggested that the heart rate decreased enormously with increased doses of X-ray radiation. In the control group, at every 24 h interval, the heart rate was seen to increase (Figure 3B). However, at each time point, the heart rate decreased with an increased radiation dose. However, the embryos exposed to 5 GY and 10 GY showed no significant differences until day 5 post-fertilization. Severe cardiovascular deformation is suspected in embryos exposed to 15 GY and 20 GY radiation as the heartbeat dropped enormously (Figure 3B). As discussed earlier, different phenotypic and developmental defects are depicted and evaluated for embryos exposed to varying doses of radiation at different time points (Figure 3C and Figure 4).
Figure 1: Stages of zebrafish embryo development. Representative images of different stages of early zebrafish development. Stages up to 75% epiboly (8 hpf) are covered. Embryos at shield stage; 6 hpf (green color) is used for radiation standardization. Scale bar = 276.4 µm. Please click here to view a larger version of this figure.
Figure 2: Generalized outline of the protocol. (A) Breeding, collection of embryos, and staging. (B) Experimental setup: seeding embryos in well-plates and drug treatment. (C) Embryos of required stages exposed to radiation and phenotypical changes observed following radiation. (D) An outline of the X-ray machine and its setup. (E) Observations, data acquisition, and imaging. Please click here to view a larger version of this figure.
Figure 3: Effect of different doses of X-ray irradiation on 6 hpf zebrafish embryos. (A) Survival curve showing the total surviving fraction of zebrafish embryos exposed to individual radiation doses starting from 2 GY to 20 GY. (B) Heartbeat count per minute of zebrafish embryos exposed to different doses of X-ray radiation at 6 hpf on subsequent days of fertilization. (C) Representative images of zebrafish embryos exposed to varying doses of radiation (from 2 GY to 20 GY), irradiated at 6 hpf. Please click here to view a larger version of this figure.
Figure 4: Representation of different morphological abnormalities due to radiation-induced toxicity. (A) A control zebrafish embryos at 72 hpf and (B) radiated embryos at 72 hpf; the upper embryo shows moderate deformities, while the lower embryo has severe deformities. Please click here to view a larger version of this figure.
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Zebrafish are used as valuable models in many studies, including several types of cancer research. This model provides a useful platform for large-scale drug screening67,68. Like any other toxicity evaluation method, the quantitative evaluation of the major biological changes upon radiation and/or drug treatment is the most crucial part of this protocol. In these kinds of studies, survival must not be the only criteria to observe toxicity; it needs to be supported with the evaluation of physical or developmental defects by proper scoring systems. In this case, also, up to 72 hpf, the survival in embryos is not much different among the groups in which embryos are exposed to X-ray radiation doses of 5 GY, 10 GY, and 20 GY; however, when the overall morphology and phenotypes of the embryos are checked at these particular doses, it is clear that the X-ray toxicity is more severe than it appears through the survival graph. The severity of morphological deformity in embryos at these doses is very high, reflecting changes in their overall body size, defects in development, the malformation of vital organs, and changes in their overall activity. Even in the group of 15 GY and 20 GY, the embryos cannot even hatch out of their chorion and exhibit ample deformities in a dose-dependent manner. Thus, for evaluating the effect of toxic substances, including highly fatal X-ray radiation, the scoring of morphological, developmental, and physiological defects must be included in all possible ways, and that should be used to evaluate the overall response of zebrafish embryos or different drugs administered in the course of the experiment.
Although there are no definitive scoring systems to evaluate radiotoxicity in the zebrafish embryo model specifically, the overall survival and/or morphological changes like bending in the body, pericardial edema, changes in the yolk sac, microcephaly, changes in the swim bladder and eye, heartbeat changes, and defects in locomotion have been taken into consideration in various studies62,63,64. The survival of embryos could be evaluated based on the heartbeat or the evaluation of apical endpoints as described in OECD guidelines. At the same time, the morphological abnormalities observed in such experiments could be scored individually; e.g., the scoring of tail bending has been adopted by multiple investigators61.
