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Occupational exposure in copper smelters may produce various adverse health effects including cancer which, according to available epidemiologic data, is associated mainly with exposure to arsenic. Despite a number of well-documented studies reporting an increased risk of cancer among copper smelters workers, the data on genotoxic effects in this industry are scarce. In view of the above, an assessment of micronuclei (MN) frequency in peripheral blood lymphocytes and buccal epithelial cells from copper smelter workers was undertaken. Additionally, the clastogenic/aneugenic effect in lymphocytes was assessed with the fluorescence in situ hybridization (FISH). The study was conducted in three copper smelters in southwestern Poland. The subjects (n = 72) were enrolled among male workers at departments where As concentration in the air was up to at 80 microg/m(3). Exposure was assessed by measurement of arsenic concentration in urine and toenail samples. The control group (n = 83) was recruited from healthy male individuals living in central Poland who did not report any exposure to known genotoxins. The results of our study showed a significant increase in MN frequency in peripheral blood lymphocytes and in buccal epithelial cells of smelter workers, compared to the controls (7.96 +/- 4.28 vs. 3.47 +/- 1.70 and 0.98 +/- 0.76 vs. 0.50 +/- 0.52, respectively). The FISH technique revealed the presence of clastogenic and aneugenic effects in peripheral blood lymphocytes in both groups. The clastogenic effect was slightly more pronounced in the smelter workers; however, the difference was not statistically significant. The mean arsenic concentrations in urine (total arsenic species) and in toenail samples in the exposed group were 54.04 +/- 42.26 microg/l and 7.63 +/- 7.24 microg/g, respectively, being significantly different from control group 11.01 +/- 10.84 microg/l and 0.51 +/- 0.05 microg/g. No correlation between As content in urine or toenail samples and the genotoxic effect was found under study.

Nucleoid sedimentation, single-cell gel electrophoresis (comet assay) and premature chromosome condensation (PCC) technique were utilized to estimate the involvement of DNA strand breaks and chromosomal damage in radio-adaptive response of stimulated human lymphocytes. Conditioning of cells with 0.02 Gy X-rays rendered them more resistant to single- and double-strand DNA breaks produced by 1 Gy challenging treatment as revealed by the sedimentation behaviour of the nucleoids and the comet assay. Nucleoid sedimentation also demonstrated that adaptive reaction towards X-ray-induced DNA damage is favoured in the presence of oxygen. A concomitant decrease in the amount of interphase chromosomal breaks visualized by PCC under the same experimental conditions was observed. Data indicate that adaptation of human lymphocytes to X-rays is tightly linked to the reduced susceptibility towards generation of DNA and chromosomal breaks. It is proposed that the very persistence of DNA strand discontinuities might serve as a triggering signal for the adaptation of human lymphocytes against ionizing radiation exposure.

The aim of the present study was to investigate whether chromosome 16p presents breakpoint regions susceptible to radiation-induced rearrangements. The frequencies of translocations were determined by fluorescence in situ hybridization (FISH) using cosmid probes C40 and C55 mapping on chromosome 16p, and a chromosome 16 centromere-specific probe (pHUR195). Peripheral lymphocytes were collected from normal individuals and from seven victims of 137Cs in the Goiania (Brasil) accident (absorbed doses: 0.8-4.6 Gy) 10 years after exposure. In vitro irradiated lymphocytes (3 Gy) were also analyzed. The mean translocation frequency/cell obtained for the 137Cs exposed individuals was 2.4-fold higher than the control value (3.6 x 10(-3) +/- 0.001), and the in vitro irradiated lymphocytes showed a seven-fold increase. The genomic translocation frequencies (FGs) were calculated by the formula Fp = 2.05 fp(1-fp)FG (Lucas et al., 1992). For the irradiated lymphocytes and victims of 137Cs, the FGs calculated on the basis of chromosome 16 were 2- to 8-fold higher than those for chromosomes 1, 4 and 12. Our results indicate that chromosome 16 is more prone to radiation-induced chromosome breaks, and demonstrate a non-random distribution of induced aberrations. This information is valuable for retrospective biological dosimetry in case of human exposure to radiation, since the estimates of absorbed doses are calculated by determining the translocation frequency for a sub-set of chromosomes, and the results are extrapolated to the whole genome, assuming a random distribution of induced aberrations. Furthermore, the demonstration of breakpoints on 16p is compatible with the reports about their involvement in neoplasias., (Copyright 2005 S. Karger AG, Basel.)

