Radiation induced strand breakage analyzed by tunel technique

Abstract

The objective of this research is to fully characterize the effectiveness and limits of using the terminal deoxynucleotidyl transferase mediated biotin-dUTP nick end labeling (TUNEL) technique for analysis of radiation induced strand breakage. If the TUNEL technique is found valuable, it could be applied to develop a biodosimetry protocol, primarily useful for individuals exposed in radiological accidents. Several techniques currently in use include fluorescent in-situ hybridization, the comet assay and the dicentric assay, yet each has drawbacks such as limited sensitivity or considerable preparation time. Recently, the TUNEL assay has been used in studies by Harvey and Ford (1997) to investigate chromatid breaks due to restriction enzymes. This research uses similar protocols to examine breaks due to radiation. Chinese hamster ovary (CHO) cells were cultured and exposed to X rays, receiving a dose ranging from 0 to 2 Gy. Slides were created using a standard metaphase chromosome preparation technique, followed by the TUNEL reaction to highlight chromosome breaks. The results were used to build a dose response curve. Although the expected increase in TUNEL positives per metaphase cell with increased x-ray dose was seen, large errors were associated with the results rendering TUNEL assay less than ideal for biodosimetry purposes. Additionally, TUNEL is not very effective at high doses because each TUNEL positive becomes indistinguishable from neighboring positives due to the high number of positives on each chromosome.