Abstract
Whole-chromosome painting (WCP) typically involves the fluorescent staining of a small number of chromosomes. Consequently, it is capable of detecting only a fraction of exchanges that occur among the full complement of chromosomes in a genome. Mathematical corrections are commonly applied to WCP data in order to extrapolate the frequency of exchanges occurring in the entire genome [whole-genome equivalency (WGE)]. However, the reliability of WCP to WGE extrapolations depends on underlying assumptions whose conditions are seldom met in actual experimental situations, in particular the presumed absence of complex exchanges. Using multi-fluor fluorescence in situ hybridization (mFISH), we analyzed the induction of simple exchanges produced by graded doses of 137Cs gamma rays (0–4 Gy), and also 1.1 GeV 56Fe ions (0–1.5 Gy). In order to represent cytogenetic damage as it would have appeared to the observer following standard three-color WCP, all mFISH information pertaining to exchanges that did not specifically involve chromosomes 1, 2, or 4 was ignored. This allowed us to reconstruct dose–responses for three-color apparently simple (AS) exchanges. Using extrapolation methods similar to those derived elsewhere, these were expressed in terms of WGE for comparison to mFISH data. Based on AS events, the extrapolated frequencies systematically overestimated those actually observed by mFISH. For gamma rays, these errors were practically independent of dose. When constrained to a relatively narrow range of doses, the WGE corrections applied to both 56Fe and gamma rays predicted genome-equivalent damage with a level of accuracy likely sufficient for most applications. However, the apparent accuracy associated with WCP to WGE corrections is both fortuitous and misleading. This is because (in normal practice) such corrections can only be applied to AS exchanges, which are known to include complex aberrations in the form of pseudosimple exchanges. When WCP to WGE corrections are applied to true simple exchanges, the results are less than satisfactory, leading to extrapolated values that underestimate the true WGE response by unacceptably large margins. Likely explanations for these results are discussed, as well as their implications for radiation protection. Thus, in seeming contradiction to notion that complex aberrations be avoided altogether in WGE corrections – and in violation of assumptions upon which these corrections are based – their inadvertent inclusion in three-color WCP data is actually required in order for them to yield even marginally acceptable results.
Highlights
Whole-chromosome painting (WCP) involves the labeling of a few select chromosomes of the genome, thereby producing discrete changes in fluorescent color patterns that accompany the junctions of exchange breakpoints
Whole-chromosome painting data can be extrapolated in order to approximate the total number of exchanges that would have been detected if all homologous chromosome pairs would have been painted a unique color, as in the combinatorial painting technologies of multi-fluor fluorescence in situ hybridization [1] or spectral karyotyping (SKY) [2]
The overarching first issue is whether such a multiplicative factor even exits that can bring three-color data into registry with multi-fluor fluorescence in situ hybridization (mFISH) data over a range of relevant doses
Summary
Whole-chromosome painting (WCP) involves the labeling of a few select chromosomes of the genome, thereby producing discrete changes in fluorescent color patterns that accompany the junctions of exchange breakpoints. These include junctions between the painted and unpainted chromosomes, and between the painted chromosomes themselves. Converting WCP data to that of whole-genome equivalency (WGE) provided by mFISH or SKY makes use of relationships similar to that developed by Lucas and colleagues [3] These consider exchanges between painted and unpainted (counterstained) chromosomes, adjusting for unseen exchanges presumed to have occurred between unpainted chromosomes. After three-color WCP was introduced, subsequent refinements were made to accommodate exchanges occurring among the individually painted chromosomes as well [4, 5]
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