Purpose: To determine the frequency of true incomplete chromosome exchanges in human lymphocytes after exposure to high-LET neutrons using chromosome painting in combination with centromeric and telomeric probes in one FISH assay., Materials and Methods: Human lymphocytes were exposed in vitro to 1 MeV neutrons at a dose of 1 Gy (dose-rate 0.1Gy x min(-1)). Chromosome aberrations were analysed in the first mitosis after irradiation using a FISH technique that combined whole chromosome-specific DNA probes (for chromosomes 4 and 8), human pan-centromeric DNA and telomeric PNA probes., Results: The frequency of true incomplete exchanges induced by 1 MeV neutron irradiation was <5% in chromosomes 4 and 8. Comparison of the frequency of true incompleteness obtained in the present experiment with a previous study that used 4 Gy X-rays showed no striking differences between X-rays and neutrons in incomplete exchange patterns but differences in the spectrum of induced aberrations were detected. Simple exchanges were more frequent with X-rays, whereas complex types were significantly commoner following neutron irradiation (41 and 23% respectively). Differences were also found for complex rearrangements: both the number of these and their complexity increased after neutron-irradiation., Conclusion: The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The application of telomeric probes to analyse chromosome aberrations has demonstrated that true incompleteness is a rare event (approximately 5%) following exposure to high-(neutron) as well as to low-(X-rays) LET radiation.

Purpose: To study the cytogenetic effects of fractionated radiotherapy in peripheral blood lymphocytes of five cancer patients. In vitro experiments were performed in parallel using the same dose range and a comparison was made of the induced frequencies of stable and unstable chromosome aberrations. The object was to clarify the use of an in vitro calibration curve for immediate and retrospective dosimetry in cases of radiation accidents., Materials and Methods: Patients were exposed to 60Co gamma-rays at a single dose of 11.5 cGy each day up to a total dose of 57.5 cGy, given in 5 days. For measurement of chromosome aberrations, blood was collected from patients before irradiation and after each exposure. Blood taken before treatment was used as a control and for in vitro irradiation experiments in the dose range 8-50 cGy. Chromosome aberration frequency (stable as well as unstable) was determined using fluorescence in situ hybridization (FISH) assay with specific DNA libraries for chromosomes 1, 4 and 8 and a pancentromertic probe for the whole genome. Giemsa-stained preparations were used to score unstable aberrations following in vivo and in vitro exposure., Results: A linear dose-response curve was determined for both dicentrics and translocations. The in vivo frequency of translocations was higher than for dicentrics. Dose-response curves generated for translocations following in vivo and in vitro irradiation yielded similar frequencies. In contrast, for dicentrics, in vitro irradiation yielded a higher frequency when compared with data generated following in vivo exposure., Conclusions: For dose reconstruction purposes, translocations frequency seems to be a more adequate end-point than the scoring of dicentrics. The established in vitro calibration curve for dicentrics may underestimate absorbed radiation dose in cases of protracted exposure.

Frequencies of chromosomal translocations in human peripheral blood lymphocytes irradiated in vitro by 200 kV X rays have been estimated by the fluorescence in situ hybridisation (FISH) technique. Probes specific for whole chromosomes 1, 3 and 4 were labelled with, biotin 16 dUTP + digoxigenin 11 dUTP; biotin 16 dUTP and digoxigenin 11 dUTP respectively, to achieve three colour FISH to distinguish exchanges involving individual chromosomes. The translocation frequencies for individual chromosomes suggested a relationship between the response and the genomic content of the chromosomes studied. Dose-response curves derived from translocations involving all the three painted chromosomes (22.3% of the genome) showed a linear-quadratic response with alpha and beta coefficients of 0.027 +/- 0.009 Gy-1 and 0.033 +/- 0.004 Gy-2, corresponding coefficients for the response curves extrapolated to the entire genome are 0.072 +/- 0.026 Gy-1 and 0.075 +/- 0.011 Gy-2 respectively. Dose-response curves for the induction of dicentrics involving painted chromosomes also showed a similar response, but the dicentric frequencies were consistently lower than the translocation frequencies for all the doses studied. Dose fractionation resulted in a small but significant reduction in the yield of exchanges, thus suggesting repair of some radiation damage during the period between the two fractions. Retrospective biological dosimetry was attempted with FISH assay of five radiation workers with chronically accumulated doses of approximately 500 mSv received over a period of 2-3 decades. Among the 2421 metaphases scored in these subjects 28 translocations involving the painted chromosomes were detected. The dose estimates based on FISH assay ranged from 0.215 +/- 0.116 Gy to 0.635 +/- 0.292 mGy, thus suggesting the usefulness of this assay in retrospective biological dosimetry.

