From DNA damage to chromosome aberrations: joining the break.

Despite many years of experimental studies on radiation-induced chromosomal aberrations, and the recent progress in elucidating the molecular mechanisms of the DNA damage response, the link between DNA double-strand break repair and its expression as microscopically visible chromosomal rearrangements remains, in many ways, obscure. Some long standing controversies have partially been resolved to the satisfaction of most investigators, including the linearity of the dose-response for DNA double-strand break induction, the necessity of pairwise interaction of radiogenic damaged sites in the formation of exchange aberrations, and the importance of proximity between lesions in misrejoining. However, the contribution of different molecular DNA repair mechanisms (e.g., alternative end-joining pathways) and their impact on the kinetics of aberration formation is still unclear, as is the definition of "complex" radiogenic damaged sites - in either the chemical or spatial sense - which ostensibly lead to chromosome rearrangements. These topics have been recently debated by molecular biologists and cytogeneticists, whose opinions are summarized in this paper.

Chromosomal aberrations in peripheral blood lymphocytes exposed to a mixed beam of low energy neutrons and gamma radiation.

Cells exposed to thermal neutrons are simultaneously damaged by radiations with high and low linear energy transfer (LET). A question relevant for the assessment of risk of exposure to a mixed beam is whether the biological effect of both radiation types is additive or synergistic. The aim of the present investigation was to calculate whether the high and low LET components of a thermal neutron field interact when damaging cells. Human peripheral blood lymphocytes were exposed to neutrons from the HB11 beam at the Institute for Energy and Transport, Petten, Netherlands, in a 37 °C water phantom at varying depths, where the mix of high and low LET beam components differs. Chromosomal aberrations were analysed and the relative biological effectiveness (RBE) values as well as the expected contributions of protons and photons to the aberration yield were calculated based on a dose response of aberrations in lymphocytes exposed to (60)Co gamma radiation. The RBE for 10 dicentrics per 100 cells was 3 for mixed beam and 7.2 for protons. For 20 dicentrics per 100 cells the respective values were 2.4 and 5.8. Within the limitations of the experimental setup the results indicate that for this endpoint there is no synergism between the high and low LET radiations.

DNA double strand breaks and chromosomal aberrations.

DNA double strand breaks (DSBs) are ultimate lesions for the formation of chromosomal aberrations (CAs). The formation of CAs is dependent on many factors; some of these are discussed in this review. FISH methodologies have uncovered CA types which cannot be seen with the classical staining methods, and thereby widened our understanding of the origin of CAs. The mobility of DSBs in interphase nuclei is limited. This makes it especially difficult to understand the origin of complex CAs involving many chromosomes. Even using high-resolution mBAND FISH to analyze CAs, the ratio of inter-/intrachromosomal CAs is higher than 1. From this it was postulated that only a subset of DSBs, namely, complex or clustered DSBs give rise mainly to interchromosomal CAs. The finding that endonucleases induce CAs does not fit the idea of complex DSBs being responsible for CA. Probably it is the proximity and not the complexity of DSBs which leads to CA.

The cyto- and genotoxicity of organotin compounds is dependent on the cellular uptake capability.

Organotin compounds have been widely used as stabilizers and anti-fouling agents with the result that they are ubiquitously distributed in the environment. Organotins accumulate in the food chain and potential effects on human health are disquieting. It is not known as yet whether cell surface adsorption or accumulation within the cell, or indeed both is a prerequisite for the toxicity of organotin compounds. In this study, the alkylated tin derivatives monomethyltin trichloride (MMT), dimethyltin dichloride (DMT), trimethyltin chloride (TMT) and tetramethyltin (TetraMT) were investigated for cyto- and genotoxic effects in CHO-9 cells in relation to the cellular uptake. To identify genotoxic effects, induction of micronuclei (MN), chromosome aberrations (CA) and sister chromatid exchanges (SCE) were analyzed and the nuclear division index (NDI) was calculated. The cellular uptake was assessed using ICP-MS analysis. The toxicity of the tin compounds was also evaluated after forced uptake by electroporation. Our results show that uptake of the organotin compounds was generally low but dose-dependent. Only weak genotoxic effects were observed after exposure of cells to DMT and TMT. MMT and TetraMT were negative in the test systems. After forced uptake by electroporation MMT, DMT and TMT induced significant DNA damage at non-cytotoxic concentrations. The results presented here indicate a considerable toxicological potential of some organotin species but demonstrate clearly that the toxicity is modulated by the cellular uptake capability.

Space radiation does not induce a significant increase of intrachromosomal exchanges in astronauts' lymphocytes.

