Incomplete Exchanges Research Articles

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin in BrdU-labeled human chromosomes

The BrdU-Hoeschst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs)_induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combinant of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significance increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of sce might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.

Role of sister chromatid exchanges in chromatid aberration formation.

MECHANISMS of formation of chromatid aberrations are not fully understood. The involvement of chromosome breakage and reunion1 or exchange2 has been suggested. DNA repair is a possible cause of aberrations and exchanges3–5, but a causal relationship between the two phenomena is not clear6,7. It is not known whether chromatid deletions derive from unrepaired chromatid breaks or from incomplete exchanges between sister chromatids. The origin of chromatid deletions can be studied by examining whether or not they occur just at the points of sister chromatid exchanges (SCE) (ref. 6). Differential labelling of sister chromatids with 5-bromodeoxyuridine (BUdR) has shown that X-ray-induced chromatid deletions are only infrequently associated with SCE, indicating that most chromatid deletions are not incomplete SCE but simple chromatid breakage8. On the other hand, the parallel distribution of SCE and chromosome aberrations induced by two different hydrocarbon carcinogens, 7,12-dimethylbenz (a) anthracene and 7,8,12-trimethylbenz (a) anthracene has been reported, suggesting that aberrations are incomplete SCE (ref. 9). The results presented here indicate that almost no chromatid deletions or gaps induced by irradiation of BUdR-substituted chromosomes with visible light represent incomplete SCE.

The induction of chromatid deletions in accord with the breakage-and-reunion hypothesis

In the present experiments it has been possible to study large numbers of X-ray induced chromatid deletions, or breaks, in Chinese hamster chromosomes and to discern whether or not a sister chromatid exchange also occurs at the point of breakage. Chromatid deletions are only infrequently associated with a sister chromatid exchange. This is contrary to the expectations derived from the exchange hypothesis of Revell. On the basis of this hypothesis, in which chromatid deletions are considered to be incomplete exchanges that occur in the necks of little loops in the chromosomes, 40% of the chromatid breaks are expected to be associated with sister chromatid exchanges. The present data are in accord with the conclusions drawn from the earlier autoradiographic experiments of Heddle and Bodycote, and show that chromatid breaks can be accounted for on the basis of the breakage-and-reunion hypothesis, with the majority being simple breaks and some being incomplete exchanges between two such breaks.

The induction of chromatid deletions in accord with the breakage-and-reunion hypothesis

In the present experiments it has been possible to study large numbers of X-ray induced chromatid deletions, or breaks, in Chinese hamster chromosomes and to discern whether or not a sister chromatid exchange also occurs at the point of breakage. Chromatid deletions are only infrequently associated with a sister chromatid exchange. This is contrary to the expectations derived from the exchange hypothesis of Revell. On the basis of this hypothesis, in which chromatid deletions are considered to be incomplete exchanges that occur in the necks of little loops in the chromosomes, 40% of the chromatid breaks are expected to be associated with sister chromatid exchanges. The present data are in accord with the conclusions drawn from the earlier autoradiographic experiments of Heddle and Bodycote, and show that chromatid breaks can be accounted for on the basis of the breakage-and-reunion hypothesis, with the majority being simple breaks and some being incomplete exchanges between two such breaks.

A model for the production of chromosome damage by mitomycin C.

A model is presented, which is based on the idea that the chromosome damage induced by Mitomycin C results directly from repair or misrepair of DNA molecules responsible for the linear continuity of the chromosomes. Testing the model with human cells confirms the prediction that exchanges with complete joining occur between chromosome regions containing homologous, repetitive DNA. Most probably incomplete exchanges involve homologous, but unique DNA sequences. — Prerequisites determining the MC-induced aberration patterns are the distribution of the chemical due to compartmentalization, the somatic pairing of chromosomes, and the occurrence of repeated or unique DNA sequences. — The scoring of different classes of MC-induced chromatid aberrations (attenuation, constriction, gap, break) in alcohol/acetic acid-fixed chromosome has a limited value.

On the formation of chromosomal aberrations

The exchange hypothesis and the breakage-first hypothesis are the 2 major hypotheses that describe how chromosomal aberrations might be produced. A critical test has shown that one important aspect of the exchange hypothesis is correct, namely that some aberrations that appear to be simple chromatid deletions are actually incomplete exchanges. Furthermore, the relative frequency of those deletions that could be positively identified as incomplete exchanges was very close to that predicted from the exchange hypothesis. Because the sample was small and because a mixture of 2 deletion types (true, non-exchange, deletionsand incomplete exchanges between sister chromatids) could have given the same results, further experiments were performed. The results show that chromatid deletions are indeed of 2 types, since the relative proportion of exchange deletions is not the same in Chinese hamster cells as in rat kangaroo cells, nor is it the same in S and in G 2 of Chinese hamster cells. Thus, some chromatid deletions are simple deletions arising from single lesions as expected on the basis of the breakage-first hypothesis and others are incomplete exchanges between sister chromatids as expected on the exchange hypothesis. It is clear, then, that neither hypothesis, as usually interpreted, is entirely correct nor incorrect, but rather that deletions are of 2 types according to mode of origin. Attempts to identify the 2 types morphologically have failed. The data also suggest that some of the chromosomal changes that we and others have considered to be gaps, are actually true deletions. This is important because many of the changes produced by chemicals in cultured mammalian cells are of this type.

