A.T. Natarajan

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.

Chromosome aberrations: past, present and future

Spontaneous and induced chromosome aberrations have been studied over more than a century. The resolution of detection of aberrations has depended on the improvement of available techniques. An overview on the major high lights in this area of research, from the time of solid staining to fluorescence in situ hybridization technique is presented in this review.

Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe

PURPOSEnTo discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay.nnnMATERIALS AND METHODSnHuman lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes.nnnRESULTSnThe combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%.nnnCONCLUSIONnThe combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.Purpose : To discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay. Materials and methods : Human lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes. Results : The combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%. Conclusion : The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.

Detection of incomplete exchanges and interstitial fragments in X-irradiated human lymphocytes using a telomeric PNA probe

PURPOSEnThe detection of incomplete exchanges and interstitial fragments by fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe.nnnMATERIALS AND METHODSnIsolated human lymphocytes were exposed in vitro to X-rays at a dose of 3 Gy. Aberrations were analysed in the first mitosis after irradiation using a telomeric PNA probe.nnnRESULTSnAfter an acute dose of 3 Gy, only about 16% of the cells contained a pair of incomplete chromosome elements with telomeric signals at only one terminal end. Acentric interstitial fragments, lacking telomeric signals, were observed with a frequency of 0.56 per cell, which was very similar to the dicentric frequency (0.61 per cell).nnnCONCLUSIONSnThe low frequency of incompleteness suggests that most of the non-reciprocal interchanges observed using chromosome painting must originate from terminal exchanges rather than from incomplete exchanges. Furthermore, it was estimated that about 78% of excess acentric fragments originate from interstitial fragments and only 22% from terminal fragments.

Analysis of radiation-induced chromosomal aberrations using telomeric and centromeric PNA probes.

Purpose : To generate dose-response curves for X-ray-induced chromosomal aberrations analysed in human blood lymphocytes using telomeric and centromeric peptide nucleic acid (PNA) probes. Materials and methods : Isolated human lymphocytes were X-irradiated with doses of 0, 1, 2, 3, 4 and 6 Gy. Aberrations were analysed in the first post-irradiation metaphases using telomeric and centromeric PNA probes. Results : Similar to the dose-response curves for the yield of dicentrics and centric rings, the dose-response curves for interstitial fragments and incomplete elements (derived from either terminal deletions or incomplete exchanges) follow a linear-quadratic function. Furthermore, it was estimated that 76% of excess acentric fragments originate from complete exchanges (interstitial deletions) and only 24% from incomplete exchanges or terminal deletions. Conclusions : Interstitial fragments form a major class of radiation-induced chromosomal aberrations. They are induced about half as frequently as dicentrics over the whole dose range investigated. The comparable trend of the dose-response curve for the different aberrations, including incomplete elements, indicates that all detected aberrations are formed by a similar underlying mechanism. It also suggests that the ratio between non- or incomplete repair (leading to open ends of broken chromosomes) and incorrect repair (leading to exchange aberrations) is independent of dose.PURPOSEnTo generate dose-response curves for X-ray-induced chromosomal aberrations analysed in human blood lymphocytes using telomeric and centromeric peptide nucleic acid (PNA) probes.nnnMATERIALS AND METHODSnIsolated human lymphocytes were X-irradiated with doses of 0, 1, 2, 3, 4 and 6 Gy. Aberrations were analysed in the first post-irradiation metaphases using telomeric and centromeric PNA probes.nnnRESULTSnSimilar to the dose-response curves for the yield of dicentrics and centric rings, the dose-response curves for interstitial fragments and incomplete elements (derived from either terminal deletions or incomplete exchanges) follow a linear-quadratic function. Furthermore, it was estimated that 76% of excess acentric fragments originate from complete exchanges (interstitial deletions) and only 24% from incomplete exchanges or terminal deletions.nnnCONCLUSIONSnInterstitial fragments form a major class of radiation-induced chromosomal aberrations. They are induced about half as frequently as dicentrics over the whole dose range investigated. The comparable trend of the dose-response curve for the different aberrations, including incomplete elements, indicates that all detected aberrations are formed by a similar underlying mechanism. It also suggests that the ratio between non- or incomplete repair (leading to open ends of broken chromosomes) and incorrect repair (leading to exchange aberrations) is independent of dose.

Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation

Whole blood samples from two healthy donors were cultured in the presence of 5-bromo-2-deoxyuridine (BrdU) for a total of 107 h following in vitro X-irradiation with a dose of 2 Gy. Starting from 35 h after culture initiation, every subsequent 12 h a sample was taken from each culture and grown in the presence of demecolcine for another 12 h. At each sampling time, the aberrations involving chromosomes 1 and 4 were analysed using dual-colour fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. Following differential staining of sister chromatids, the analysed cells were identified to be either in their first, second or third etc. mitosis after irradiation. Cells within the same postirradiation division contained higher frequencies of aberrations when derived from later sampling times, indicating a delay in progression of aberrant cells to mitosis. In contrast, when the aberration frequencies are calculated by sampling time (i.e. independent of the cell cycle) minimal effect of sampling time could be seen. This observation held true for all types of chromosomal aberrations. Analysis of about 2250 first-division cells for each donor (derived from all sampling times) indicates a relative overrepresentation of chromosome 4 in the formation of exchange aberrations/colour junctions. Whereas dicentric frequencies for chromosomes 1 and 4 were close to the expected values based on the DNA content of these chromosomes, frequencies of reciprocal translocations showed a clear overinvolvement of chromosome 4. This resulted in a distinct difference in the reciprocal translocation to dicentric ratio, being 1.12 for chromosome 1 and 2.09 for chromosome 4. These results indicate a non-DNA-proportional distribution of radiation-induced chromosome rearrangements in cultured human lymphocytes.

Formation of DNA adducts and induction of mutagenic effects in rats following 4 weeks inhalation exposure to ethylene oxide as a basis for cancer risk assessment.

Ethylene oxide (EO) is mutagenic in various in vitro and in vivo test systems and carcinogenic in rodents. EO forms different adducts upon reaction with DNA, N7-(2-hydroxyethyl)guanine (N7-HEG) being the main adduct. The major objectives of this study were: (a) to determine the formation and persistence of N7-HEG adducts in liver DNA of adult male rats exposed to 0, 50, 100 and 200 ppm by inhalation (4 weeks, 5 days/week, 6 h/day) and (b) to assess dose-response relationships for Hprt gene mutations and various types of chromosomal changes in splenic lymphocytes.N7-HEG adducts were measured 5, 21, 35 and 49 days after cessation of exposure. By extrapolation, the mean concentrations of N7-HEG immediately after cessation of exposure (day 0) to 50, 100 and 200 ppm were calculated as 310, 558 and 1202 adducts/10(8) nucleotides, respectively, while the mean concentration in control rats was 2.6 adducts/10(8) nucleotides. At 49 days, N7-HEG values had returned close to background levels. The mean levels of N-(2-hydroxyethylvaline) adducts in haemoglobin were also determined and amounted 61.7, 114 and 247 nmol/g globin, respectively. Statistically significant linear relationships were found between mean N7-HEG levels (day 0) and Hprt mutant frequencies at expression times 21/22 and 49/50 days and between mean N7-HEG (day 0) and sister-chromatid exchanges (SCEs) or high frequency cells (HFC) measured 5 days post-exposure. At day 21 post-exposure, SCEs and HFCs in-part persisted and were significantly correlated with persistent N7-HEG adducts. No statistically significant dose effect relationships were observed for induction of micronuclei, nor for chromosome breaks or translocations. In conclusion, this study indicates that following sub-chronic exposure, EO is only weakly mutagenic in adult rats. Using the data of this study to predict cancer risk in man resulting from low level EO exposures in conjunction with other published data, i.e., those on (a) genotoxic effects of EO in humans and rats, (b) DNA binding of other carcinogens, (c) natural background DNA binding and (d) genotoxic potency of low energy transfer (LET) radiation, it is not expected that long term occupational exposure to airborne concentrations of EO at or below 1 ppm EO produces an unacceptable increased risk in man.

Impact of radiation quality on the spectrum of induced chromosome exchange aberrations.

Purpose: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation. Materials and methods: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes. Results: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations. Conclusions: Despite the reduced rejoining eyciency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.

Translocation analysis by the FISH-painting method for retrospective dose reconstruction in individuals exposed to ionizing radiation 10 years after exposure.

Fluorescence in situ hybridization (FISH) is a powerful method largely used for detecting chromosomal rearrangements, translocations in particular, which are important biomarkers for dose assessment in case of human exposure to ionizing radiation. To test the possibility of using the translocation analysis by FISH-painting method in retrospective dose assessment, we carried out in vitro experiments in irradiated human lymphocytes, in parallel with the analysis of translocations in lymphocytes from 10 individuals, who were exposed to 137cesium in the Goiânia (Brazil) accident (samples collected 10 years after exposure). The in vitro dose-response curve for the genomic translocation frequencies (FGs) fits a linear quadratic model, according to the equation: Y=0.0243X(2)+0.0556X. The FG values were also calculated for the individuals exposed to 137cesium, ranging from 0.58 to 5.91 per 100 cells, and the doses were estimated and compared with the results obtained by dicentric analysis soon after the accident, taking the opportunity to test the validity of translocation analysis in retrospective biodosimetry. A tentative of retrospective dosimetry was performed, indicating that the method is feasible only for low level exposure (below 0.5Gy), while for higher doses there is a need to apply appropriate correction factors, which take into consideration mainly the persistence of chromosomal translocations along with time, and the influence of endogenous and exogenous factors determining the inter-individual variability in the cellular responses to radiation.

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.