John R.K. Savage

Chromosomal aberrations: formation, identification and distribution

Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.

FISH "painting" patterns resulting from complex exchanges.

Many of the unusual patterns seen when irradiated chromosomes are FISH painted arise from Complex interchanges. In this paper, we present a method for classifying chromosome-type complex exchanges. Every possible exchange configuration for the first nine complex families has been determined, and for each of these, the expected metaphase pattern forms seen if any one of the participating chromosomes is painted, has been worked out. A total of 65 recognisably different pattern forms emerge, and a simple classification of these is provided for scoring and discussion purposes. 47 of these patterns are unique (Tell-tale), in that they are found in, and are diagnostic for, only one complex family or sub-family (of the sub-set analyzed). Analysis of the contingency tables indicates that: (a) The same individual complex exchange can appear in different pattern forms, depending upon which of the participating chromosomes is painted, and the converse-different forms can represent the same exchange. (b) Many different complex exchange configurations, from different families, can give rise to identical pattern forms. (c) If obvious complex forms are at all frequent in a sample, then a lot of the exchanges being scored as simple are not. These observations will have considerable repercussions on the numerical assessment of damage when using painted chromosomes at higher radiation doses.

Frequency and distribution studies of asymmetrical versus symmetrical chromosome aberrations

Two aspects of the relationship between Asymmetrical (A) and Symmetrical (S) radiation-induced chromosomal aberrations are considered in this paper. (1) Are A and S truly alternative modes of lesion interactions? Relative frequencies for chromatid-type and chromosome-type are examined, and new lymphocyte data using banding is used to look at this, and also for parallelism in chromosome participation of the two forms for various aberration categories. All the tests applied suggest that A and S are alternative interaction modes. (2) The long-term survival characteristics of A and S are discussed, and the differences in expected frequencies of derived S per surviving cell from chromosome-type and chromatid-types are stressed. Since many in vivo tissues have varying mixtures of potential chromatid and chromosome aberration-bearing target cells, ultimate cell survival and derived S frequencies may differ between tissues for the same absorbed dose. An Appendix gives Relative Corrected Lengths (RCL) for chromosomes of the human karyotype which should be used when testing the various exchange aberration categories for random chromosome participation.

On the scoring of FISH-"painted" chromosome-type exchange aberrations

The effectiveness of FISH painting for easy detection of radiation-induced chromosome aberrations lies, principally, in the fact that only part of any exchange event is highlighted. However, this partial detection imposes certain limitations when the technique is applied to fundamental studies with primary aberrations, particularly at higher doses, when Complex exchanges are present. Some of these limitations are discussed in this paper.

A brief survey of aberration origin theories.

The salient points of three currently debated theories for chromosomal aberration origins (the Classic Breakage-and-Reunion theory, the Exchange theory, and the Molecular theory) are outlined, and some comments are made on each in the light of recent research.

The effect of variable G2 duration upon the interpretation of yield-time curves of radiation-induced chromatid aberrations

Abstract A variable G2 + prophase (G2P) transit time markedly influences the precision with which cells observed in a metaphase sample can be assigned to various positions in the compartment G2P, the profile of the yield time curve for radiation-induced chromatid aberrations, and the form of the curve of frequency of tritiated thymidine labelled metaphases (FLM). In hypothetical populations uncomplicated by extended metaphase durations and radiation induced cell perturbation, precision is poor when the parameters of a log-normal transit-time distribution are comparable to those found in experimental populations. To facilitate calculation, a very simple “all or nothing” radiosensitivity situation is assumed for G2P, namely that observable chromatid aberrations can only be produced in a cell during 10% of its transit of G2P, and not otherwise. When the position of this sensitive region is fixed, any alteration in transit-time distribution parameters modifies the profiles of both the yield-time aberration and FLM curves. Changes in the yield-time curve profile are even more dramatic if the position of the sensitive region in G2P is altered, whilst other parameters are held constant. We conclude that (1) neither the peak aberration yield, nor the integrated area under the yield-time curve are valid measures of radiation effect; (2) the profile of the yield-time curve does not reflect, in any simple manner, the pattern of intrinsic radiosensitivity throughout G2P; (3) the position of any observed aberration peak in relation to the known modal G2P duration is seldom reliable as an indication of the sensitive region location; (4) the true form of the transit-time distribution cannot be derived from the ascending limb of the FLM curve. Consideration is given to real populations where colchicine-extended metaphase, and treatment-induced cell perturbation produce substantial changes in the transit-time distribution, thus aggravating the already complex problem of interpretation.

