Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Direct analysis of the clastogenic effect of formaldehyde in unstimulated human lymphocytes by means of the premature chromosome condensation technique

The cytogenetic effect of formaldehyde (FA) on unstimulated human lymphocytes was studied by means of conventional chromosome analysis and the premature chromosome condensation (PCC) technique. In first post-treatment metaphases no significantly increased yields of chromosomal changes could be observed. The analysis of PCCs, however, showed high yields of chromosome fragments. Bleomycin (BLM) used as positive control was also highly clastogenic in PCCs and resulted in significantly increased yields of chromosome-type aberrations. As recently argued, a premitotic selection against heavily damaged cells could be an explanation for the discrepancy between the chromosome findings in metaphase and PCC analysis after FA treatment. In addition, a differential effectiveness may exist in unstimulated lymphocytes to convert multiple fragmentation into chromatid- or chromosome-type aberrations through S-phase-dependent or S-phase-independent mechanisms.     

Chromosome aberration measurements in mitotic and G<Subscript>2</Subscript>-PCC lymphocytes at the standard sampling time of 48 h underestimate the effectiveness of high-LET particles

The relationship between heavy-ion-induced cell cycle delay and the time-course of aberrations in first-cycle metaphases or prematurely condensed G₂-cells (G₂-PCC) was investigated. Lymphocytes of the same donor were irradiated with X-rays or various charged particles (carbon, iron, xenon, and chromium) covering an LET range of 2–3,160 keV/μm. Chromosome aberrations were measured in samples collected at 48, 60, 72, and 84 h postirradiation. Linear-quadratic functions were fitted to the data, and the fit parameters α and β were determined. At any sampling time, α values derived from G₂-cells were higher than those from metaphases. The α value derived from metaphase analysis at 48 h increased with LET, reached a maximum around 155 keV/μm, and decreased with a further rise in LET. At the later time-points, higher α values were estimated for particles with LET > 30 keV/μm. Estimates of α values from G₂-cells showed a similar LET dependence, yet the time-dependent increase was less pronounced. Altogether, our data demonstrate that heavily damaged lymphocytes suffer a prolonged G₂-arrest that is clearly LET dependent. For this very reason, the standard analysis of aberrations in metaphase cells 48 h postirradiation will considerably underestimate the effectiveness of high-LET radiation. Scoring of aberrations in G₂-PCC at 48 h as suggested by several authors will result in higher aberration yields. However, when particles with a very high-LET value (LET > 150 keV/μm) are applied, still a fraction of multiple damaged cells escape detection by G₂-analysis 48 h postirradiation.     

The kinetics of postirradiation chromatin restitution as revealed by chromosome aberrations detected by premature chromosome condensation and fluorescence in situ hybridization

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.     

Genetic damage induced by in vitro irradiation of human G0 lymphocytes with low-energy protons (28 keV/microm): HPRT mutations and chromosome aberrations

Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G0 phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/microm) in the dose range 0.125-2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0-1.5 Gy, the alpha parameters of the curves were 0.42/Gy and 3.6 x 10(-6) mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (alpha = 0.62 aberrations per cell Gy(-1)). By irradiating G0 lymphocytes from a single subject with 28 keV/microm protons and gamma rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Biodosimetry estimate for high-LET irradiation

The purpose of this paper is to prepare for an easy and reliable biodosimeter protocol for radiation accidents involving high-linear energy transfer (LET) exposure. Human peripheral blood lymphocytes were irradiated using carbon ions (LET: 34.6 keV μm⁻¹), and the chromosome aberrations induced were analyzed using both a conventional colcemid block method and a calyculin A induced premature chromosome condensation (PCC) method. At a lower dose range (0–4 Gy), the measured dicentric (dics) and centric ring chromosomes (cRings) provided reasonable dose information. At higher doses (8 Gy), however, the frequency of dics and cRings was not suitable for dose estimation. Instead, we found that the number of Giemsa-stained drug-induced G2 prematurely condensed chromosomes (G2-PCC) can be used for dose estimation, since the total chromosome number (including fragments) was linearly correlated with radiation dose (r = 0.99). The ratio of the longest and the shortest chromosome length of the drug-induced G2-PCCs increased with radiation dose in a linear-quadratic manner (r = 0.96), which indicates that this ratio can also be used to estimate radiation doses. Obviously, it is easier to establish the dose response curve using the PCC technique than using the conventional metaphase chromosome method. It is assumed that combining the ratio of the longest and the shortest chromosome length with analysis of the total chromosome number might be a valuable tool for rapid and precise dose estimation for victims of radiation accidents.     

Analysis of X-ray induced aberrations in mammalian chromosomes by electrofusion induced premature chromosome condensation

