Chromosome breakpoint distribution of damage induced in peripheral blood lymphocytes by densely ionizing radiation.

PURPOSE: To assess the chromosomal breakpoint distribution in human peripheral blood lymphocytes (PBL) after exposure to a low dose of high linear energy transfer (LET) alpha-particles using the technique of multiplex fluorescence in situ hybridization (m-FISH). MATERIALS AND METHODS: Separated PBL were exposed in G0 to 0.5 Gy 238Pu alpha-particles, stimulated to divide and harvested approximately 48 - 50 hours after exposure. Metaphase cells were assayed by m-FISH and chromosome breaks identified. The observed distribution of breaks were then compared with expected distributions of breaks, calculated on the assumption that the distribution of breaks is random with regard to either chromosome volume or chromosome surface area. RESULTS: More breaks than expected were observed on chromosomes 2 and 11, however no particular region of either chromosome was identified as significantly contributing to this over-representation. The identification of hot or cold chromosome regions (pter,p,cen,q,qter) varied depending on whether the data were compared according to chromosome volume or surface area. CONCLUSIONS: A deviation from randomness in chromosome breakpoint distribution was observed, and this was greatest when data were compared according to the relative surface area of each individual chromosome (or region). The identification of breaks by m-FISH (i.e., more efficient observation of interchanges than intrachanges) and importance of territorial boundaries on interchange formation are thought to contribute to these differences. The significance of the observed non-random distribution of breaks on chromosomes 2 and 11 in relation to chromatin organization is unclear.

Increased complexity of radiation-induced chromosome aberrations consistent with a mechanism of sequential formation.

Complex chromosome aberrations (any exchange involving three or more breaks in two or more chromosomes) are effectively induced in peripheral blood lymphocytes (PBL) after exposure to low doses (mostly single particles) of densely ionising high-linear energy transfer (LET) alpha-particle radiation. The complexity, when observed by multiplex fluorescence in situ hybridisation (m-FISH), shows that commonly four but up to eight different chromosomes can be involved in each rearrangement. Given the territorial organisation of chromosomes in interphase and that only a very small fraction of the nucleus is irradiated by each alpha-particle traversal, the aim of this study is to address how aberrations of such complexity can be formed. To do this, we applied theoretical "cycle" analyses using m-FISH paint detail of PBL in their first cell division after exposure to high-LET alpha-particles. In brief, "cycle" analysis deconstructs the aberration "observed" by m-FISH to make predictions as to how it could have been formed in interphase. We propose from this that individual high-LET alpha-particle-induced complex aberrations may be formed by the misrepair of damaged chromatin in single physical "sites" within the nucleus, where each "site" is consistent with an "area" corresponding to the interface of two to three different chromosome territories. Limited migration of damaged chromatin is "allowed" within this "area". Complex aberrations of increased size, reflecting the path of alpha-particle nuclear intersection, are formed through the sequential linking of these individual sites by the involvement of common chromosomes.

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

PURPOSE: To 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. MATERIALS AND METHODS: Metaphase 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. RESULTS: From 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. CONCLUSION: The 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.

Cancer survival in the USA, 1973-1990: a statistical analysis.

Relative survival rates in the National Cancer Institute (NCI) 'SEER' review of cancer in the USA are fitted by a model which can be used to estimate median survival time in any calendar year. It is argued that median survival times (MSTs) are better indicators of survival than 5-year relative survival rates (RSRs), especially when survival times are short.

An investigation of LET 'finger-prints' in Tradescantia.

Chromosome-type aberration scores, obtained in the Tradescantia microspore system following exposure to radiation, have been extracted from all significant papers since 1940, and augmented with unpublished results from this laboratory. The data include aberrations produced by X-, gamma-rays and neutrons, and cover an enormous range of qualities, doses, dose-rates and ambient gas conditions. Two proposed LET 'finger-print' ratios were examined: Dicentrics/Centric-rings (D/R or F-ratio) and Total deletions/Dicentrics+Centric-rings. There was no significant effect of LET on either of these. Further, D/R was independent of dose, whereas Deletions/D+R showed, as expected, a positive dose effect. In a very small subset of experiments, sizes of interstitial deletions had been recorded, and a significant reduction in modal size was observed for neutrons compared to X-rays. Even if this observation is confirmed by future work, it will not provide a usable 'finger-print' for long-term studies since, being acentric, deletions are rapidly eliminated from a dividing cell population.

Effectiveness of 0.28 keV carbon K ultrasoft X-rays at producing simple and complex chromosome exchanges in human fibroblasts in vitro detected using FISH.