Chromosome aberration analysis in astronauts has been used to provide direct, biologically motivated estimates of equivalent doses and risk associated to cosmic radiation exposure during space flight. However, the past studies concentrated on measurements of dicentrics and translocations, while chromosome intrachanges (inversions) have never been measured in astronauts' samples. Recent data reported in the literature suggest that densely ionizing radiation can induce a large fraction of intrachanges, thus leading to the suspicion that interchanges grossly underestimate the cosmic radiation-induced cytogenetic damage in astronauts. We have analyzed peripheral blood lymphocytes from 11 astronauts involved in short- or long-term space flights in low-Earth orbit using high-resolution multicolor banding to assess the frequency of intrachromosomal exchanges in both pre- and post-flight samples. We did not detect any inversions in chromosome 5 from a total of 2800 cells in astronauts' blood. In addition, no complex type exchanges were found in a total of 3590 astronauts' lymphocytes analyzed by multifluor fluorescence in situ hybridisation. We conclude that, within the statistical power of this study, the analysis of interchanges for biological dosimetry in astronauts does not significantly underestimate the space radiation-induced cytogenetic damage, and complex-type exchanges or intrachanges have limited practical use for biodosimetry at very low doses.

Chromosomal intrachanges induced by swift iron ions.

We measured the induction of structural aberrations in human chromosome 5 induced by iron ions using the novel technique of multicolor banding in situ hybridization (mBAND). Human lymphocytes isolated from whole blood were exposed in vitro to 500 MeV/n (LET=200 keV/micrometers, doses 1 or 4 Gy) Fe nuclei at the HIMAC accelerator in Chiba (Japan). Chromosomes were prematurely condensed by calyculin A after 48 h in culture and slides were painted by mBAND. We found a frequency of 0.11 and 0.57 residual breakpoints per chromosome 5 after 1 and 4 Gy Fe-ions, respectively. Inter-chromosomal exchanges were the prevalent aberration type measured at both doses, followed by terminal deletions, and by intra-chromosomal exchanges. Among intra-chromosomal exchanges, intra-arm events were more frequent than inter-arm, but a significant number of intra-changes was associated to inter-changes involving the same chromosome after 4 Gy of iron ions. These events show that the complexity of chromosomal exchanges induced by heavy ions can be higher than expected by previous FISH studies.

Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells.

Humans are exposed to arsenic and their organic derivatives, which are widely distributed in the environment, via food, water, and to a lesser extent, via air. Following uptake, inorganic arsenic undergoes biotransformation to mono- and dimethylated metabolites. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In the present study, the genotoxic effects and the cellular uptake of inorganic arsenic [arsenate, As(i)(V); arsenite, As(i)(III)] and the methylated arsenic species monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], trimethylarsenic oxide [TMAO(V)] were investigated in Chinese hamster ovary (CHO-9) cells. The chemicals were applied at different concentrations (0.1 microM to 10 mM) for 30 min and 1 h, respectively. Cytotoxic effects were investigated by the trypan blue extrusion test and genotoxic effects by the assessment of micronucleus (MN) induction, chromosome aberrations (CA), and sister chromatid exchanges (SCE). Intracellular arsenic concentrations were determined by ICP-MS techniques. Our results show that MMA(III) and DMA(III) induce cytotoxic and genotoxic effects to a greater extent than MMA(V) or DMA(V). Viability was significantly decreased after incubation (1 h) of the cells with > or = 1 microM As(i)(III), > or = 1 microM As(i)(V), > or = 500 microM MMA(III), > or = 100 microM MMA(V), and 500 microM DMA(V) and > or = 0.1 microM DMA(III). TMAO(V) was not cytotoxic at concentrations up to 10 mM. A significant increase of the number of MN, CA and SCE was found for DMA(III) and MMA(III). As(i)(III + V) induced CA and SCE but no MN. TMAO(V), MMA(V) and DMA(V) were not genotoxic in the concentration range tested (up to 5 mM). The nuclear division index (NDI) was not affected by any of the tested arsenic compounds after a recovery period of 14 to 35 h. When the uptake of the chemicals was measured by ICP-MS analysis, it was found that only 0.03% MMA(V) and DMA(V), and 2% MMA(III), As(i)(III) and (V) were taken up by the cells. In comparison, 10% of the DMA(III) dose was taken up. The total intracellular concentration of all arsenic compounds increased with increasing arsenic concentrations in the culture medium. Taken together, these data demonstrate that arsenic compounds in the trivalent oxidation state exhibit the strongest genotoxic effects. Trivalent organoarsenic compounds are more membrane permeable than the pentavalent species. The potency of the DNA damage decreases in the order DMA(III) > MMA(III) > As(i)(III and V) > MMA(V) > DMA(V) > TMAO(V). We postulate that the induction of genotoxic effects caused by the methylated arsenic species is primarily dependent upon their ability to penetrate cell membranes.