Summary Premature chromosome condensation (PCC) was induced by electrofusion of metaphase cells of an Ehrlich ascites tumor cell line with interphase cells of a Muntjac cell line or of a Chinese Hamster subline. Electrofusion was performed by cell alignment in a weakly inhomogeneous a.c. field of 200 V/cm amplitude (peak-to-peak value) and of 1.7 MHz frequency, followed by the application of a series of breakdown (fusion) pulses of 5 kV/cm strength and 15 µs duration. Most of the PCC's were of the G2 type despite the large proportion of G1 and S cells in the suspension. The number of chromatid aberrations observed in electrofused cells which had not been subjected to irradiation was not significantly above the spontaneous level. This indicates that electrofusion, at least as used here, did not lead to lesions expressed as structural aberrations. When interphase cells were irradiated by X-ray doses below 3 Gy before electrofusion PCC analysis showed chromosome damage consisting mainly of breaks and gaps. The frequency of aberrations recorded by PCC was 6 to 40 fold larger than that seen in conventional metaphase analysis. This large increase probably arose because of an effective suppression of the G2 repair of chromosomal lesions by the fast condensation process which took place within about 30 min. This assumption was supported by PCC experiments in which the time between X-irradiation and fusion with subsequent chromosome condensation was varied. The results demonstrated that G2 repair of chromosomal lesions was not detectable until 20 min after fusion with a half-time of the repair kinetics of about 1.5 h. The selectivity of premature chromosome condensation in G2 cells is discussed in terms of the differences between electrofusion and chemically or virally induced fusion. It is assumed that the concentration and the transfer rate of the chromosome condensation factor from the metaphase to the interphase cell are the limiting factors in achieving PCC. This is because the localised permeabilisation of the membrane and the dominance of two-cell fusions are characteristic of electrofusion.     

Chromosome aberrations in the blood lymphocytes of astronauts after space flight.

Cytogenetic analysis of the lymphocytes of astronauts provides a direct measurement of space radiation damage in vivo, which takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. Chromosome exchanges were measured in the blood lymphocytes of eight crew members after their respective space missions, using fluorescence in situ hybridization (FISH) with chromosome painting probes. Significant increases in aberrations were observed after the long-duration missions. The in vivo dose was derived from the frequencies of translocations and total exchanges using calibration curves determined before flight, and the RBE was estimated by comparison with individually measured physical absorbed doses. The values for average RBE were compared to the average quality factor (Q) from direct measurements of the lineal energy spectra using a tissue-equivalent proportional counter (TEPC) and radiation transport codes. The ratio of aberrations identified as complex was slightly higher after flight, which is thought to be an indication of exposure to high-LET radiation. To determine whether the frequency of complex aberrations measured in metaphase spreads after exposure to high-LET radiation was influenced by a cell cycle delay, chromosome damage was analyzed in prematurely condensed chromosome samples collected from two crew members before and after a short-duration mission. The frequency of complex exchanges after flight was higher in prematurely condensed chromosomes than in metaphase cells for one crew member.     

Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH)

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

Induction of chromosome aberrations and micronuclei in human peripheral blood lymphocytes at low dose of radiation

The chromosome damage induced by the doses of y-irradiation 6)Co in peripheral blood lymphocytes was studied using different cytogenetic assays. Isolated lymphocytes were exposed to 0.01-1.0 Gy, stimulated by PHA, and analysed for chromosome aberrations at 48 h postirradiation by metaphase method, at 49 h--by the anaphase method, at 58 h by micronucleus assay with cytochalasin B and, additionally, micronuclei were counted at 48 h on the slides prepared for the metaphase analysis without cytochalasin B. Despite of the quantitative differences in the amount of chromosome damage revealed by different methods all of them demonstrated complex nonlinear dose dependence of the frequency of aberrant cells and aberrations. At the dose range from 0.01 Gy to 0.05-0.07 Gy the cells had the highest radiosensitivity mainly due to chromatid-type aberration induction. With dose increasing the frequency of the aberrant cells and aberrations decreased significantly (in some cases to the control level). At the doses up to 0.5-0.7 Gy the dose-effect curves have become linear with the decreased slope compare to initial one (by factor of 5 to 10 for different criteria) reflecting the higher radioresistance of cells. These data confirm the idea that the direct linear extrapolation of high dose effect to low dose range--the procedure routinelly used to estimate genetic risk of low dose irradiation--cannot be effective and may lead to underestimation of chromosome damage produced by low radiation doses. Preferences and disadvantages of used cytogenetic assays and possible mechanisms of low ionising radiation doses action were discussed.     

The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells

The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (⁶⁰Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

High LET heavy ion radiation induces lower numbers of initial chromosome breaks with minimal repair than low LET radiation in normal human cells

We investigated the earliest possible chromosome break and repair process in normal human fibroblasts irradiated with low and high LET (linear energy transfer) heavy ion radiation using the modified premature chromosome condensation (PCC) technique utilizing wortmannin (WM) during the fusion incubation period [M. Okada, S. Saito, R. Okayasu, Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation, Mutat. Res., 562 (2004) 11-17]. The initial numbers of breaks were approximately 10/cell/Gy in X-irradiated samples, followed by carbon (LET: 70 keV/microm), neon, and the number was around 5/cell/Gy in silicon (LET: 70 and 200 keV/microm) and iron (LET: 200 keV/microm) samples. If WM was not used, the initial numbers of breaks with silicon and iron were higher than those of X-rays. To quantify these data, we used initial repair ratio (IRR) defined as the number of G1 PCC breaks with WM divided by the number of breaks without WM. X-irradiation gave the maximum IRR ( approximately 2.0), while iron as well as silicon irradiation showed the minimum IRR ( approximately 1.0), suggesting almost no rejoining at the initial stage. Although there is a comparatively good correlation between the IRR value and the cell survival, the survival fraction with the repair data at 2 or 6h correlates better statistically. Our data indicate that high LET heavy ion irradiation induces a lower number of initial chromosome breaks with minimal repair when compared with low LET irradiation. These results at the chromosome level substantiate and extend the notion that high LET radiation produces complex-type DNA double strand breaks (DSBs).