Variations in radiosensitivity during the cell cycle in a marsupial cell line

A permanent marsupial cell line, with 96% diploidy, of the male Potorous tridactylus (2n = 13) was irradiated in different stages of the cell cycle. The results for first-division postirradiation show that cells in G 2 had more chromatid deletions and more total damage than cells in late G 1 and all parts of S. The unusual length of S has enabled a more detailed examination of this stage of the cell cycle. The number on total aberrations decreased with progress through S. Contrary to other reports on animal cells, the frequency of isochromatid deletions was found to vary considerably in S and G 2. The number of isochromatid aberrations in early S was at least twice the number found in late S for 4 different series of irradiation experiments. It was also shown that isochromatid deletions respond to a fractionated dose as the accepted two-hit aberrations do. These data suggest that isochromatid deletions may, in part at least, originate from two-hit events. This finding can be interpreted as lending support to Revell's 21,22 contention that all chromatid aberrations result from incomplete exchanges and should be regarded as multi-hit phenomena.

Changes in chromosome structure induced by radiations: a test of the two chief hypotheses.

Two hypotheses have been advanced to suggest how radiation may produce chromosome aberrations. The classical “breakage-first” hypothesis due to Sax1 and others (reviewed in ref. 2) is that the initial lesion is a break in the chromosome. The two ends produced may then (1) rejoin in the original configuration (restitution); (2) rejoin “illegitimately” with other ends produced by another break to form an exchange; or (3) remain unjoined to form a terminal deletion. Revell's exchange hypothesis3 is that all chromatid aberrations are exchanges produced by the interaction of two lesions (not breaks). The aberrations involving only one chromosome are produced only at the point of overlap where the chromosome is looped back on itself. The two proposals differ principally in that the exchange hypothesis proposes (1) that the initial lesion is not a break, (2) that all aberrations are exchanges involving two lesions, and (3) that all aberrations except exchanges between different chromosomes are formed at the point of overlap where the chromosome is in a loop. Chromatid deletions, which are unrejoined breaks on the breakage-first hypothesis, in Revell's hypothesis result from the failure to complete an exchange at the point of overlap of a loop in the way known to occur frequently when exchanges are produced between chromosomes. Attempts to distinguish between these two hypotheses have centred on the relative frequencies of the different kinds of aberrations and on the dose response relationships. Results have been equivocal: some of the data do not agree with the simple statements of either the breakage-first4–7 or the exchange hypotheses8–12. We report here a more direct test of the differences between the two hypotheses. We have not explored the nature of the initial lesion, but have shown that many terminal deletions are actually incomplete exchanges. The data agree both qualitatively and quantitatively with Revell's exchange hypothesis.

On the biological effectiveness of intermediate neutrons: Induction of chromosomal aberrations

Seeds of Nigella damascena and excised embryos of barley were irradiated with neutrons of maximum energies 507, 250 and 76 keV produced by (p,n) reaction with a thick Li target, and with somewhat moderated fission neutrons in the R-1 reactor, Stockholm. Neutrons of the lower energy were found to induce chromosome aberrations (minutes, dicentrics and rings, observed in the first metaphase in roots after onset of germination) with a reduced effectiveness compared to 507 keV and fission neutrons. This reduced RBE at lower neutron energy is suggested to be due to a shorter track length in the sense of N eary et al. rather than to a changed LET. In Nigella simple chromosome breaks were induced at an increasing effectiveness with decreasing neutron energy. This relatively rare aberration type (not determinable in barley) is possibly a consequence of incomplete exchanges. Elastic nuclear collisions are not expected to play a role in the effects observed. Further approaches to study the action of neutrons at the still lower energies where this mechanism of energy dissipation predominates are discussed.

The exchange hypothesis and chromosome-type aberrations

Regression analyses of the dose-response curves of radiation-induced chromosome aberrations in blood cells of Wallabia bicolor (2 n = 11) show that the kinetics of yield of terminal deletions, as well as classical two-lesion aberrations (rings, dicentrics, and intercalary deletions), best fit the model, Y = a + cD 2 . In addition, all aberrations, including terminal deletions, respond to a fractionated dose in the way expected of two-hit aberrations. These observations suggest that terminal deletions originate from two-hit events. It is proposed that they result from incomplete exchanges, in accord with Revell's 18–21 explanation of the origin of chromatid deletions, rather than from single breaks as proposed on the “breakage first” 28 model.