Name: measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation
Uploaded: 2015-07-03T17:00:00.000+00:00
Duration: 11 min 24 s
Description: 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

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 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
<ol>
	<li>Animal Grouping
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Chronic Exposure of Mice to Internal &#946;-Radiation
	<ol>
		<li>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.</li>
		<li>Validate the final concentrations of tritium in the drinking water by measuring radioactivity using a liquid scintillation counter. Adjust concentrations, if required.</li>
		<li>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.</li>
		<li>Treat sham-irradiated control mice identically. Keep them in a separate &#8220;clean&#8221; room or on a separate &#8220;clean&#8221; rack under positive air pressure to avoid tritium cross-contamination from the air.</li>
	</ol>
	</li>
	<li>Chronic Exposure of Mice to External &#947;-Radiation 
	Note: &#947;-irradiation facility/equipment will vary depending on the location.</li>
	<li>Using available methods of dosimetry, determine the distances from a &#947;-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 &#956;Gy/h is equivalent to a dose rate produced by 1 MBq/L tritium in the body.</li>
	<li>Expose mice by placing mouse cages at the distances determined in step 1.3.1 for one month. Minimize daily interruption of &#947;-irradiation to 30 min. Use thermoluminescence dosimeters to measure total absorbed doses.</li>
</ol>
2. Sacrifices and Sampling
<ol>
	<li>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.</li>
	<li>Dispense 5 ml of RPMI media into each 60 mm petri dish.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Grasp the abdominal wall with forceps and cut along the median axis of the muscular wall to open up the abdominal cavity.</li>
	</ol>
	</li>
	<li>Excise the spleen by lightly gripping it with sterile forceps and gently pulling on it while simultaneously cutting away the connective tissue with scissors.</li>
	<li>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 &#176;C for supplementary assays.</li>
	<li>Place the remainder of the spleen into a cell strainer inside a 60 mm petri dish with 5 ml of pre-dispensed RPMI media.</li>
	<li>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.</li>
</ol>
3. Preparation of Splenocyte Cultures
<ol>
	<li>Homogenize the spleen by mincing inside a cell strainer with sterile forceps with a curved end.</li>
	<li>Remove the cell strainer and collect the filtered cell suspension from the 60 mm dish into a 15 ml tube.</li>
	<li>Rinse the cell strainer with 5 ml of media to collect the remainder of the cells.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at RT.</li>
	<li>Decant supernatant and re-suspend pellet in 10 ml of RPMI.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at RT.</li>
	<li>Decant supernatant and re-suspend pellet in 5 ml of RPMI.</li>
	<li>Transfer 4 ml of cell suspension into a 25 ml tissue culture flask and remove to a CO2 incubator (37 &#176;C, 5% CO2, 80% humidity)</li>
</ol>
4. Challenging Irradiation and Fixing
<ol>
	<li>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 &#176;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.</li>
	<li>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).</li>
	<li>Centrifuge tubes at 300 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 300 &#956;l of TBS.</li>
	<li>While mixing on a vortex at low speed, add 700 &#956;l of -20 &#176;C 100% ethanol. Close the lid and invert a few times to mix.</li>
	<li>Store the samples at -20 &#176;C. Splenocytes fixed in ethanol can be stored at -20 &#176;C for at least 12 months without any noticeable loss in the &#947;H2AX signal.</li>
	<li>Irradiate the splenocyte cultures from step 3.8 with a challenging &#947;-radiation dose of 2 Gy at a dose rate &#8805; 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.</li>
	<li>Immediately return the cultures to a CO2 incubator.</li>
	<li>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.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 1 ml of TBS.</li>
	<li>Repeat steps 4.3 - 4.5.</li>
	<li>24 hrs after the challenging 2 Gy &#947;-irradiation, harvest and fix a t = 24 hrs sample as described in step 4.8 &#8211; 4.10.</li>
</ol>
5. Immunofluorescent Labeling with Anti-&#947;H2AX 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&#160;for further details.
<ol>
	<li>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 (&#8220;2nd&#160;Ab only&#8221;). Use this historical control sample in every flow cytometry run within a study.</li>
	<li>Add 0.5 ml of ice-cold TBS to each tube.</li>
	<li>Remove fixed splenocytes from -20 &#176;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 &#176;C storage and keep as a backup sample.</li>
	<li>Centrifuge splenocytes at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS containing 1% FBS</li>
	<li>Centrifuge cells at 300 x g for 5 min at 4 &#176;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.</li>
	<li>Centrifuge cells at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 200 &#956;l of primary anti-&#947;H2AX antibody diluted 1:150 in TST buffer. Use 200 &#956;l of TST for the &#8220;2nd&#160;Ab only&#8221; control sample.</li>
	<li>Position tubes on a rotating shaker at an angle of 45 - 60&#176; and shake for 1.5 h at 300 x g at RT.</li>
	<li>While tubes are incubating, prepare a sufficient volume (200 &#956;l/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.</li>
	<li>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 &#176;C. Decant the supernatant and gently re-suspend pellet in 1 ml of TBS containing 2% FBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 200 &#956;l of the secondary antibody solution from step 5.8.</li>
	<li>Incubate the samples on a shaking platform for 1 hr as in step 5.7.</li>
	<li>Add 1 ml of ice-cold TBS containing 1% FBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 0.5 ml of TBS containing 50 &#956;g/ml propidium iodide.</li>
	<li>Incubate for 5 min at RT protected from light.</li>
	<li>Analyze samples on a flow cytometer using instrument-specific protocols16. Read at least 10,000 cells per sample.</li>
</ol>

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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><tbody><tr><td>HTO: tritiated water, [3H]</td><td>locally obtained from a nuclear reactor&amp;nbsp;</td><td></td><td>stock activity 3.7 GBq/mL;&amp;nbsp; 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>1 mCi/ml (185 MBq)</td></tr><tr><td>OBT: Glycine, [2-3H]: organically bound tritium</td><td>Perkin Elmer</td><td>NET004005MC</td><td>1 mCi/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>1 mCi/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&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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>1 mg/ml</td></tr><tr><td>ethanol</td><td>Commercial Alcohols</td><td>P006-EAAN</td><td>500 ml 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 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 12 cm sharp/sharp</td></tr><tr><td>forceps, straight</td><td>Fine Science Tools</td><td>11008-13</td><td>Semken forceps 13 cm, 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 60 x 15 mm</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>PBS recipe: 1 tablet dissolved in 200 ml 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</td><td></td><td></td><td></td></tr><tr><td>30 g TRIS HCl</td><td>Sigma Aldrich</td><td>T3253-1KG</td><td>Trizma hydrochloride</td></tr><tr><td>88 g NaCl</td><td>Fisher Scientific</td><td>S271-500</td><td>Sodium Chloride</td></tr><tr><td>2 g KCl</td><td>Fisher Scientific</td><td>P217-500</td><td>Potassium Chloride</td></tr><tr><td>Dissolve in 1 L 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; ...

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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 ...

measuring-dna-damage-repair-mouse-splenocytes-after-chronic-vivo

Research

JoVE Journal

Biology

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. 

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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Genetics

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Bioengineering

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Immunology and Infection

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Neuroscience

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Medicine

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

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

Summary

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Chapters in this video

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Quantitation of γH2AX Foci in Tissue Samples

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis