Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop III

Abstract

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Title: The Induction of Truly Simple Exchanges Is Not Independent of Dose Rate.

Authors: B.D. Loucas1 , S.M. Bailey2, E.H. Goodwin3 and M.N. Cornforth1.

Institutions: 1Dept. Radiation Oncology, Univ. Texas Medical Branch,
2Dept. Radiol. Health Sciences, Colorado State Univ.,
3Biosciences Division, Los Alamos National Laboratory.

For many years it was assumed that virtually all radiation-induced exchange aberrations were “simple”, arising through the pairwise rejoining of two breaks. Subsequent whole chromosome painting studies led to the realization that exchanges are frequently complex, involving the interaction of three (or more) damaged breaks distributed among two (or more) chromosomes. Because such studies typically involve painting only a small number of select chromosomes, ambiguities arise in the resulting staining patterns that confound attempts to estimate the frequency and extent of complex rearrangements. Many complex xchanges produce pseudosimple staining patterns, meaning they only appear to be simple. And while other exchanges can often be identified as being complex by their staining patterns, the number of chromosomes and breakpoints which can be deduced to have participated in the exchange often severely underestimates that which has actually occurred. These ambiguities are largely overcome through the use of modern combinatorial painting techniques, such as mFISH or SKY, that allow the identification of each homologous chromosome pair in the human karyotype.

An astonishing prediction that arose from the analysis of earlier whole chromosome painting data is that the characteristic curvature in the low LET dose response for chromosome aberrations derives principally (if not solely) from complex aberrations, leaving the dose response for simple exchanges with an apparently linear shape. This prediction was later experimentally verified by our own mFISH studies on human lymphocytes and fibroblasts. These results have been viewed as a challenge to the usual cytogenetic viewpoint that exchanges involve the interaction of two (or more) damaged sites, via a molecular process employing nonhomologous endjoining. Thus, such interaction is fundamentally two-hit in nature. That the dose response for simple exchanges has a linear shape has been interpreted by some investigators to support the alternative notion that exchanges occur when a single radiation-induced chromosome break enters into an exchange with an undamaged chromosome via a one-hit process, a notion seemingly compatible with repair processes utilizing homologous/homeologous recombination.

The issue is of primary relevance to low dose effects, because if a one-hit mechanism is really responsible for the formation of simple exchanges, then the linearity in question defines a dose response that, by definition, can be extrapolated with confidence to arbitrarily low (e.g., sub-rad) doses. Significantly, a one-hit mechanism also predicts that the results of such an extrapolation would be the same, irrespective of radiation intensity. In other words, the dose response for simple exchanges should be identical, regardless of the rate at which low LET radiation is delivered. To test this prediction we irradiated noncycling G0 human fibroblasts with 137Cs g–rays under conditions of limiting low dose rate (LLDR). [LLDR is defined here as a dose rate for which further reduction in dose rate does not lead to additional reduction in the frequency of chromosome aberrations]. Results were compared to those derived from cells receiving comparable doses given at high (acute) dose rates.

To address the issue definitively, we employed mFISH, which allowed us to distinguish unequivocally the truly simple exchanges from pseudosimple exchanges. Our previously reported preliminary results showed that the acute (high dose-rate) dose response (slope) for true simple exchanges was significantly steeper than that obtained under LLDR. We now extend these results to include the analysis of additional cells, including those from acutely irradiated cultures that were given the benefit of full postirradiation recovery prior to release from density inhibition (i.e., PLDR). This was deemed necessary in order to make a more meaningful comparison to cells exposed at LLDR, as the vast majority of damage under these conditions is subject to full PLDR. We can now state with considerable confidence that the dose response for simple exchanges from acute radiation exposures is fully five-fold higher than that produced under conditions of LLDR. Consequently, while the acute dose response for simple exchanges is, in fact, largely linear in shape, it is unlikely that this linearity derives from a one-hit process. Instead, we argue that the process of complex exchange formation competes for broken chromosome ends that might otherwise become involved in the formation of simple exchanges. Competition for reactive breaks causes warpage in the shape of dose response for simple exchanges which, over a limited range of doses, gives the appearance of linearity with dose.

Thus, the data do not support the contention that the apparent linearity observed in the acute dose response for simple exchanges derives from a one-hit interaction process. On the other hand, the data are generally consistent with the predictions of the classical two-hit model. An interesting observation less easily explained by either model relates to the appearance of complex aberrations under LLDR. We now estimate that roughly ten percent of all exchange breakpoints derive from complexes under LLDR, a value that should hold for situations of extremely low doses delivered acutely. For both models, the contribution of complexes to overall cytogenetic damage would be expected to (and does) fall with decreasing dose and decreasing dose rate. But since aberrations produced under LLDR are assumed to arise exclusively by single track action for both models, it is surprising that any complex aberrations would be produced at all.