PURPOSE: To study the effects of carbon K ultrasoft X-rays, which produce a single photoelectron with a track length of < 7 nm, on the production of structural chromosome-type changes. MATERIALS AND METHODS: Untransformed human fibroblasts (HF12) were irradiated in G1 phase. Aberrations were analysed using fluorescence in situ hybridization using multi-coloured chromosome specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe. RESULTS: CK X-rays have a high efficiency per unit absorbed dose for producing simple and complex exchanges. Mean absorbed doses of 0.33-1.31 Gy produce simple exchanges with a predominantly linear dose dependency, and visibly complex exchanges increased by more than the power 2 of the dose, with no evidence of a linear component. The proportion of exchanges that are visibly complex ranged from 9% to 46%. CONCLUSIONS: The linear response for simple exchanges provides further support to the hypothesis that damaged DNA may be able to interact with undamaged DNA. The high proportion of complex exchanges may be due to the increased efficiency of double-strand break induction and to the high density of tracks per unit absorbed dose targeting pre-existing sites, some of which may be close to the incident nuclear membrane.

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.

Comment on the ratio of chromosome-type dicentric interchanges to centric rings for track-clustered compared with random breaks.

Evidence has been published suggesting that the ratio of chromosome-type dicentric interchanges to centric rings (D/R or F) is significantly lower for neutrons than for X or gamma rays, and it is proposed that a low D/R could be used as a "fingerprint" for high-LET radiations. One explanation offered for this observation is that the closely spaced, clustered breaks confined to linear tracks will favor intrachanges, as opposed to interchanges, leading to a lower D/R, while the more scattered, random breaks of low-LET radiations will favor the reverse situation and elevate the D/R. We have tested this suggestion empirically by constructing various modeled tracks and grids of breaks which satisfy the proposed conditions. These have then been superimposed, in random orientation, on an array of hexagons, representing a planar section through the interphase arm domains of 14 polarized, metacentric, G1-phase chromosomes, and the D/R computed from the interaction of break clusters with the arms. The ratios recovered were essentially the same for the four different break distributions tested, and we conclude that, for this simple model, the determinant of the D/R is the arm arrangement in the array, rather than the disposition of the breaks.

Retinoblastoma: a model for deriving the mutation rate without using any estimate of the size of the population at risk.

Paediatric cancer of the retinae arises from a transient population of stem cells whose growth and decay are responsible for the development of the fully differentiated photo-receptors and nerve cells. A model is described which fits the data on the incidence of bilateral retinoblastoma and gives the somatic mutation rate without using an estimate of the size of the population at risk. It also gives the shape of the retinoblast growth/decay curve. The model has been tested on two independent sets of data from the USA, and given that both sets are representative of the USA as a whole, there seems to have been little change in the somatic mutation rate over the last 30 or so years. For a total retinoblast formation of 4 x 10(6) cells, the average mutation rate is 2.94 x 10(-7) per cell per year.

A method for comparing DNA replication programmes at the level of the chromosome bands.

During DNA synthesis, each pair of homologous chromosomes replicates its bands in a precise order and at a specific time. When using asynchronous cell populations, this replication programme has to be reconstructed from a series of "stills"--serial samples taken at intervals through S-transit. Obviously, the result obtained is dependent upon the kinetic progression of cells through the cycle, and any perturbation of the cycle. A difficulty arises when we wish to compare the replication programme of a chromosome in two different cultures (e.g. cells from different origins, or after different treatments). Kinetic differences between cultures make it almost impossible to obtain two samples for analysis containing the same "mixture" of cells. Thus, a false programme difference could be introduced, or a real one masked. In this paper, we present a method of comparison that overcomes this problem. It is based upon the observation that with serial sampling of steady-state cell populations through S-phase, band appearance curves are sigmoidal and are very well approximated by cumulative Normal distributions with very similar standard deviations. If a family of such curves, closely spaced in time, is sampled twice, the two observed frequencies for each curve are related, their probits all lying on a single straight line. This line has a slope of 45 degrees and its displacement from the origin is a function of the time interval between the two samples. Given two identical families of such curves, exactly the same relationship will hold if one sample is drawn from each. If, however, the two families differ (in order, spacing, standard deviation etc.), the probit/probit plot will deviate in various ways from a straight line with 45 degrees slope. Any two subsets of chromosome replication-band frequencies can be regarded as derived from a family of cumulative Normal curves and probit/probit comparisons used to test the similarity of their replication programmes.

Replication kinetics of X chromosomes in fibroblasts and lymphocytes.

The kinetics of replication for early and late replicating X chromosomes in karyotypically normal fibroblasts and lymphocytes was studied using terminal bromodeoxyuridine (BrdU) treatment followed by Hoechst/light/Giemsa staining. Although the order of band appearance differs between the two tissues, the programme (order and interval between band appearances) for early replicating bands (dark R-bands) is identical in the two homologues. This is probably also the case for later replicating bands (dark G-bands) though the criteria for determining mean band appearance times are less reliable for these bands when terminal BrdU treatment is used. This means that the late X has a delayed start but thereafter proceeds at the same pace as its early counterpart.

The dosimetry of 224Ra in mouse bone.

