Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for autom ated image analysis and data processing for Comet assay using human periphe ral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA da mage in mouse peripheral blood leucocytes using identical doses and experim ental conditions, e.g. lyses, electrophoretic conditions and duration of el ectrophoresis which are known to affect tail moment (TM) and tail length (T L) of comets. Human and mouse whole blood samples were irradiated with diff erent doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668 Gy/mi n between 0 and 4 degreesC in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodid e. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluoresc ence, tail fluorescence and tail length were measured. Increase in TM and T L was considered as the criteria of DNA damage. Analysis of data revealed h eterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observ ed in control and irradiated groups of all the three donors. Data were anal yzed for statistical significance using one-way ANOVA. Though a small Varia tion in basal level of TM and TL was observed amongst human and mouse contr ols, the differences were not statistically significant. A dose-dependent i ncrease in TM (P < 0.001) and TL (P < 0.001) was obtained at all the radiat ion doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leuco cytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes , a sharp increase in TM and TL was observed only up to 4 Gy, and there aft er saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r(TM) = 0.999 and r(TL) = 0.999), wher e as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. Fro m the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radia tion damage than humans. (C) 2001 Elsevier Science B.V. All rights reserved .