Distribution of breakpoints and fragment sizes in human chromosome 5 after heavy-ion bombardment.

PURPOSE: To measure the location of heavy ion-induced residual breakpoints in human chromosome 5 and the size distribution of chromosome fragments involved in inter- or intrachromosomal exchanges. MATERIALS AND METHODS: Human peripheral blood lymphocytes were exposed to 4 Gy accelerated 56Fe (iron) ions (500 MeV per nucleon (MeV n(-1)), linear energy transfer=200 keV microm(-1)). Cells were then stimulated to grow in vitro for 48 h, and chromosomes were prematurely condensed by calyculin A. Chromosome 5 was painted using high-resolution multicolour banding. The location of the observed residual breakpoints and the size of all chromosome 5 fragments involved in structural aberrations were measured using dedicated image analysis software. RESULTS: Mapping of 283 breakpoints revealed a slight deviation from randomness, with an excess of breakpoints clustered in two small bands and an under representation of breaks at the telomeric end in the q-arm. Breakpoints per unit length were similar in p- and q-arms. The distribution of chromosome fragments has a maximum for very small fragments (< 10% of the chromosome size), indicating a severe fragmentation of chromosome 5 after heavy-ion bombardment. Only fragments < 40% of the chromosome size were involved in intrachromosomal exchanges (interstitial deletions or inversions), whereas fragments up to 75% of the whole chromosome 5 were found in interchromosomal exchanges. CONCLUSIONS: Residual breakpoints after exposure to high-energy iron ions were not distributed randomly along chromosome 5, although the p- and q-arms displayed similar radiosensitivity. Large fragments are either restituted or misrejoined to other chromosome ends, whereas small intrachromosomal fragments can produce either inter- or intrachromosomal exchanges.

Pathways of DNA double-strand break repair and their impact on the prevention and formation of chromosomal aberrations.

DNA double-strand breaks (DSB) are considered the critical primary lesion in the formation of chromosomal aberrations (CA). DSB occur spontaneously during the cell cycle and are induced by a variety of exogenous agents such as ionising radiation. To combat this potentially lethal damage, two related repair pathways, namely homologous recombination (HR) and non-homologous DNA end joining (NHEJ), have evolved, both of which are well conserved from bacteria to humans. Depending on the pathway used, the underlying mechanisms are capable of eliminating DSB without alterations to the original genomic sequence (error-free) but also may induce small scale mutations (base pair substitutions, deletions and/or insertions) and gross CA (error-prone). In this paper, we review the major pathways of DSB-repair, the proteins involved therein and their impact on the prevention of CA formation and carcinogenesis.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

Complex chromosomal rearrangements induced in vivo by heavy ions.

It has been suggested that the ratio complex/simple exchanges can be used as a biomarker of exposure to high-LET radiation. We tested this hypothesis in vivo, by considering data from several studies that measured complex exchanges in peripheral blood from humans exposed to mixed fields of low- and high-LET radiation. In particular, we studied data from astronauts involved in long-term missions in low-Earth-orbit, and uterus cancer patients treated with accelerated carbon ions. Data from two studies of chromosomal aberrations in astronauts used blood samples obtained before and after space flight, and a third study used blood samples from patients before and after radiotherapy course. Similar methods were used in each study, where lymphocytes were stimulated to grow in vitro, and collected after incubation in either colcemid or calyculin A. Slides were painted with whole-chromosome DNA fluorescent probes (FISH), and complex and simple chromosome exchanges in the painted genome were classified separately. Complex-type exchanges were observed at low frequencies in control subjects, and in our test subjects before the treatment. No statistically significant increase in the yield of complex-type exchanges was induced by the space flight. Radiation therapy induced a high fraction of complex exchanges, but no significant differences could be detected between patients treated with accelerated carbon ions or X-rays. Complex chromosomal rearrangements do not represent a practical biomarker of radiation quality in our test subjects.

Insights into the mechanisms of sister chromatid exchange formation.

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies.

Chromosomal aberrations in uranium and coal miners.

PURPOSE: To compare chromosomal aberrations in peripheral lymphocytes of Wismut uranium miners (WUM) and Ruhr coal miners (RCM). MATERIALS AND METHODS: Peripheral lymphocytes from 66 WUM and 29 RCM were cultured and analysed for structural chromosomal aberrations in Giemsa-stained M1 metaphases. Cytogenetic data from 23 male white-collar workers from public services were used as a historical control group. RESULTS: The frequencies of chromosomal aberrations and sister chromatid exchanges in WUM and RCM were quite similar. Compared with public services workers, WUM and RCM had significantly higher frequencies of chromosomal aberrations. CONCLUSIONS: Chromosomal aberrations in WUM are not induced by radioactive particles inhaled during underground mining but as in RCM rather result from factors such as age, lifestyle, illnesses, medications and diagnostic irradiations.

