Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Summary

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Abstract

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of γ- and β-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called γH2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled γH2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Introduction

Significant controversy over the potential harmful effects of either low or very low doses of ionizing radiation and public’s fear of radiation, driven by images of the Hiroshima and Nagasaki atomic bombings and of rare nuclear plant accidents (exacerbated by mass media), has led to very strict radiation protection regulation and standards that are potentially not scientifically justified. In the last three decades, numerous reports have documented both the lack of harmful and the presence of potentially beneficial biological effects induced by low dose radiation^1-4. The major radiation health risk factor is the probability of cancer, estimated based on epidemiological studies of atomic bomb survivors receiving high or medium doses of radiation. Linear extrapolation of these data (so called linear-no-threshold or LNT model) is used to estimate risks of cancer at low doses. However, this approach has not received world-wide scientific acceptance and is heavily debated⁵.

It is evident that more studies are required to clarify this issue and possibly improve radiation protection standards. Such studies should involve chronic treatments (best approximation of environmental and occupational exposures), in vivo animal models (best for extrapolating effects to human) and such end-points as DNA damage rates, DNA repair and mutagenesis. It is known that DNA is the primary target for damaging radiation effects and incomplete or mis-repair may lead to mutagenesis and cancer development⁶.

DNA double-strand breaks (DSB) are one of the most deleterious types of DNA lesions and may lead to cell death and tumorigenesis⁷. It is, therefore, important to be able to reliably and accurately measure the level of DSB after exposure to low dose radiation and/or other stressors, such as chemical pollutants. One of the most sensitive and specific markers of DNA DSB is phosphorylated histone H2AX, called γH2AX⁸, yet other markers and methods have been suggested^9,10. It is estimated that thousands of H2AX molecules, in the vicinity of an induced DSB, are involved in the formation of γH2AX enabling the detection of individual DSB by immunofluorescent labeling with an anti-γH2AX antibody and fluorescence microscopy¹¹. The response is very quick, reaching its maximum between 30 and 60 min. Evidence exists that γH2AX facilitates repair of DNA DSB by attracting other repair factors to the sites of breaks and by modifying chromatin structure to anchor broken DNA ends and provide access for other repair proteins (reviewed in ¹²). Upon completion of repair of DNA DSB, γH2AX gets de-phosphorylated and/or undergoes degradation, and newly synthesized H2AX molecules replace γH2AX in the affected areas of chromatin¹⁰. Monitoring formation and loss of γH2AX can, therefore, provide an accurate estimate of DNA DSB repair kinetics. This approach has been used to study repair of DSB in various human tumor cell lines irradiated with high doses of radiation and its rate and residual DSB levels have been shown to correlate with radiosensitivity^13-15.

We modified this experimental approach and applied it to an in vivo mouse study to examine the effects of low doses of chronic γ- and β-irradiation on DNA DSB levels and repair (Figure 1). Firstly, we demonstrate a method to perform a long-term chronic exposure of mice to β-radiation either emitted by tritium (hydrogen-3) in the form of tritiated water (HTO) or as organically bound tritium (OBT) dissolved in drinking water. The two forms are expected to accumulate and/or distribute differently in the body and therefore, produce different biological effects. Both forms are potential hazards in nuclear industry. This treatment is paralleled by chronic exposure to γ-radiation at an equivalent dose rate to allow a correct comparison of the two radiation types, which is crucial for the evaluation of their relative biological effectiveness. Beta-radiation is composed of electrons, making it very different from the γ-radiation, high energy photons. Due to this difference, Β-radiation represents mostly internal health hazard and may produce different biological effects compared to γ-radiation. This complication resulted in significant controversies over the regulation of exposure to β-radiation emitted by HTO. Thus, regulatory levels of HTO in drinking water for the public vary from 100 Bq/L in Europe to 75,000 Bq/L in Australia. It is, therefore, important to compare biological effects of HTO to equivalent doses of γ-radiation. Secondly, the rate of DNA DSB is measured in isolated splenocytes upon the completion of chronic exposures using immunofluorescently labeled γH2AX detected by flow cytometry. This allows the evaluation of the extent of DNA damage inflicted by the in vivo exposures. However, it is sensible to expect that such low level exposures may not produce any detectable rates of DNA DSB; instead, some hidden changes/responses may be expected that would affect the cells’ ability to repair DNA damage. These changes, if found, may be either stimulatory (producing a beneficial effect) or inhibitory (producing a deleterious effect). The proposed protocol allows revealing such changes by challenging the extracted splenocytes with a high dose of radiation that produces a significant amount of damage (e.g., 2 Gy producing approximately 50 DNA DSB per cell or a 3 - 5 fold increase in total γH2AX level). Subsequently, the formation and loss of γH2AX, reflecting the initiation and completion of DSB repair, is monitored by flow cytometry. In this way, not only basal and treatment-induced levels of DNA DSB can be measured, but also its potential effect on the cells’ ability to respond and repair DNA damage induced by much higher stressor levels.

