The authors would like to acknowledge the contribution of our colleagues Sandrine Roch-Lefevre and Eric Gregoire of the Institute of Radioprotection and Nuclear Safety (Paris, France). This work was supported by the Government of Canada Science and Technology program at Canadian Nuclear Laboratories (Chalk River, Ontario, Canada), the CANDU Owners Group (Toronto, Ontario, Canada), the Canadian Nuclear Safety Commission and by the Institute of Radioprotection and Nuclear Safety (Paris, France).

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 &#947;-irradiation, handling radioactive animal tissues, bedding waste) should adhere to r...

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	<li>Mitchel, R. E., Jackson, J. S., McCann, R. A., Boreham, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptive+response+modifies+latency+for+radiation-induced+myeloid+leukemia+in+CBA/H+mice.">The adaptive response modifies latency for radiation-induced myeloid leukemia in CBA/H mice.</a> Radiat Res. 152 (3), 273-279 (1999).</li><li>Shadley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+adaptive+response+in+human+lymphocytes.">Chromosomal adaptive response in human lymphocytes.</a> Radiat Res. 138 (Suppl 1), S9-12 (1994).</li><li>Wiencke, J. K., Afzal, V., Olivieri, G., Wolff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+[3H]thymidine-induced+adaptive+response+of+human+lymphocytes+to+subsequent+doses+of+X-rays+involves+the+induction+of+a+chromosomal+repair+mechanism).">Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism).</a> Mutagenesis. 1 (5), 375-380 (1986).</li><li>Mitchel, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dose+window+for+radiation-induced+protective+adaptive+responses.">The dose window for radiation-induced protective adaptive responses.</a> Dose-Response. 8 (2), 192-208 (2010).</li><li>Tubiana, M., Feinendegen, L. E., Yang, C., Kaminski, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J Cell Biol. 151 (7), 1381-1390 (2000).</li><li>Kinner, A., Wu, W., Staudt, C., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+in+recognition+and+signaling+of+DNA+double-strand+breaks+in+the+context+of+chromatin.">Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.</a> Nucleic Acids Res. 36 (17), 5678-5694 (2008).</li><li>Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G., Bonner, W. 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L., Banath, J. P., Sinnott, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+histone+H2AX+in+spheroids,+tumors,+and+tissues+of+mice+exposed+to+etoposide+and+3-amino-1,2,4-benzotriazine-1,3-dioxide.">Phosphorylated histone H2AX in spheroids, tumors, and tissues of mice exposed to etoposide and 3-amino-1,2,4-benzotriazine-1,3-dioxide.</a> Cancer Res. 64 (15), 5363-5369 (2004).</li><li>Banath, J. P., Klokov, D., MacPhail, S. H., Banuelos, C. A., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+gammaH2AX+foci+as+an+indication+of+lethal+DNA+lesions.">Residual gammaH2AX foci as an indication of lethal DNA lesions.</a> BMC Cancer. 10, 4 (2010).</li><li>Blimkie, M. S., Fung, L. C., Petoukhov, E. S., Girard, C., Klokov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+double-strand+breaks+is+not+modulated+by+low-dose+gamma+radiation+in+C57BL/6J+mice.">Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.</a> Radiat Res. 1815 (5), 548-559 (2014).</li><li>Runge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+interpretation+of+ionizing+radiation-induced+gammaH2AX+foci+by+the+novel+pattern+recognition+system+AKLIDES(R).">Fully automated interpretation of ionizing radiation-induced gammaH2AX foci by the novel pattern recognition system AKLIDES(R).</a> Int J Radiat Biol. 88 (5), 439-447 (2012).</li><li>Jucha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FociCounter:+A+freely+available+PC+programme+for+quantitative+and+qualitative+analysis+of+gamma-H2AX+foci.">FociCounter: A freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.</a> Mutat Res. 696 (1), 16-20 (2010).</li><li>Garty, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RABIT:+a+rapid+automated+biodosimetry+tool+for+radiological+triage.">The RABIT: a rapid automated biodosimetry tool for radiological triage.</a> Health Phys. 98 (2), 209-217 (2010).</li><li>Kovalchuk, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+changes+in+DNA+repair+in+radiation-exposed+mice.">Age-dependent changes in DNA repair in radiation-exposed mice.</a> Radiat Res. 182 (6), 683-694 (2014).</li><li>Rube, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+in+the+context+of+chromatin:+new+molecular+insights+by+the+nanoscale+detection+of+DNA+repair+complexes+using+transmission+electron+microscopy.">DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy.</a> DNA Repair. 10 (4), 427-437 (2011).</li><li>Amundson, S. A., Do, K. T., Fornace, A. 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The authors declare that there are no known competing financial interests.

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 ...

