Detection and quantification of 8-hydroxydeoxyguanosine adducts in peripheral blood of people exposed to ionizing radiation.

Ionizing radiation produces a variety of damaging insults to nucleic acids, including the promutagenic lesion 8-hydroxydeoxyguanosine. In the present study, the 8-hydroxydeoxyguanosine content of peripheral blood leukocyte DNA isolated from individuals exposed to therapeutic doses of ionizing radiation was determined by a HPLC-coupled 32P-postlabeling assay. Peripheral blood leukocyte DNA from individuals irradiated with 180-200 cGy were observed to contain 2-4.5 times as much 8-hydroxydeoxyguanosine as that from unexposed individuals. These results were confirmed by the use of a HPLC-coupled electrochemical detection system. Thus, human exposure to ionizing radiation significantly increased the circulating leukocyte DNA content of 8-hydroxydeoxyguanosine. ImagesFIGURE 2.


Introduction
Ionizing radiation, such as that used in radiation therapy, causes a wide variety of DNA damage, ranging from singleand double-strand breaks to DNA-protein cross-links, 8-hydroxydeoxyguanosine , thymidine glycol, and other oxidative and free-radical damage (1). Aside from single-strand breaks, 8-OHdG appears to be the major adduction product of radiation damage to cellular DNA (1,2). This lesion, 8-OHdG, is known to be removed from DNA by repair mechanisms (3,4), which is not surprising considering that it is a promutagenic lesion (5,6). Thus, the ability to monitor individuals for 8-OHdG levels would be valuable to the assessment of DNA damage caused by ionizing radiation and other oxidative and free-radical inducing agents.
Techniques have recently been developed by us and others (3,(7)(8)(9) for the sensitive detection and quantitation of 8-OHdG residues in biological samples. This paper describes the quantitation of this oxidative DNA damage in circulating, nucleated blood cells in humans exposed to ionizing radiation by the use of
water, applied to prewashed PEI cellulose plates, and the nucleotides resolved by to4imensional TLC (Fig. 2). The appropriate spots meescraped and the n1dioactivity determined by liquid scintillation counting. The level of 8-OHdG was observed to be greater in irradiated than in unexposed individuals ( Table 1). ..~~~~~~~~~~b e detected by UV mm at 254mm were also spotted on these TLC plates). 01~~~~~~~The spotted plates were developed in 1 M ammonium isobutyrate, pH 7/10% isopropanol (DI) and washed in MeOH and dried before turning the plate 90~,~~.

8-HYDROXYDEOXYGUANOSINE IN RADIATION-EXPOSED SUBJECTS
described above, except that the procedure was performed under helium. Samples of DNA (100 ug) were enzymatically digested as described above. The digests were filtered through Millipore Ultrafree MC (10,000 molecular weight cutoff) filters and lyophilized to dryness. Each sample was dissolved in 30 yL of HPLC eluant and aliquots analyzed by HPLC with electrochemical detection. Samples were isocratically eluted with 20 mM sodium phosphate, pH 5.5 / 5 % MeOH, at a flow rate of 1.0 mL/min. Again, increased levels of 8-OHdG were observed in irradiated patients (Table 1).

Discussion
The results indicate that people exposed to a therapeutic dose of ionizing radiation have higher levels of 8-OHdC in their circulating blood leukocyte DNA than nonirradiated individuals. HPLC/32P-postlabeling and TLC analysis of human peripheral blood DNA samples from exposed individuals showed between 2 and 4.4 times the content of this oxidative damage found in control samples. Similar increases in 8-OHdG levels in irradiated patients were also observed using electrochemical detection techniques. However, the basal level of 8-OHdG in circulating nucleated blood cells was approximately 5 times higher than previously suggested (3,7,8). The protracted time frame of the DNA isolation, HPLC fractionation, postlabeling, and TLC procedures may have allowed excessive oxidation of the deoxyguanosine to have occurred, forming an abnormally high background.
Future studies ofpatients will require optimization ofconditions, both in the choice of patient (field ofexposure) and in the time frame of sample procurement and processing. First, the vascularity and the volume ofblood irradiated varies with tissue and tumor, so that the actual dose of radiation that peripheral blood cells receive may vary from person to person. Second, the pharmacokinetics of 8-OHdG formation and removal in circulating nucleated cells in humans following exposure to ionizing radiation is not known. Thus, the optimal time of sampling from patients needs to be discerned. And last, the optimal conditions and time frame for processing blood samples needs to be determined to avoid spurious increases in in vitro oxidative production of 8-OHdG.