Protocol

All mouse handling and treatment procedures should adhere to rules set forth by the legislature and/or animal care programs and approved by a local animal care committee. All methods described in this protocol were performed in accordance with the guidelines of the Canadian Council on Animal Care with the approval of the local animal care committee.

CAUTION: All work with radioactivity (including, but not limited to handling of tritium, external γ-irradiation, handling radioactive animal tissues, bedding waste) should adhere to rules set forth by the legislature and/or radiation protection authorities and be performed by authorized personnel in a certified laboratory and/or facility.

1. Animal Work

1. Animal Grouping
   1. If mice are arriving from an external organization, acclimatize animals for at least 10 days in the animal facility. If the study includes multiple treatment groups, ensure that the animal supplier can send all animals as a single batch.
   2. Randomly allocate mice to treatment groups of 10 mice per group. If using male mice of the C57BL/6 strain, have the animal supplier pre-arrange randomization of very young C57BL/6 males in their facility to mitigate aggression issues.
2. Chronic Exposure of Mice to Internal β-Radiation
   1. Prepare working concentrations of tritium (10 kBq/L and 1 MBq/L) in mouse drinking water by diluting the original stock of HTO and OBT (a mixture of tritiated proline, alanine and glycine amino acids) solutions.
   2. Validate the final concentrations of tritium in the drinking water by measuring radioactivity using a liquid scintillation counter. Adjust concentrations, if required.
   3. Treat animals for one month by providing access ad libitum to the tritium water. If treatment water gets low during the two week period, add more water. Otherwise, change bottles at two weeks.
   4. Treat sham-irradiated control mice identically. Keep them in a separate “clean” room or on a separate “clean” rack under positive air pressure to avoid tritium cross-contamination from the air.
3. Chronic Exposure of Mice to External γ-Radiation
   Note: γ-irradiation facility/equipment will vary depending on the location.
4. Using available methods of dosimetry, determine the distances from a γ-radiation source that have radiation fields producing the desired dose rates. Ensure homogeneity of the radiation fields within the areas covering the animal cages.
   Note: 1.7 μGy/h is equivalent to a dose rate produced by 1 MBq/L tritium in the body.
5. Expose mice by placing mouse cages at the distances determined in step 1.3.1 for one month. Minimize daily interruption of γ-irradiation to 30 min. Use thermoluminescence dosimeters to measure total absorbed doses.

2. Sacrifices and Sampling

1. Prepare sterile surgical instruments, 60 mm petri dishes with inserted 70-micron cell strainers, 15 ml and 1.5 ml tubes, RPMI media supplemented with 5% fetal bovine serum (FBS) and place all within a biological safety cabinet. Ensuring sterility of the work is crucial.
2. Dispense 5 ml of RPMI media into each 60 mm petri dish.
3. Sacrifice mice using a method approved by the local animal care committee. Cervical dislocation is a good choice for the splenocyte work described in this protocol. However, if blood samples for other assays (e.g., for the mFISH, the cytochalasin block micronucleus or other common genetoxicity assays) are required, use intra-cardiac puncture after anaesthesia with isoflurane.
4. Place the animal in dorsal recumbency, with the head pointing away from you. Spray the mouse down with 70% ethanol. Using forceps, grab the skin anteriorly to the urethral opening and make a small incision with scissors in the perineal region.
   1. From this incision, cut along the ventral midline to the chest cavity, being careful to only cut the skin and not the muscle wall underneath. Dissect the skin away from the midline.
   2. Grasp the abdominal wall with forceps and cut along the median axis of the muscular wall to open up the abdominal cavity.
5. Excise the spleen by lightly gripping it with sterile forceps and gently pulling on it while simultaneously cutting away the connective tissue with scissors.
6. Cut a small piece of the spleen (approximately 1/10 of the spleen) and place in a 1.5 ml tube. Snap freeze in liquid nitrogen for subsequent storage at -80 °C for supplementary assays.
7. Place the remainder of the spleen into a cell strainer inside a 60 mm petri dish with 5 ml of pre-dispensed RPMI media.
8. Proceed to the next mouse.
   Note: Extracted spleens can be kept in 60 mm dishes with media (for up to 2 hrs) while the rest of the mice are sacrificed. Depending on the number of people participating, between 5 and 15 mice can be processed in one day.

