Chromosomal aberrations induced by defined DNA double-strand breaks: the origin of achromatic lesions.

The mechanisms of formation of chromosomal aberrations are poorly understood, despite the common use of aberrations as a measure of the genetic effects of physical and chemical agents. We have used restriction endonucleases to introduce defined DNA double-strand breaks into mammalian cells, and measured chromosomal aberration formation relative to the activity of the endonuclease. The endonucleases AluI and Sau3AI remain active for a relatively short time under simulated cellular conditions and induce achromatic lesions ('gaps') in chromatids only within the first hour or two following treatment. In contrast, the endonuclease MboI (an isoschizomer of Sau3AI) is active for an extremely long time and continues to produce chromatid gaps during the whole 12 hr sampling period. This observation strongly suggests that the aberrations classified as gaps are a manifestation of unrejoined DNA double-strand breaks. The formation of gaps may relate to the opportunities for repair of DNA breaks in relation to cell-cycle position. It is more difficult to relate the formation of structural chromatid aberrations to the endonuclease activity, although at relatively low concentrations all 3 endonucleases gave similar levels of structural aberrations.

Investigating the nature of chromatid breaks produced by restriction endonucleases.

It is a basic assumption of the breakage-and-reunion theory that the majority of open chromatid breaks seen at metaphase are the residue of unrejoined primary breaks that have neither restituted nor rejoined illegitimately to form exchange aberrations. If Chinese hamster chromosomes with BrdU sister-chromatid differentiation are irradiated, and chromatid aberrations scored from G2 cells, some 15-20% of open breaks show a colour-jump at the point of discontinuity, indicating a two-lesion intrachange origin. Since we see complete forms of several intrachanges whose incomplete forms will also look like breaks, but devoid of a colour-jump, it appears that a substantial proportion of observed breaks are intrachange derived. Experiments to date show that the colour-jump proportion is constant, irrespective of radiation dose, radiation quality, BrdU concentration and hamster cell origin. It is the same for the very low "spontaneous' breaks found in control samples. Restriction endonucleases (RE) can be introduced into cells by various poration methods, and are highly efficient at producing all types of aberrations. This is taken as strong evidence that DNA dsb are significant lesions triggering aberrations. One might anticipate, therefore, that observed breaks will be predominantly unrejoined dsb, and the proportion of colour-jump break correspondingly low. We tested this supposition using three RE; Alu 1, a blunt-end cutter, Sau3A 1, a cohesive-end cutter, both with a short life-time in vivo, and Mbo 1, an isoschizomer of Sau3A 1, which has a long cutting life-time in vivo. Although there were differences in absolute yields of breaks, and of relative frequencies of aberration types recovered, the proportion of colour-jump breaks was as high as that in a parallel X-ray experiment, and fell well within the range encountered in all our previous experiments. It is difficult to reconcile this universal constancy of colour-jump breaks with the expectations of breakage-and-reunion theory, where the occurrence of two-break events must be a treatment variable. Rather, our results suggest that most open breaks are secondary, resulting from a regular intrachange processing mechanism.

Chromatid damage induced by 238Pu alpha-particles in G2 and S phase Chinese hamster V79 cells.

The comparative induction of chromatid aberrations by 238Pu alpha-particles, or by 250 kVp X-rays was investigated in V79 Chinese hamster cells. Metaphases were sampled at hourly intervals postirradiation up to 8 h and BrdU/FPG staining methods were used to distinguish G2, S and G1 phase cells. Two experiments were performed. In the first, an alpha-particle dose of 0.41 Gy was compared with an X-ray dose of 1.5 Gy used in a previously published study. In the second, an X-ray dose of 1.2 Gy was used in parallel with 0.41 Gy of alpha-particles to produce a similar overall frequency of interchanges, and allow comparative ratios to be derived for other aberration types. At these isoexchange doses, alpha-particles produce relatively less gaps and breaks, particularly in late G2, and significantly more isochromatid deletions. A very high proportion of the isochromatid deletions were incomplete after alpha-particles compared with X-rays, but no difference in incompleteness was found for interchanges. With X-rays, about 6% of interchanges are complex intra-interchange forms. At similar exchange frequencies this increases to 26.7% for alpha-irradiation, suggesting increased multiple lesion interaction. Differences in dose distribution between alpha-particles and X-rays are discussed and mitotic delay is examined after separation of the analysed cells into damaged and undamaged classes.

Electroporation and streptolysin O--a comparison of poration techniques.

CHO (Chinese hamster ovary), xrs5 (X-ray sensitive Chinese hamster) and HF19 (untransformed human fibroblast) cells, were exposed to a lethal dose of the restriction enzyme Pvu II during electroporation or poration with the bacterial toxin streptolysin O. The uptake of the exclusion dye trypan blue was used as a measure of poration and compared with survival as measured by subsequent colony formation. It was assumed that any surviving cells had not been permeabilized and therefore did not receive any restriction enzyme. Electroporation alone proved to be more cytotoxic to the cells, whilst streptolysin O was more efficient at permeabilizing both hamster and human cells.

A case of caffeine-mediated cancellation of mitotic delay without enhanced breakage in V79 cells.

Chinese hamster cells (V79 379A) were grown for 17 h in the presence of 10 micrograms/ml bromodeoxyuridine (BrdU) to obtain cells with potential sister-chromatid differentiation. At this time batches were irradiated (1.5 Gy, 250 kVp X-rays) and the medium of all flasks replaced with one containing 10 micrograms/ml thymidine with or without 400 micrograms/ml caffeine. Metaphases from irradiated and unirradiated batches were sampled every hour for 7 h and FPG stained. All categories of chromatid-type aberrations were scored in G2 and S phase cells. As expected, mitotic delay in the presence of caffeine, (measured by a shift in the portion of the fraction of differentially stained metaphases (FDM) curve and a reduced fall in the mitotic index) was largely cancelled, but there was a negligible increase in chromatid-aberration frequency (all categories), only achieving significance if the consistency of the whole 7-h sampling period was considered. Caffeine had no effect on the frequencies of light/dark chromatid involvement, nor in the completeness of chromatid interchanges. We conclude that the enhanced breakage frequency often observed with post-irradiation caffeine treatment is not necessarily causally related to the cancellation of delay.

Revell revisited.

The controversy of the Classic versus the Exchange theories for the origin of simple chromatid breaks is outlined. Using BrdU harlequin sister-chromatid differentiation four Revell ratios can be defined and these have been obtained and tested as a block in V79 hamster cells. The values are quite different from the simple predictions. However, values similar to those observed (taken as a block) can be readily simulated from Revell theory by making the assumption that intra-chromatid events are dominant (0.7:0.3). They can also be obtained from contingency modelling of Classic theory using the same assumption plus the additional constraint that there is no contribution from isolated single lesions (Poisson class 1). If this latter assumption is correct, then the frequency of breaks involving a colour-jump (ratio III) should not decline to zero as the dose falls. A dose-response experiment shows that it does not, but remains approximately constant at about 12%, even in the unirradiated control. An added complication arises when we discover for the TB/BB situation, that whilst neither breaks nor gaps show any excess BB involvement (sensitisation), lesions involved in interchanges show at least a 2-fold BB excess. Clearly, the chromatid discontinuities we are scoring are not behaving as would be expected of a residue of unrejoined primary breaks (Classic theory) and we infer also that they are not 'simple'.