Intra-individual variation in G2 chromosomal radiosensitivity

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TThhee ccoommbbinineedd ddaatata frfroomm aalll titmimee ppeerrioioddss eexxhhibibitietedd aa mmeenntatall pprorototoccool,l, ddirireecctt ccoommppaarirsisoonn ooff sstutuddyy rreessuultlsts isis rraarreelyly o w n - a portable incubator (M-Tech Diagnostics, Warrington, UK) at 37 C and irradiated (or sham irradiated) with 0.5 Gy, 300 kV X-rays (Aegleteq Ltd, Bucks, UK). Following a 30-min recovery time, 0.2 ml colcemid (10 lg/ml) (Invitrogen Limited) was added and at 90 min after irradiation, the contents of the culture flasks were transferred to centrifuge tubes (Sterilin Ltd, Carerphilly, UK) and placed on ice. Subsequent centrifugation, hypotonic treatment [0.075 M potassium chloride (KCl)] (VWR International) and fixation (methanol:glacial acetic acid, 3:1) (VWR International) were carried out at 4 C. Fixed cells were stored at 20 C overnight, or longer, prior to slide making. Metaphase slides were made according to standard procedures and stained with Giemsa (VWR International). For each irradiated sample, 100 well-spread metaphases were analysed and the total number of chromatid gaps and breaks determined to give a G2 aberration frequency. Chromatid-type aberrations were scored according to previously outlined criteria (24), with breaks being defined as mis-aligned discontinuities and gaps as single aligned discontinuities wider than the width of a chromatid. Two scorers were used to score each sample to eliminate scorer bias, with each scorer analysing 50 metaphases per sample. One scorer was used for all four time periods and, whilst the second scorer remained the same for Periods 13, a different scorer was used in Time Period 4. Scoring criteria were periodically checked to ensure that there were no differences between scorers. Earlier studies using the G2 assay in our laboratory (15,24) indicated that the spontaneous aberration yield was negligible (,1.0 aberration per 100 cells) and therefore, no adjustment for spontaneous yield was made in the analysis of the results for the present study. Statistical methods The distributions of chromatid aberrations among metaphase cells in all samples from each individual were analysed for approximation to the Poisson distribution as previously described (24). If the observed distributions follow Poisson statistics, the variance and the mean of the observed distributions would be equal and a ratio value of 1.0 would be expected. If the variance is greater than the mean, aberrations are overdispersed and the Poisson distribution does not apply. Ratios of variance to mean (the mean being the number of aberrations divided by the number of metaphase cells studied) were calculated for each sample and an average value for each donor (Z) determined for each of the four time periods, as well as a combined study value for each individual (Table I). Homogeneity of repeat sampling was investigated using chi squared (v2) with the Poisson derived variance (the expected value) adjusted to account for the observed overdispersion of aberrations and thus, the formula v2 5 P(O E)2/(EZ) was adopted, where O is the observed value of aberrations per 100 cells, E is the expected value of aberrations per 100 cells and Z is the compensation factor defined above. Standard errors for mean aberration yields were calculated by adjusting for overdispersion and any additional intra-individual variation according to the formula O(number of aberrations Z Y), and then normalising to 100 cells scored. Y was estimated by summing all the chi-squared values and dividing by the total degrees of freedom. Chromatid aberration frequencies for each sample time period from each donor are presented in Figure 1. Mean induced aberration frequencies and coefficients of variation (CVs) for Ratio of variance to mean (average value) Mean aberration frequency SE per 100 cells (range) 12.03.200114.01.2002 11.03.200207.04.2003 25.05.200408.06.2004 19.06.200611.12.2006 11.03.200207.04.2003 19.06.200611.12.2006 1.1 (63113) 2.4 (91128) 12.4 (71106) 3.4 (85150) 0.7 (63150) 2.44 (59122) 5.9 (75187) 2.34 (59187) Fig. 1. Radiation-induced G2 chromatid aberration frequencies for Donors 1 and 2. the different time periods and the combined data are presented in Table I. When analysing individual time periods, Donor 1 exhibited no statistically significant intra-individual variation for Periods 1 and 2 (Period 1: v212 5 18.15, P 5 0.111 and Period 2: v25 5 5.95, P 5 0.311) although significant variation was observed in Periods 3 and 4 (Period 3: v21 5 4.72, P 5 0.030 and Period 4: v26 5 18.74, P 5 0.005). Statistically significant intra-variation was observed in both time periods involving Donor 2 (Period 2: v28 5 25.08, P 5 0.002 and Period 4: v26 5 42.95, P , 0.001). For the combined data from all time periods, intra-individual variation was found to be highly statistically significant for both individuals (Donor 1: v227 5 75.64, P , 0.001 and Donor 2: v215 5 111.57, P , 0.001). A number of studies have experienced variation in G2 radiation-induced aberration frequencies in repeat samples from some individuals and some laboratories have reported levels of variation not significantly different to inter-individual variability (8,13,2325). Baria et al. (13) recorded a CV of 18.6% for intra-individual variability from the repeated sampling of nine adult normal donors in comparison with inter-individual variation of 19.2% and thus did not demonstrate statistically significant differences between the normal donors. In contrast, previous studies by the same group reported intra-individual variation for normal donors in the range of 710% and interindividual variation in the range of 1520% (3,5,14,22). Vral et al. (23) examined 14 apparently healthy individuals over a period of 1 year, with two volunteers providing nine samples each. Intra-individual variation for these two donors (CV 5 14 and 16%, respectively) was not significantly different from the inter-individual variability observed for the group as a whole (CV 5 17%) and the (...truncated)