While working with zebrafish and conducting this protocol, one must be careful about certain considerations. These include the breeding group, which must always belong to the same strain of fish. All experiments must involve one predefined strain of zebrafish embryo. Another important factor is the embryo stage; one needs to be meticulous with the developmental stage at which the embryos are irradiated because a slight change in the timing or stage will lead to different results. Some drugs may affect the development or cause severe damage to the embryos when administered at an early stage of development, like 2 hpf. In that case, the proper sub-lethal dose of the drug must be determined, and then the screening can be carried out.
The X-ray irradiation parameters must be uniform for all the experiments performed. The three important aspects of a standard X-ray irradiator are the filter type, the radiation dose and pattern of irradiation, and the distance between the X-ray source and the object. There are primarily two types of filters used to generate X-ray beams: aluminum filters and copper filters; however, filters with varying combinations of copper and aluminum or other metals are also used to generate X-rays in other cases69. For zebrafish embryos, the cupper filter is used here to produce X-rays. The distance from the X-ray source to the experimental subject is termed as source to subject distance (SSD). In this study, the SSD was set to be 50 cm. The X-ray radiation was given using a 0.3 mm Cu filter. A single exposure of the desired dose was given at a dose rate of 140.32 cGY/min within the wavelength range 0.01-10 nm. Before carrying out any radiological experiment, the radiation dose suitable for the experiment and objective should be standardized. The purpose of the study, the timing of irradiation, and the radiation dose are the three major criteria for radiation dose standardization. The timing of radiation will include both at what stage of embryo development the radiation should be given and the time period for which the embryo will be exposed to radiation of a set dose. It is well known that, at early developmental stages, the effect of radiation is maximized. In this protocol, the embryos were irradiated at a developmental stage of 6 hpf with different doses of radiation (2 GY, 5 GY, 10 GY, 15 GY, and 20 GY) and observed for 5 days post fertilization. Any deviation from the usual protocol should be clearly defined and standardized.
This model has several advantages for studying the effect of radiosensitizers or protectors in a nearly longitudinal study, such as the ability to obtain several embryos from individual breeding, to breed every week from a single parent tank, to place a significant number of embryos in experimental groups, to observe phenotypic effects in a few days post treatment, and to see a spectrum of phenotypic variables after treatments. This model can reflect the impact of radiation almost on all systems of the embryo, and multiple drugs can be tested at a time in well-plate formats. However, this approach also faces certain limitations. For example, this model cannot recapitulate all the deformities shown by radiations in higher animals and humans. In addition, many protein-based or mechanistic studies in these fishes are limited due to reagent availability issues, such as with antibodies. However, despite these limitations, the zebrafish proves to be an excellent model for radiological studies.
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The authors have declared no competing interests.
SS's lab and RKS's lab are funded by grants from DBT and SERB, India. APM is a recipient of the ICMR fellowship, Government of India. DP is a recipient of the CSIR fellowship, Government of India. UN is a recipient of the DST-Inspire fellowship, Government of India. Figure 2 was generated using Biorender (https://biorender.com).
|6 Well plates||Corning||CLS3335||Polystyrene|
|Calcium Chloride||Fisher Scientific||10101-41-4|
|Dissecting Microscope||Zeiss||Stemi 2000|
|External Tank for the 1.0 L Breeding Tank||Tecniplast||ZB10BTE||Polycarbonate|
|GraphpadPrism||GraphPad Software, Inc.||Version 5.01|
|Kline concavity slides||Himedia||GW092-1PK||Glass|
|Methylene blue hydrate||Sigma-Aldrich||66720-100G|
|Pasteur pipettes||Himedia||PW1212-1X500NO||Polyethylene plastic|
|Perforated Internal Tank for the 1.0 L Breeding Tank||Tecniplast||ZB10BTI||Polycarbonate|
|Polycarbonate Divider for the 1.0 L Breeding Tank||Tecniplast||ZB10BTD||Polycarbonate|
|Polycarbonate Lid for the 1.0 L Breeding Tank||Tecniplast||ZB10BTL||Polycarbonate|
|Sodium hydroxide pellet||SRL||1949181|
|Stereo Microscope Leica M205FA||Leica||Model/PN MDG35/10 450 125|
|X-Rad 225 Precision X-Ray||Precision X-Ray||X-RAD 225XL|
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