Purpose: Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes., Methods and Materials: Unstimulated G0 lymphocytes were pretreated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique., Results: The results show that sodium butyrate pretreatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G0 lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations., Conclusions: Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB.

Blood samples were irradiated in vitro with alpha-rays emitted from short-lived radon decay products dissolved in the culture medium at doses between 0.03 and 41.4 mGy. The data were collected from experiments conducted during the period 1984-1992 and comprise a total of about 64000 scored metaphases. For statistical reasons, only 60,022 metaphases were used for the subsequent analysis. The results for total chromosome aberrations and dicentrics indicate a linear dose dependence in the dose range above about 10 mGy, consistent with other experimental observations. At doses below about 10 mGy, aberration frequencies cannot be linearly extrapolated from higher doses, suggesting that there is no dependence on dose within a certain low-dose range. In addition, a statistically significant minimum has been observed at a dose of about 0.03 mGy, which is consistently lower than the related control values. The behavior of the aberration frequencies in the low-dose region seems to be influenced by the control values, which also depend on the environmental radiation burdens to the donors before blood sampling and thus were significantly affected by the Chernobyl fallout.

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

The genotoxicity of fluoride in vivo in seven patients with osteoporosis was cytogenetically investigated. The patients were treated with fluoride-containing formulations (disodium monofluorophosphate and sodium fluoride) for a period of 15 months up to 49 months. Fluoride intake ranged from 22.6-33.9 mg F/day and serum fluoride concentrations were between 0.1 mg F/l and 0.2 mg F/l. Peripheral blood lymphocytes of these patients were cultured in vitro and examined for chromosomal aberrations, micronuclei in cytokinesis-blocked binucleated lymphocytes as well as cell cycle progression. When a comparison was made between patients' group and a matched control group, it was found that fluoride at the tested concentrations had no detectable genotoxic potential in human.

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times., (Copyright 1998 Elsevier Science B. V.)

Frequencies of chromosomal aberrations were estimated in X-irradiated (0; 0.5; 1; 2; 3 and 4 Gy) human lymphocytes using arm-specific probes for chromosome 1 in combination with a pancentromeric probe in a triple colour FISH procedure. This allowed the construction of dose-response curves for both interchanges as well as interarm intrachanges. We found that 11.7-18.4% of aberrant chromosomes contained interarm intrachanges. Assuming a free interaction of randomly induced exchange breakpoints throughout the whole genome, interarm intrachanges occur about 8.7 times more frequently than expected. The close proximity of the two arms of a chromosome in the interphase nucleus appears to promote the formation of interarm intrachanges. Convincing evidence was obtained, by comparing the frequency of colour junctions that occur between 1p-1q and 1p-3q (the arms 1q and 3q are of similar size). With this approach, we observed 8.8 times more colour junctions between 1p-1q than between 1p-3q, illustrating the importance of proximity in the formation of chromosomal exchange aberrations. At doses higher than 2 Gy, a saturation of simple reciprocal aberrations, both for interchanges as well as for interarm intrachanges was observed. Furthermore, we found that symmetrical and asymmetrical simple interchanges as well as interarm intrachanges occur with equal frequencies in human chromosome 1., (Copyright 1998 Elsevier Science B. V.)