Significant controversy over the potential harmful effects of either low or very low doses of ionizing radiation and public&#8217;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 radiation1-4. 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 &#947;H2AX8, yet other markers and methods have been suggested9,10. It is estimated that thousands of H2AX molecules, in the vicinity of an induced DSB, are involved in the formation of &#947;H2AX enabling the detection of individual DSB by immunofluorescent labeling with an anti-&#947;H2AX antibody and fluorescence microscopy11. The response is very quick, reaching its maximum between 30 and 60 min. Evidence exists that &#947;H2AX 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, &#947;H2AX gets de-phosphorylated and/or undergoes degradation, and newly synthesized H2AX molecules replace &#947;H2AX in the affected areas of chromatin10. Monitoring formation and loss of &#947;H2AX 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 radiosensitivity13-15.
We modified this experimental approach and applied it to an in vivo mouse study to examine the effects of low doses of chronic &#947;- and &#946;-irradiation on DNA DSB levels and repair (Figure 1). Firstly, we demonstrate a method to perform a long-term chronic exposure of mice to &#946;-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 &#947;-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 &#947;-radiation, high energy photons. Due to this difference, &#914;-radiation represents mostly internal health hazard and may produce different biological effects compared to &#947;-radiation. This complication resulted in significant controversies over the regulation of exposure to &#946;-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 &#947;-radiation. Secondly, the rate of DNA DSB is measured in isolated splenocytes upon the completion of chronic exposures using immunofluorescently labeled &#947;H2AX 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&#8217; 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 &#947;H2AX level). Subsequently, the formation and loss of &#947;H2AX, 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&#8217; ability to respond and repair DNA damage induced by much higher stressor levels.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HTO: tritiated water, [3H]</td>
			<td>locally obtained from a nuclear reactor&#160;</td>
			<td></td>
			<td>stock activity 3.7 GBq/mL;&#160; can be substituted with HTO from Perkin Elmer</td>
		</tr>
		<tr>
			<td>OBT: Alanine, L-[3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET348005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Glycine, [2-3H]: organically bound tritium</td>
			<td>Perkin Elmer</td>
			<td>NET004005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET323005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>tritiated water, [3H] (HTO)</td>
			<td>Perkin Elmer</td>
			<td>NET001B005MC</td>
			<td>substitute for HTO of local origin&#160;&#160;</td>
		</tr>
		<tr>
			<td>GammaBeam 150 irradiator</td>
			<td>Atomic Energy of Canada Limited</td>
			<td>locally manufactured</td>
			<td>can be substituted with another g-radiation source of sufficiently low activity&#160;</td>
		</tr>
		<tr>
			<td>tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Fisher Scientific</td>
			<td>SH3025501</td>
			<td>Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>F1051-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-gH2AX antibody, clone JBW301</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor-488 goat anti-mouse antibody</td>
			<td>Life Technologies (formerly Invitrogen)</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodine</td>
			<td>Sigma Aldrich</td>
			<td>P4864-10ML</td>
			<td>1mg/mL</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P006-EAAN</td>
			<td>500ml bottles Absolute Ethanol</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>2682550</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Linde</td>
			<td>P110403</td>
			<td></td>
		</tr>
		<tr>
			<td>12 x 75 mm mL uncapped glass tubes</td>
			<td>Fisher Scientific</td>
			<td>K60B1496126</td>
			<td>disposable borosilicate glass tubes with plain end</td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>VWR</td>
			<td>CA15708-120</td>
			<td>nunc tisue culture 25ml flask (supplier no. 156340)</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
			<td>Wagner scissors 12cm sharp/sharp</td>
		</tr>
		<tr>
			<td>forceps, straight</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>Semken forceps 13cm, straight</td>
		</tr>
		<tr>
			<td>forceps, curved</td>
			<td>Fine Science Tools</td>
			<td>11003-12</td>
			<td>Narrow Pattern forceps 12 cm, curved</td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td>VWR</td>
			<td>CA25382-100</td>
			<td>BD Falcon tissue culture dish 60x15mm</td>
		</tr>
		<tr>
			<td>cell strainers, 70 mm</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td>Falcon cell strainers 50/case</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS recipe: 1 tablet dissolved in 200mL of deionized water, adjust pH to 7.4 if needed.</td>
			<td>Sigma Aldrich</td>
			<td>P4417-100TAB</td>
			<td>Phosphate Buffered Saline Tablets</td>
		</tr>
		<tr>
			<td>TBS recipe: to make 10X Stock,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30g TRIS HCl</td>
			<td>Sigma Aldrich</td>
			<td>T3253-1KG</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr>
			<td>88g NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>2g KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Potassium Chloride</td>
		</tr>
		<tr>
			<td>Dissolve in 1L of deionized water, adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation

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 &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, 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 &#947;H2AX 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.

Figure 2 shows examples of flow cytometry graphs expected for splenocytes prepared using the method described here. Cells are first gated based on the &#60;electronic volume-side scatter&#62; 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 &#947;H2AX signal calculated using the FL1 channel confirms a &#62; ...

Watch this Scientific Journal Video about Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation at JoVE.com

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

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.

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

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

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11.0K Views.  Canadian Nuclear Laboratories. 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. 