Anal. Chem. 1999, 71, 4423-4426
Correspondence
Detection of Low Dose Radiation Induced DNA Damage Using Temperature Differential Fluorescence Assay Kim R. Rogers* and Alma Apostol
United States EPA, National Exposure Research Laboratory-Las Vegas, Las Vegas, Nevada 89193 Steen J. Madsen
Department of Health Physics, University of Nevada-Las Vegas, Las Vegas, Nevada 89154 Charles W. Spencer
Nevada Radiation Oncology Centers, Las Vegas, Nevada 89106
A rapid and sensitive fluorescence assay for radiationinduced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses. The double-strand, sensitive dye PicoGreen was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng of double stranded DNA (106 copies for plasmid puc 19) are detected by this method. DNA damage and repair are critical aspects of cellular function. As a result, a wide variety of methods have been reported for detection of oxidative DNA damage. General strategies that have been employed to measure damage caused by ionizing radiation or chemical modification include the use of gel electrophoresis,1 capillary electrophoresis,2 and HPLC3 as well as various optical,4 acoustic,5 and electrochemical6 methods. Because the effects of ionizing radiation on DNA have been well-described over the past several decades, ionizing radiation has often been used as a calibrator to compare various analysis methods. Ionizing radiation * Corresponding author: (mailing address) U.S. EPA, 944 E. Harmon Ave., Las Vegas, NV 89119; (tel.) (702-798-2299; (fax) 702-798-2107; (e-mail)
[email protected]. (1) Delaney, C. A.; Green, I. C.; Lowe, J. E.; Cunningham, J. M.; Butler, A. R.; Renton, L.; D’Costa, I.; Green, M. L. R. Mut. Res. Fundam. Molec. Mech. Mutagen 1997, 375, 137. (2) Le, X. C.; Xing, J. Z.; Lee, J.; Leadon, S. A.; Weinfeld, M. Science (Washington, D.C.) 1998, 280, 1066. (3) Inoue, S.; Kawanishi, S. FEBS Lett. 1995, 371, 86. (4) Prutz, W. A. J. Biochem. Biophys. Methods 1996, 32, 125. (5) Zhang, H.; Tan, H.; Wang, R.; Wei, W.; Yao, S. Anal. Chim Acta 1998, 374, 31. (6) Fojta, M.; Palacek, E. Anal. Chim Acta 1997, 342, 1. 10.1021/ac990537z CCC: $18.00 Published on Web 09/01/1999
© 1999 American Chemical Society
is known to produce a variety of changes to DNA including base alterations and loss, DNA-protein cross-links, and both single strand (ss) and double strand (ds) breaks.7 Methods reported to detect ss breaks include the ethidium bromide binding assay,4 single-cell gel electrophoresis (comet) assay,1 alkaline unwinding assay,8 and alkaline elution assay9 which have been used for naked DNA as well as DNA isolated from cells, tissues, and organisms exposed to various agents known to cause damage. These methods typically use alkaline conditions (e.g., pH 12-13) to partially denature “unwind” damaged DNA. Because the degree of denaturation under controlled conditions is dependent on the number of ss breaks, these techniques can be very sensitive for measuring this kind of damage. These methods, however, typically require the separation of ss and ds forms prior to quantitation; thus rendering them time-consuming and expensive. Techniques that use temperature-induced denaturation (i.e., melting curve analysis) monitored by absorbance (i.e., hyperchromic effect) have also been used to measure radiation-induced damage. Assays that rely on absorbance, however, are typically insensitive requiring fairly large doses of radiation (i.e., 10 Gy). They also require relatively large amounts of DNA (i.e., 0.3 mg per assay).7 In this paper, we report the use of partial temperature denaturation monitored by the use of the fluorescent ds indicator dye PicoGreen to detect low levels of radiation-induced damage to relatively small amounts of calf thymus DNA and Escherichia coli plasmid DNA. (7) von Sonntag, C. Chemical Basis of Radiation Biology; Taylor and Francis: Philladelphia, 1989; pp 194-220. (8) Tamir, S.; Burney, S.; Tannenbaum, S. R. Chem. Res. Toxicol. 1996, 9, 821. (9) Kraynak, A. R.; Storer, R. D.; Jensen, R. D.; Kloss, M. W.; Soper, K. A.; Clair, J. H.; DeLuca, J. G.; Nichols, W. W.; Eydelloth, R. S. Toxicol. Appl. Pharmacol. 1995, 135, 279.
Analytical Chemistry, Vol. 71, No. 19, October 1, 1999 4423
EXPERIMENTAL SECTION Calf thymus DNA, E. coli plasmid (puc 19, containing 5996 base pairs), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma (St. Louis, MO). PicoGreen was from Molecular Probes (Eugene, OR). All other chemicals were of reagent-grade. Radiation-induced DNA damage was indirectly measured using observed changes in temperature-induced partial strand separation. All samples were irradiated with a linear accelerator (Siemens Mevatron, New York, NY) producing X-rays of 6 MeV average energy. The samples containing DNA were placed beneath a 1.5-cmthick water-equivalent buildup material and irradiated at an X-ray source-to-sample distance of 100 cm for doses g10 cGy or 152 cm for doses