It is not yet resolved as to what type of DNA double strand break repair operates in G0 lymphocytes. We have employed Down syndrome (DS) lymphocytes with three copies of chromosome #21 to answer the question whether the presence of three copies reduces the frequency of exchange aberrations involving this chromosome in comparison to normal cells with two copies of #21. Peripheral blood lymphocytes from three DS patients and two normal individuals were X-irradiated with 1 and 3 Gy. The frequencies of unstable aberrations were found to be higher in DS lymphocytes than normal lymphocytes after 3 Gy of X-rays. FISH studies employing chromosome specific DNA libraries for chromosomes #21 and #22 indicated that the frequencies of exchange aberrations per chromosome are similar in both disomic and trisomic condition. This indicates that the presence of an extra copy of chromosome #21 does not alter the yield, suggesting that homologous recombination does not play a major role in the repair of DNA strand breaks in human G0 lymphocytes., (Copyright 1998 Elsevier Science B.V.)

Purpose: The detection of incomplete exchanges and interstitial fragments by fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe., Materials and Methods: Isolated 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., Results: After 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)., Conclusions: The 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.

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.

Frequencies of spontaneously occurring and X-ray induced, stable and unstable types of chromosome aberrations in peripheral blood lymphocytes from two groups of radiosensitive patients, i.e., aplastic anemia (AA) and Diamond-Blackfan anemia (DBA), were determined. Two types of staining methods, i.e., chromosome painting with two cocktails of chromosome-specific DNA libraries (Nos. 1, 3, X and Nos. 2, 4 and 8), as well as conventional Giemsa staining, were employed. Chromosome painting was done with single and multicolor fluorescence in situ hybridization (FISH). The frequencies of spontaneously occurring chromosome aberrations in AA and DBA patients were not significantly different from healthy individuals. Hypersensitivity to X-rays was seen both in G0 as well as in G2 phase of the cell cycle in lymphocytes from AA and DBA patients, confirming our earlier findings using micronucleus (DBA) and G2 radiosensitivity (AA) assays.

Frequencies of intra-chromosomal exchanges (pericentric inversions and centric rings) and inter-chromosomal exchanges (dicentrics and translocations) in X-irradiated (2.5 Gy) human lymphocytes have been estimated. To detect these events we employed FISH (fluorescence in situ hybridization) technique and arm specific painting probes for chromosomes #1 and #3. The ratio between centric rings and pericentric inversions was found to be about 1. For intra-changes to inter-changes, the ratio (F) was between 6 and 9. Based on the total number of colour junctions involving chromosomes #1 and #3 it was found that exchanges between the arms of the same chromosome occur about 8.7 times more than inter-chromosomal exchanges calculated on the basis of the DNA content of the chromosomes and random induction of aberrations in the total genome. Chromosomal organization in interphase nucleus appears to promote the formation of more intra-changes than inter-changes following X-irradiation, most probably due to close proximity of the two arms of a chromosome.

In human hepatoma (Hep G2) cells and peripheral blood lymphocytes (HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization (FISH) assays were applied to study aneugenic and clastogenic potentials of X-rays, directly and indirectly acting chemicals. Induction of MN was studied in vitro following treatment with X-rays, directly acting chemicals, such as methylmeth-anesulphonate (MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastine sulphate (VBS), and indirectly acting agents, such as cyclophosphamide (CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on the presence of the fluorescent signal in the MN following FISH with a human DNA centromeric probe, MN in the binucleated Hep G2 cells and lymphocytes were scored as centromere-positive or centromere-negative, representing an aneugenic and clastogenic event respectively. In the controls approximately 50% of spontaneously occurring MN were centromere-positive. Treatment of human hepatoma cells and HPBL (in vitro) with potent aneugens such as COL, VCS and VBS increased the number of MN in a dose-dependent manner; of these 75-93% were centromere-positive. X-irradiation induced MN in a dose-related manner in binucleated Hep G2 cells and HPBL, of which 33-40% were centromere-positive, which demonstrates the significant aneugenic potentials of X-rays. Strong clastogenic activity was observed with MMS and frequency of centromere-positive MN was low: approximately 20 and 30% for HPBL and Hep G2 cells respectively. In Hep G2 cells significant aneugenic activity was found with indirectly acting promutagens/procarcinogens such as HMPA and 2-AAF, in contrast to CP, which came out as a potent clastogen. The non-carcinogen 4-AAF was not able to induce an increase in the frequency of MN in Hep G2 cells. All indirectly acting chemicals tested came out negative when HPBL were used as targets for DNA damage. The results presented correlate positively with data from in vivo assays and indicate that the Hep G2 cell system is a suitable bioactivation system (in vitro) for evaluating the clastogenic and aneugenic potentials of chemicals which require exogenous metabolic activations in order to exert their mutagenic potential.