Calculations are described, based on experimental findings, which show the variation of absorbed dose from 224Ra in bone marrow of CBA/H mice. These calculations indicate that, following an injection of a leukaemogenic amount of 16 kBq 224Ra into these mice, most marrow cells in the cancellous bone of femur ends are killed but most marrow cells in the femur shaft survive. The calculations also suggest that the mean leukaemogenic absorbed dose of about 1.5 Gy is received by a population of marrow cells about 30 microns from bone surface in the femur shaft.

Risk factors for radiogenic cancer: a comparison of factors derived from the Hanford survey with those recommended by the ICRP.

A model for cancer induction in man exposed to low doses of radiation and based on the analysis of a survey of workers from a nuclear fuel processing plant is examined and compared with that adopted by the ICRP to limit risks to radiation workers. It is shown that claims that ICRP has significantly underestimated the risk apply primarily to those exposed in later life, and arise from assumptions regarding the age dependence of sensitivity to radiation which are questionable. A preliminary attempt is made to test the proposed model using the United Kingdom luminiser population. Deaths from cancers in four tissues believed to be sensitive to radiation induced carcinogenesis are examined and the observed number of deaths in the study population is compared with the number predicted by the model. Taken individually, only one of these sites rejects the model but taken together these four sites, which comprise about half the cancer observed in the luminiser population, provide a conclusive rejection of the model.

Man's sensitivity to bone marrow failure following whole body exposure to low LET ionising radiation: inferences to be drawn from animal experiments.

Evidence concerning the sensitivity of man to bone marrow failure following exposure to brief but substantial doses of ionising radiation is sparse. There is, however, a relatively substantial body of information on such effects in large animals. Reported experiments on six species where exposure to low LET radiation was uniform to the whole body and of brief duration (exposure times of the order of one hour or less) have been reanalysed both in terms of exposure and of midline tissue dose. The results indicate a marked lack of homogeneity among values for LD50 within species thus questioning the applicability of LD50 as a species dependent constant. It is, however, suggested that on a purely empirical basis these large animal data suggest that the dose killing 'most' (where 'most' is between 90 and 95 per cent) is about twice that killing 'few' (where 'few' is between 5 and 10 per cent). For man, where there is evidence that the dose killing few is unlikely to be less than 3 Gy, this relationship might indicate a gradient of mortality with dose between 3 and 6 Gy.

Specific-locus mutation response to unequal, 1 + 9 Gy X-ray fractionations at 24-h and 4-day fraction intervals.

The specific-locus mutation frequency obtained from mouse spermatogonial stem cells following unequal, 1 + 9 Gy X-ray fractionation with a 24-h fractionation interval is low, and consistent with the two fractions acting additively. The response is therefore markedly different from the augmented mutation frequencies obtained with 500 + 500 R and 100 + 500 R, 24-h fractionations. The lower yield compared with the 100 + 500 R response also indicates a clear difference from the translocation data which demonstrate increases in yield with increasing second dose over the same dose range. The decline in specific locus mutation yield with the increase in the second dose from 500 R to 9 Gy suggests that the stem cells surviving the first fraction are heterogeneous in their sensitivities to this class of genetic damage. A similar, additive specific locus mutation frequency is obtained with unequal, 1 + 9 Gy X-irradiation when the interval between fractions is 4 days. This is consistent with 500 + 500 R, 4-day and 7-day interval responses obtained previously but again differs from the sub-additive translocation responses obtained with such X-ray fractionation. Taken together with the data from previous studies the present results suggest that (1) 24 h after the first fraction, (a) the surviving stem cell have two components; survivors of the formerly radiosensitive, cycling component of the normal stem cell population and the formerly radioresistant, G0 or arrested G1 cells, which are being 'triggered' into a rapid cell cycle to achieve repopulation of the testis; (b) these two components are of near-equal sensitivity to translocation induction and cell killing, hence the additive translocation yields with equal X-ray fractionations and yields consistent with those extrapolated from lower doses with higher, unequal fractionations, e.g. 1 + 7 Gy, 1 + 9 Gy; but (c) the formerly radioresistant, triggered component is much more sensitive than the surviving cycling component to specific locus mutation and cell killing, hence the augmented mutation response with 500 + 500 R fractionation and the drop in yield with 1 + 9 Gy compared with 100 + 500 R X-irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The uptake and turnover of 90Sr in the human skeleton.

In the United Kingdom, measurements have been made of the concentration of 90Sr in human bone from 1955 to 1970, and in human diet since 1958. A correlation of these two series of observations has enabled estimates to be made of (i) the fraction of the dietary intake of 90Sr that reaches the skeleton, (ii) the rate of turnover of 90Sr in the skeleton, and (iii) the way in which both these parameters vary with age. The results may be used to predict future levels of 90Sr in human bone from measurements of the radionuclide in diet, and also to calculate the radiation doses received by tissues in bone from intakes of 90Sr and 89Sr.