3. Preparation of Splenocyte Cultures

1. Homogenize the spleen by mincing inside a cell strainer with sterile forceps with a curved end.
2. Remove the cell strainer and collect the filtered cell suspension from the 60 mm dish into a 15 ml tube.
3. Rinse the cell strainer with 5 ml of media to collect the remainder of the cells.
4. Centrifuge tubes at 300 x g for 5 min at RT.
5. Decant supernatant and re-suspend pellet in 10 ml of RPMI.
6. Centrifuge tubes at 300 x g for 5 min at RT.
7. Decant supernatant and re-suspend pellet in 5 ml of RPMI.
8. Transfer 4 ml of cell suspension into a 25 ml tissue culture flask and remove to a CO₂ incubator (37 °C, 5% CO₂, 80% humidity)

4. Challenging Irradiation and Fixing

1. Transfer the remaining 1 ml of cell suspension from step 3.8 into a 1.5 ml tube on ice and centrifuge at 300 x g for 5 min at 4 °C. DNA damage measured in this sample will represent both treatment-induced damage and t = 0 data-point for the construction of a DNA repair curve.
2. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 1 ml of TBS buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2.7 mM KCl).
3. Centrifuge tubes at 300 x g for 5 min at 4 °C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 300 μl of TBS.
4. While mixing on a vortex at low speed, add 700 μl of -20 °C 100% ethanol. Close the lid and invert a few times to mix.
5. Store the samples at -20 °C. Splenocytes fixed in ethanol can be stored at -20 °C for at least 12 months without any noticeable loss in the γH2AX signal.
6. Irradiate the splenocyte cultures from step 3.8 with a challenging γ-radiation dose of 2 Gy at a dose rate ≥ 200 mGy/min using available irradiation device.
   Note: The purpose of this irradiation is to induce DNA DSB to challenge the repair machinery. X-rays can also be used for this purpose.
7. Immediately return the cultures to a CO₂ incubator.
8. 1 hr after the challenging 2 Gy irradiation, remove the cultures from the incubator to a biological safety cabinet. To collect a representative sample aliquot, gently re-suspend cells using a pipettor and transfer 1 ml of the cell suspension to a 1.5 ml tube on ice. Return the cell cultures to a CO₂ incubator.
9. Centrifuge tubes at 300 x g for 5 min at 4 °C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 1 ml of TBS.
10. Repeat steps 4.3 - 4.5.
11. 24 hrs after the challenging 2 Gy γ-irradiation, harvest and fix a t = 24 hrs sample as described in step 4.8 – 4.10.

5. Immunofluorescent Labeling with Anti-γH2AX Antibody

Note: Typically, 15 - 30 samples per day can be immunofluorescently labeled and assayed by flow cytometry in a single run. To ensure an accurate comparison between treatment groups, include sample(s) from each group. See also Reference ¹⁶ for further details.

1. Prepare a labeled set of tubes for the number of samples to be processed and place on ice. Use 12 x 75 mm uncapped glass tubes. Sterility is not required for this work. Include one additional historical control sample for labeling with secondary antibody only (“2nd Ab only”). Use this historical control sample in every flow cytometry run within a study.
2. Add 0.5 ml of ice-cold TBS to each tube.
3. Remove fixed splenocytes from -20 °C, vortex for 5 sec and transfer a 0.5 ml aliquot to prepared tubes with TBS. Place the remainder of samples back into -20 °C storage and keep as a backup sample.
4. Centrifuge splenocytes at 300 x g for 5 min at 4 °C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS containing 1% FBS
5. Centrifuge cells at 300 x g for 5 min at 4 °C. Decant the supernatant and gently re-suspend pellet in 1 ml of TST buffer (0.05% Triton X-100, 2% FBS in TBS). Incubate for 20 min on ice.
6. Centrifuge cells at 300 x g for 5 min at 4 °C. Decant the supernatant and gently re-suspend pellet in 200 μl of primary anti-γH2AX antibody diluted 1:150 in TST buffer. Use 200 μl of TST for the “2nd Ab only” control sample.
7. Position tubes on a rotating shaker at an angle of 45 - 60° and shake for 1.5 h at 300 x g at RT.
8. While tubes are incubating, prepare a sufficient volume (200 μl/sample) of the goat anti-mouse secondary antibody conjugated with Alexa-488 at a 1:200 dilution in TST buffer.
   Note: All work involving Alexa-488 conjugated antibody (steps 5.10 to 5.16 below) should be done under dim light conditions and labeled samples should be protected from light by wrapping tubes in aluminum foil.
9. Once the 1.5 hrs incubation is completed, add 1 ml of ice-cold TBS containing 2% FBS to each tube and centrifuge at 300 x g for 5 min at 4 °C. Decant the supernatant and gently re-suspend pellet in 1 ml of TBS containing 2% FBS.
10. Centrifuge at 300 x g for 5 min at 4 °C. Decant supernatant and gently re-suspend pellet in 200 μl of the secondary antibody solution from step 5.8.
11. Incubate the samples on a shaking platform for 1 hr as in step 5.7.
12. Add 1 ml of ice-cold TBS containing 1% FBS.
13. Centrifuge at 300 x g for 5 min at 4 °C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS.
14. Centrifuge at 300 x g for 5 min at 4 °C. Decant the supernatant and gently re-suspend pellet in 0.5 ml of TBS containing 50 μg/ml propidium iodide.
15. Incubate for 5 min at RT protected from light.
16. Analyze samples on a flow cytometer using instrument-specific protocols¹⁶. Read at least 10,000 cells per sample.