Chromosomal aberrations in human lymphocytes were analyzed by fluorescence in situ hybridization (FISH) in the first 3 postirradiation (0 and 2 Gy) divisions. Cells were grown in the presence of BrdU, collected at different sampling times (47, 70 and 91 h) and analyzed using an alphoid centromeric probe and PCR amplified DNA libraries for chromosomes 2 and 8. Following differential staining of sister chromatids, the analyzed cells were identified to be either in the first, second or third mitosis after irradiation. The frequencies of both dicentrics and fragments showed a reduction of about 50% after each cell generation, whereas translocations were more persistent. Cells within the same postirradiation division showed higher aberration frequencies when derived from later sampling times, indicating a delay in progression of aberrant cells. As a result, the frequencies for dicentrics and fragments remained rather constant at different sampling times if the cell cycle parameter was not taken into account. Thus, the average generation time of the lymphocytes had a clear effect on the obtained aberration frequencies. The described method allows the study of the persistence of chromosome damage using the FISH technique during 3 subsequent cell divisions in vitro.

The influence of smoking on the spontaneous frequencies of unstable and stable chromosomal aberrations in stimulated lymphocytes using conventional Giemsa staining and fluorescence in situ hybridization (FISH) was studied. The study groups consisted of 12 smokers and 30 nonsmokers. The frequency of translocations was determined using nine different chromosome-specific DNA libraries, representing approximately 50% of the human genome, usually in a combination of two to three chromosomes in one cocktail. Using DNA libraries for specific chromosomes and FISH also made it possible to detect aneuploid cells. No significant difference was observed between smokers and nonsmokers in the frequencies of unstable and stable type of chromosomal aberrations. However, a significantly (P < or = 0.05) higher frequency of hyperploidy was evident in smokers compared with nonsmokers.

Following the radiological accident which occurred in the city of Goiania (Brazil), in September 1987, a cytogenetic follow-up of 15 exposed patients was started, aiming to observe the mean lifetime of lymphocytes containing dicentric and ring aberrations. The results suggest that the disappearance rate of unstable aberrations follows a two-term exponential function. Up to 470 days after exposure, there is a rapid fall in the aberration frequency. After 470 days, the disappearance rate is very slow. These results may reflect different subpopulations of human lymphocytes, with different lifespans. The estimated average half-time of elimination of dicentrics and rings among the highly exposed group (doses above 1 Gy) was 110 days for the initial period after the exposure (up to 470 days). This value is significantly shorter than the usually accepted value of 3 years reported in the literature. Statistical analysis of possible correlations between the individual half-times and biological parameters, such as sex, age, leukopenia level shown during the critical period, absorbed dose (initial frequency of chromosomal aberrations) and the administration of the bone marrow stimulating factor (rHuGM-CSF) was performed. None of these parameters showed a correlation with the half-time of disappearance of chromosomal aberrations. For the individuals who had received less than 1 Gy the disappearance of aberrations was slower, with a half-time of 160 days during the period up to 470 days after exposure. Mean disappearance functions of unstable chromosome aberrations were inferred, to be applied in accident situations in which there is a blood sampling delay.

We have made use of the cytokinesis-block micronucleus method to evaluate the adaptive response in human lymphocytes from two donors conditioned with low doses of hydrogen peroxide before irradiation with either 1.5 or 3.0 Gy of X-rays. A protective effect of pre-exposure to H2O2 against radiation damage detected as micronuclei in binucleate cells was evident in cells allowed to recover for different periods after X-ray exposure, though cells challenged with 3.0 Gy of X-rays showed the adaptive response at later fixation time than those exposed to the lower dose (1.5 Gy). We propose this protocol as an interesting alternative to the single fixation method to score chromosomal aberrations at metaphase for the study of the adaptive response.

