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 ....

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 fa....

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