X-ray-induced simple, pseudosimple and complex exchanges involving two distinctly painted chromosomes

PURPOSEnTo detect simple, pseudosimple and complex chromosome exchanges in X-ray-induced aberrations involving two distinctly painted chromosomes. Each visibly complex two-paint exchange was analysed to determine the number of breaks and chromosomes necessary to derive the pattern. In addition, the number of associated paint junctions was scored to assess the frequency of non-reciprocal exchanges.nnnMATERIALS AND METHODSnMetaphase spreads were prepared from a human primary fibroblast cell line irradiated with 2, 4 and 6 Gy 250kV X-rays. FISH-painting was performed with distinctly labelled probes for chromosomes 1 and 2, and a pancentromeric probe.nnnRESULTSnFrom a total of 78 two-paint exchanges observed, 35 were apparently simple, with no additional counterstain chromatin, and 43 were visibly complex with two-colour painting, of which 23 contained at least one pseudosimple exchange. A detailed analysis of the number of two-paint colour junctions showed that at least 50% of the visibly complex exchange patterns involved non-reciprocal exchanges. The simple and complex exchange dose-response curves were considered to be linear and curvilinear respectively.nnnCONCLUSIONnThe frequency of non-reciprocal rejoining events within complex exchanges is consistent with an interaction model based on the free exchange of multiple break-ends. In addition, the simple and complex exchanges have distinct dose-response curves, in agreement with previous data for single-painted exchanges corrected for pseudosimples.

Analysis of lymphocytes from uranium mineworkers in Namibia for chromosomal damage using fluorescence in situ hybridization (FISH).

Workers in the open pit uranium mine in Namibia appear to suffer from health problems including malignant diseases at a much higher prevalence when compared with the general population. The objective of the present study was to determine whether long-term exposure to low-dose uranium increases the risk of biological radiation damage which could lead to malignant diseases. In order to investigate this risk, we measured the relative frequency of chromosome alterations using Fluorescence in situ hybridization (FISH). A representative cohort of 11 non-smoking miners, were compared to a control group of 9 individuals with no occupational history in mining. We determined a significant increase in chromosome aberrations in the circulating lymphocytes of miners versus the non-smoking controls (p = 0.0000096). Therefore, we concluded that these uranium exposed miners are at an increased risk to acquire genetic damage, which may be associated with an increased risk for malignant transformation.

The transmission of FISH-painted patterns derived from complex chromosome exchanges.

The first 26 families of complex chromosome-type exchanges (from three breaks in two chromosomes to five breaks in five chromosomes) have been evaluated, and the 15,060 exchanges resulting from unrestricted restitution or rejoining of the break ends yields 203 distinctive patterns, if a single participating chromosome is FISH-painted. If we assume that any exchange that produces an acentric fragment of any sort (compound-terminal or interstitial) will be ultimately eliminated in a continuously dividing cell population, then, irrespective of family origin, only 17 of these patterns (approximately 8%) will be transmitted, long-term. The 17 are illustrated, and the implications briefly discussed.

Subdivision of S-phase and its use for comparative purposes in cultured human cells

Intranuclear DNA synthesis and concomitant chromosome duplication occur during a discrete period of the cell cycle termed S-phase. Using replication-banding and serial time sampling in asynchronous cell populations, it is possible to subdivide the S-phase into four or five chronological compartments termed subphases. This paper discusses methods for analysing the sampling data to obtain the average duration of these subphases and the positions within S of the borders between them. Such information not only allows a more detailed analysis of the cell cycle, but also provides parameters which can be used for rigorous comparisons of cell populations from different sources and experimental conditions. Examples are given of application of the method to normal and chromosomally abnormal primary human fibroblasts and lymphocytes growing in short-term in vitro culture.