This paper presents results of a collaborative experiment between six laboratories which examined the yields of unstable chromosomal aberrations in human lymphocytes induced in vitro by X-rays over the dose range 0-300 mGy. The work included data points of nominal doses of 0, 3, 5, 6, 10, 20, 30, 50 and 300 mGy. Cells from 24 donors were examined and a total of about 300,000 metaphases were scored. The work was undertaken to determine the limits of sensitivity of the system taking into account variations in scoring data due to inter-donor sample and inter-laboratory effects. Despite the existence of these effects, aberration yields significantly in excess of control values were seen at doses greater than 20 mGy and these were consistent with a linear extrapolation from higher doses. Below 20 mGy the observed dicentric yields were generally lower than background, but not significantly so. Excess acentric aberrations, on the other hand, and centric rings, were higher than the controls but the increase was usually not significant. It is concluded that the statistical uncertainties are such that below 20 mGy this technique cannot distinguish between a linear or a threshold model.

The premature chromosome condensation (PCC) technique and conventional chromosome analysis were applied to examine the kinetics of radiation-induced primary chromosome breaks, their rejoining and formation of dicentrics in human peripheral blood lymphocytes (PBLs). Numbers of chromosomal elements and dicentrics per cell were analyzed for each dose. Dose-dependent increases were observed for chromosome fragments (linear) and dicentrics (linear/quadratic). For an assessment of repair kinetics, numbers of breaks and dicentrics were estimated immediately and at several recovery periods after irradiation, using the PCC technique. It was found that chromosome fragments restitute with time, whereas the dicentrics are formed very quickly and their frequency remains the same, despite the decline in the number of chromosome breaks at later recovery times. Fractionation experiments were conducted to study the time-dependent interaction of primary breaks in the formation of dicentric chromosomes. PBLs were irradiated with 3 Gy X-rays split into 2 equal fractions separated by different intervals up to 5 h. No marked difference was observed in the yield of dicentrics following the different fractionation protocols, except that the mean levels of dicentrics declined when the fraction interval was 4 h or more. It appears that most of the dicentrics are formed by misrepair of strand breaks, produced directly by radiation and not resulting from rejoining of existing breaks during the slow repair process which follows. We also studied the role of the chromatin configuration at the time of irradiation on the yield of chromosome fragments and dicentrics. Highly condensed chromatin due to pretreatment with 0.3 M NaCl was found to reduce the frequency of radiation-induced chromosomal aberrations.

An investigation has been carried out to determine whether chromosome aberrations in peripheral blood lymphocytes reflect the elevated environmental dose of low-LET ionising radiation, mainly due to radiocesium from Chernobyl fallout, to the population living in Salzburg city. Sixteen volunteers were sampled 1 year after the Chernobyl accident. Two of these persons were also sampled before the accident, and then in 1988 and 1990. The radioactive environment of Salzburg city and the radiation burden of its inhabitants have been frequently determined before and after the accident. The Cs-137 content of the volunteers was measured by whole-body counting. The additional external plus internal radiation doses in the year 1987 to the tested individuals ranged between 15 and 68% of the former normal environmental burden. The aberration frequencies showed a sharp increase of about a factor 6 from the pre-Chernobyl dose rate (0.9. mGy/year) to the post-Chernobyl dose rate (about 2 mGy/year total) but then decreased again with higher additional dose. In the two persons analysed before and up to 4 years after the accident the aberration yield showed a significant increase from 1984/85 to 1987, a decrease in 1988 and a further decrease in 1990. If these last 2 values are plotted against additional dose they fit the curve of the pooled 1987 values. The dose-effect curves revealed the same tendency as we found in various previous investigations and support the assumption that repair enzymes could be triggered by a certain amount of damage to the DNA.