Results

Figure 2 shows examples of flow cytometry graphs expected for splenocytes prepared using the method described here. Cells are first gated based on the scatter plot (Figure 2A and 2D; electronic volume is equivalent to forward scatter). FL3/propidium iodine histograms (Figure 2B and 2E) confirm normal cell cycle distribution. Mean γH2AX signal calculated using the FL1 channel confirms a > 2-fold i...

Discussion

The protocol presented in this paper is useful for conducting large scale mouse in vivo studies examining the genotoxic effects of low levels of various chemical and physical agents, including ionizing radiation. Our unique specific pathogen-free animal facility equipped with a GammaBeam 150 irradiator and a 30 meter long irradiation hall allows conducting life-long studies involving low or very low dose rate irradiations on hundreds or even thousands of mice. Smaller irradiation facilities for chronic low dose ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HTO: tritiated water, [3H]	locally obtained from a nuclear reactor		stock activity 3.7 GBq/mL;  can be substituted with HTO from Perkin Elmer
OBT: Alanine, L-[3-3H]: organically bound tritium (OBT)	Perkin Elmer	NET348005MC	1 mCi/ml (185 MBq)
OBT: Glycine, [2-3H]: organically bound tritium	Perkin Elmer	NET004005MC	1 mCi/ml (185 MBq)
OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT)	Perkin Elmer	NET323005MC	1 mCi/ml (185 MBq)
tritiated water, [3H] (HTO)	Perkin Elmer	NET001B005MC	substitute for HTO of local origin
GammaBeam 150 irradiator	Atomic Energy of Canada Limited	locally manufactured	can be substituted with another g-radiation source of sufficiently low activity
Tween-20	Sigma Aldrich	P1379-500ML
RPMI	Fisher Scientific	SH3025501	Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL
fetal bovine serum	Sigma Aldrich	F1051-100ML
anti-gH2AX antibody, clone JBW301	Millipore	05-636
Alexa fluor-488 goat anti-mouse antibody	Life Technologies (formerly Invitrogen)	A21121
propidium iodine	Sigma Aldrich	P4864-10ML	1 mg/ml
ethanol	Commercial Alcohols	P006-EAAN	500 ml bottles Absolute Ethanol
1.5 ml tubes	Fisher Scientific	2682550	microcentrifuge tubes
15 ml tubes	Fisher Scientific	05-539-5	sterile polypropylene centrifuge tubes
liquid nitrogen	Linde	P110403
12 x 75 mm uncapped glass tubes	Fisher Scientific	K60B1496126	disposable borosilicate glass tubes with plain end
T25 flasks	VWR	CA15708-120	nunc tisue culture 25ml flask (supplier no. 156340)
scissors	Fine Science Tools	14068-12	Wagner scissors 12 cm sharp/sharp
forceps, straight	Fine Science Tools	11008-13	Semken forceps 13 cm, straight
forceps, curved	Fine Science Tools	11003-12	Narrow Pattern forceps 12 cm, curved
60 mm petri dishes	VWR	CA25382-100	BD Falcon tissue culture dish 60 x 15 mm
cell strainers, 70 mm	Fisher Scientific	08-771-2	Falcon cell strainers 50/case
PBS recipe: 1 tablet dissolved in 200 ml of deionized water, adjust pH to 7.4 if needed.	Sigma Aldrich	P4417-100TAB	Phosphate Buffered Saline Tablets
TBS recipe: to make 10x stock
30 g TRIS HCl	Sigma Aldrich	T3253-1KG	Trizma hydrochloride
88 g NaCl	Fisher Scientific	S271-500	Sodium Chloride
2 g KCl	Fisher Scientific	P217-500	Potassium Chloride
Dissolve in 1 L of deionized water, adjust pH to 7.4