Human lymphocytes irradiated with graded doses of up to 5 Gy of 150 kV X rays were fused with mitotic CHO cells after delay times ranging from 0 to 14 h after irradiation. The yields of dicentrics seen under PCC conditions, using C-banding for centromere detection, and of excess acentric fragments observed in the PCC experiment were determined by image analysis. At 4 Gy the time course of the yield of dicentrics shows an early plateau for delay times up to 2 h, then an S-shaped rise and a final plateau which is reached after a delay time of about 8 to 10 h. Whereas the dose-yield curve measured at zero delay time is strictly linear, the shape of the curve obtained for 8 h delay time is linear-quadratic. The linear yield component, alpha D, is formed entirely in the fast process manifested in the early plateau, while component beta D2 is developed slowly in the subsequent hours. Analysis of the kinetics of the rise of the S-shaped curve for yield as a function of time leads to the postulate of an "intermediate product" of pairwise DNA lesion interaction, still fragile when subjected to the stress of PCC, but gradually processed into a stable dicentric chromosome. It is concluded that the observed difference in the kinetics of the alpha and beta components explains a number of earlier results, especially the disappearance of the beta component at high LET, and opens possibilities for chemical and physical modification of the beta component during the extended formation process after irradiation observed here.
Repair kinetics observable at the level of exchange-type chromosomal aberrations (dicentric chromosomes), using fractionation and delayed-plating techniques, have been compared with repair kinetics of radiation-induced DNA double-strand breaks, measured with PFGE, and with repair kinetics of all strand breaks, measured with the alkali-unwinding technique. Only data from quiescent or proliferating CHO K1 cells obtained in the same laboratory were used. We determined repair kinetics in terms of the time constant tau (equal to half-time/log(e)2). The repair kinetics (tau approximately 11-14 min) observed in the split-dose formation of dicentric chromosomes agrees with fast repair kinetics of double-strand breaks (tau approximately 11-13 min), thus permitting us to identify the latter as the 'primary lesions' whose pairwise interaction leads to the beta D2 yield term of the aberrations. The repair kinetics observed for dicentric chromosomes formed under delayed-plating conditions (tau approximately 75 min), which mainly affects the alpha D yield term, is attributed to an intermediate interchromosomal product temporarily existing in the course of aberration formation; it is suggested that this product is mechanistically correlated with the slow repair kinetics of 'clustered damage' to DNA seen with the applied molecular methods (tau approximately 90 min).
In a study of X-ray-induced chromosome aberrations in human G(0) lymphocytes irradiated with 4 Gy using premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH), the time-dependent pattern of chromosome fragments and interchromosomal exchanges involving chromosome 4 was recorded after postirradiation incubation times varying from 0.5 to 46.5 h. Unattached acentric fragments and incomplete interchromosomal exchanges have high initial yields, followed by an exponential decrease, while complete interchromosomal exchanges have almost zero initial yield with a subsequent increase in their number. Plateau values of all yields are reached after about 25 h. This temporal variation of aberration yields can consistently be explained by the competition of disruptive PCC stress with the progress of postirradiation structural restitution at the sites of radiation-induced chromatin instabilities. Details of the temporal pattern of incomplete exchanges reflect the different kinetics of the alpha and beta components of the yield of aberrations. The observed large difference between late-PCC and metaphase yields of unattached acentric fragments and the almost perfect conversion from incomplete prematurely condensed chromosomes into complete metaphase exchanges are explained by a difference in the magnitude of chromosome condensation stress between PCC and mitotic conditions. Chromatin sites prone to fragmentation and incompleteness under conditions of PCC can therefore persist as genetic instabilities hidden during mitosis.
In their recent paper on modelling the kinetics of chromosome exchange formation [1], Moiseenko et al. comment upon the observation of Vyas et al. [2] made at doses of 2 and 3 Gy that the yield of radiation-induced dicentric chromosomes in human interphase lymphocytes seen under conditions of premature chromosome condensation (PCC) at fusion delay times up to 2 h was similar to the yield observed in the first metaphase after stimulation with phytohemagglutinin (PHA). Moiseenko et al. think that this lack of a detectable difference was the reason why Vyas et al. did not increase the delay time further. However, in our subsequent study [3] at a dose of 4 Gy, a significant additional yield contribution solely ascribable to the βD 2 component was observed at longer delay times up to 14 h [3]. This contrast is interesting, especially with regard to the design of further experiments, because of the great influence of the chosen dose level upon the detectability of the delayed yield increase.In order to illustrate this, we have plotted in Fig. 1 the dose-dependent yield of dicentrics seen under PCC conditions for fusion delay times t D of 0 and 8 h (taken from [3]) and the yields observed in the first metaphase after PHA stimulation (taken from [4] and new unpublished data, R. P. Virsik-Peuckert). It is true that at D = 2 Gy and even at D = 3 Gy, statistical limits of uncertainty may prevent the detection of any yield difference between the observation at t D = 0 h and the two other conditions. However, at D = 4 Gy, where we studied the change of the dicentric yield with fusion delay time [3], the yield difference cannot be masked by measurement uncertainty, and it would be even more prominent in an experiment performed at 5 Gy. We conclude that the yield of radiation-induced dicentrics seen under PCC conditions at long fusion delay times closely resembles the yield seen in first metaphase, while the yield for short delay times is significantly smaller at doses exceeding 3 Gy.It is worth noting that the linear term at t D = 8 h is only about two-thirds of that seen at t D = 0 h [3], a reduction of opposite tendency to the increase of the dose-squared term with increasing delay time. This partial compensation contributes to the difficulties in detecting any yield differences at smaller doses. From a mechanistic point of view, this reduction of the linear term with increasing delay time in contrast to the increase of the dose-squared term may be an indication of a fundamental difference in the formation processes of the linear and dose-squared components [3, 5, 6]. Fig. 1 Dose-response curves for the yield of radiation-induced dicentric chromosomes (x-rays, 150 kV) in human peripheral blood lymphocytes seen in interphase under PCC conditions after fusion delay times of t D = 0 h ( c ) and t D = 8 h ( a ) [3] and in first metaphase after PHA stimulation ( D from [4], B from new experiments, R. P. Virsik-Peuckert) References 1. Moiseenko VV, Edwards AA, Nikjoo N (1996) Modelling the kinetics of chromosome exchange formatio...
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