The presence of micronuclei (MN) in binucleated peripheral blood lymphocytes of 37 testicular carcinoma patients was studied 1.5 to 9.3 years following curative chemotherapy. All patients received cisplatinum, 35 patients also received bleomycin. In addition, most patients were treated with another cytotoxic drug, i.e., vinblastine (n = 23) and/or etoposide (n = 24). The mean time interval between cessation of chemotherapy and the micronucleus assay was 4.6 years. The median frequency of MN in binucleated cells in treated patients (0.059) was significantly higher than that in 12 untreated cancer patients (0.036; P = 0.003) or that in 26 healthy age-matched controls (0.034; P less than 0.001). Frequencies of MN in the 12 untreated cancer patients (including 7 patients with disseminated testicular carcinoma) did not differ from those in the 26 healthy controls (P = 0.890), suggesting that chromosomal damage in lymphocytes of treated testicular cancer patients must be attributed to the chemotherapy. Results indicate that lymphocytes containing chemically induced chromosomal damage persist for up to 9.3 years following cessation of chemotherapy. The implications of these findings with regard to the increased risk for secondary tumors are discussed.

Heparinized whole-blood samples from 4 normal donors were treated with resorcinol 24 h after initiation of cultures. Then, after being cultured for 24 and 48 h, the lymphocytes were fixed and the frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined. The results from two different experiments indicate that, even at a low concentration of 0.002%, resorcinol was capable of increasing the frequency of chromosomal aberrations. Lymphocytes fixed at 48 h after the start of culture had a lower frequency of aberrations induced by resorcinol than those fixed at 72 h. In the SCE test in human lymphocytes, resorcinol was negative. Parallel experiments with Chinese hamster ovary (CHO) cells indicate that resorcinol is not effective in inducing either chromosomal aberrations or SCEs in these cells. The induced chromosomal aberrations were analysed as induced micronuclei in bone marrow of mice; and in a test in vitro with human diploid fibroblasts, resorcinol was also negative.

The yield of radiation-induced micronuclei in human lymphocytes was assessed by two methods, i.e., by incorporating bromodeoxyuridine or by inhibiting cytokinesis by cytochalasin for identification of cells which have undergone one cell division. The cytochalasin block method was found to be more efficient with a capacity to detect between 60 and 90% of the induced fragments. Dose-response characteristics and the results of fractionation experiments indicate that the yield of micronuclei reflects both classes of acentric fragments, i.e., those associated and independent of exchange type of aberrations.

A method suitable for performance of liquid-holding (LH) experiments with human lymphocytes in vitro is described. The lymphocytes are stimulated with concanavalin A (Con A) and after washing off the Con A with a sugar-containing medium are liquid-held in Con A-free medium for different times, after which they are restimulated with Con A. The BUdR-labeling method was used to determine the proportion of first-, second-, and third-division cells. With this system we were able to show that the frequencies of X-ray-induced chromosomal aberrations are not influenced by liquid holding, supporting the idea that these aberrations are fixed shortly after irradiation by misrepair. This sytem may prove useful for investigation of specific problems in mutagenicity research, especially those involving mutagens, which induce delayed formation of chromosome aberrations.

When lymphocytes from human peripheral blood were labeled with In-111 oxinate, several of their properties appeared to be affected. The spontaneous release of the radionuclide was found to be relatively high. Labeled lymphocytes showed a decreased proliferative capacity, dependent on the dose of the label. Cytogenetic studies revealed that In-111 oxinate induces severe chromosomal aberrations. These results emphasize the need for great caution in the use of the In-111 label for studies on lymphocyte traffic in humans.

In a coordinated research programme sponsored by the International Atomic Energy Agency, the frequencies of chromosomal aberrations induced in peripheral blood lymphocytes (in vitro) by 250 kV X-rays at low doses (0.4, 1, 2, 3, 5, 10 and 30 rad) were determined. Blood from 2 donors was used to conduct one master experiment at these dose levels. The culture time used was 48 h and all samples including the controls were processed according to a standard protocol. The coded slides were scored by investigators from 10 participating laboratories. The main results are the following: (1) the frequencies of all types of chromosome aberrations at 0.4 rad are significantly lower than the control values; (2) there is no increase in the frequencies of dicentrics up to 2 rad and in those of terminal deletions up to 5 rad; (3) the mean frequencies of all aberrations considered together are not significantly different from one another at 1, 2 and 3 rad (P = 0.05); and (4) over the entire dose range the dose-effect relationship is clearly non-linear. A fit of these data to a linear quadratic model (E(D) = c + alpha D + beta D2) showed that the observed total aberration frequencies at doses 1, 2, 3 and 5 rad are below the curve defined by the model. The deviations can be explained by an altered kinetics of aberration production at very low doses probably due to DNA repair mechanisms operating these cells.