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome.

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

Biological dosimetry in Russian and Italian astronauts.

Large uncertainties are associated with estimates of equivalent dose and cancer risk for crews of long-term space missions. Biological dosimetry in astronauts is emerging as a useful technique to compare predictions based on quality factors and risk coefficients with actual measurements of biological damage in-flight. In the present study, chromosomal aberrations were analyzed in one Italian and eight Russian cosmonauts following missions of different duration on the MIR and the international space station (ISS). We used the technique of fluorescence in situ hybridization (FISH) to visualize translocations in chromosomes 1 and 2. In some cases, an increase in chromosome damage was observed after flight, but no correlation could be found between chromosome damage and flight history, in terms of number of flights at the time of sampling, duration in space and extra-vehicular activity. Blood samples from one of the cosmonauts were exposed in vitro to 6 MeV X-rays both before and after the flight. An enhancement in radiosensitivity induced by the spaceflight was observed.

CABAND: Classification of aberrations in multicolor banded chromosomes.

A system is presented to describe aberrations in chromosomes painted with the mBAND methodology. The CABAND (classification of aberrations in multicolor banded chromosomes) system is based on the fact that only banded parts of aberrations and not DAPI stained parts can be described accurately.

Chromosome aberration dosimetry in cosmonauts after single or multiple space flights.

BACKGROUND AND AIMS: Cosmic radiation is one of the main hazards for manned space exploration. Uncertainty in radiation risk estimates for crews of long-term missions are very high, and direct biological measurements are necessary. We measured chromosomal aberrations in peripheral blood lymphocytes from 33 cosmonauts involved in space missions during the past 11 years. METHODS: Blood lymphocytes from the cosmonauts were stimulated to grow in vitro and were harvested at their first mitosis. Slides were either stained with Giemsa stain for dicentrics analysis, or painted with whole-chromosome DNA probes for translocation analysis (FISH). RESULTS: A statistically significant increase in the yield of chromosomal aberrations was measured following long-term space missions in lymphocytes from cosmonauts at their first flight. No significant changes in aberration frequencies were observed for short-term taxi flights. The increase in long-term missions was consistent with the values calculated from physical dosimetry data. However, for cosmonauts involved in two or more space flights, the yield of interchromosomal exchanges was not related to the total duration of space sojourn or integral absorbed dose. Indeed, the yield of aberrations at the end of the last mission was generally in the range of background frequencies measured before the first mission. CONCLUSIONS: Chromosome aberration dosimetry can detect radiation damage during space flight, and biological measurements support the current risk estimates for space radiation exposure. However, for cosmonauts involved in multiple space missions the frequency of chromosomal aberrations is lower than expected, suggesting that the effects of repeated space flights on this particular endpoint are not simply additive. Changes in the immune system in microgravity and/or adaptive response to space radiation may explain the apparent increase in radioresistance after multiple space flights.

Correlation of chromosomal aberrations and sister chromatid exchanges in individual CHO cells pre-labelled with BrdU and treated with DNaseI or X-rays.

PURPOSE: To analyse the correlation between chromosomal aberrations and sister-chromatid exchanges (SCE) in cells treated in G1 phase with X-rays or DNaseI. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells were labelled with 5'-bromodeoxyuridine (BrdU) for one round of replication and irradiated in G1 phase with 1.2, 2.4, 3.6 and 4.8 Gy X-rays or treated by electroporation with 1000, 2000, 3000 and 4000 units DNaseI per 800 microl electroporation buffer. Using a computer-aided metaphase relocation system first post-treatment mitoses were analysed for SCE and chromosomal aberrations allowing a precise investigation of correlation between both phenomena. RESULTS: A better correlation between aberrations and SCE was observed for DNaseI than for X-rays. X-rays induced more SCE than expected on the basis of aberrations, whereas the frequencies of SCE induced by DNaseI can mainly be accounted for by chromosomal aberrations. DISCUSSION: The results obtained with X-rays support an earlier observation that radiation induces both "true" SCE that are not related to chromosomal aberrations and result from radiation damage to BrdU, and "false" SCE that result from exchange-type aberrations. The high frequency of aberrations observed in cells treated with DNaseI and the good correlation between aberrations and SCE suggests that the endonuclease induces mainly "false" SCE.

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells.

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.

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.