The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored. It is concluded that using controlled experimentals conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.

Human peripheral blood lymphocytes stimulated in vitro were exposed to low level irradiation ('adaptive dose') from radioisotopes ([3H]dThd, [14C]dThd, HTO and 32P). After 50 h in culture, they were irradiated with 50 rad of X-rays ('challenge dose') and fixed 3 h later. In another series, the lymphocytes received an adaptive dose of 5 rad of X-rays at 32 h after stimulation and a challenge dose of 150 rad at 48 h; the cells were fixed at 54 h. In cells that received both the adaptive and challenge doses, the frequencies of chromosomal aberrations (chromatid and isochromatid deletions) were lower than expected on the basis of additivity of the effects of the individual treatments. These results support those published from Wolff's laboratory in showing that human lymphocytes can become 'adapted' by prior exposure to low level irradiation so that they become less sensitive to the chromosome-breaking effects of X-rays delivered subsequently. The magnitude of reduction in frequencies in the 'adapted' cells, however, varied between the blood samples from different donors.

The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal aberrations were studied in human peripheral blood lymphocytes. Unstimulated (G0) lymphocytes were pre-treated with sodium butyrate (5 mM) for 24 h, X-irradiated and then stimulated to pass through the cell cycle. Cells in their first post-radiation metaphase were scored for chromosomal aberrations. In parallel biochemical experiments nucleoid sedimentation technique was used to examine the induction and repair of DNA-strand breaks. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. We therefore suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G0 lymphocytes.

We have examined lymphocytes from human preterm (PT) and fullterm (FT) babies for an effect of gestational age (GA) on chromosomal aberrations either occurring spontaneously or induced by treatment with X-rays (1 Gy) alone; or with caffeine (1,3,7-trimethylxanthine) supplementation (5 X 10(-4) M), in comparison to the lymphocytes of healthy adults (AD). Percent of abnormal cells (%Abn) was used as an indicator of chromosome sensitivity to the different treatments. PT babies had significantly higher spontaneous and X-ray-induced %Abn values than AD, but were comparable to FT. After X-irradiation + caffeine the yield of aberrations in any 2 groups was not significantly different. Chromosomal sensitivity may result from factors other than GA. This in vitro model may permit study of the mechanisms of chromosomal damage repair and prevention of free radical damage of DNA during the perinatal period.

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.

Arsenic (As) contamination in ground water has affected more than 19 countries. Approximately 36 million people in the Bengal delta alone are exposed to this toxicant via drinking water (>50 µg/l) and are at potential health risk. Chronic ingestion of As via drinking water is associated with occurrence of skin lesions, cancer and other arsenic‐induced diseases in West Bengal, India. An in vitro cytogenetic study was performed utilizing chromosomal aberrations (CA) in lymphocytes treated with sodium arsenite (0–5 µM) in six symptomatic (having arsenic‐related skin lesions) individuals, six age‐ and sex‐matched As‐exposed asymptomatic (no arsenic‐related skin lesions) individuals and six control individuals with similar socio‐economic status residing in non‐affected districts of West Bengal with no evidence of As exposure. The mean As content in nails and hair was 9.61 and 5.23 µg/g in symptomatic, 3.48 and 2.17 µg/g in asymptomatic and 0.42 and 0.33 µg/g in the control individuals, respectively. The main aim of our study was to determine whether genotoxic effects differed in the lymphocytes of the control (no exposure to arsenic), asymptomatic and symptomatic individuals after in vitro treatment with sodium arsenite. Although both the exposed groups had chronic exposure to As through the drinking water, individuals with skin lesions accumulated more As in their nails and hair and excreted less in urine (127.80 versus 164.15 µg/l). The results show that sodium arsenite induced a significantly higher percentage of aberrant cells in the lymphocytes of control individuals than in the lymphocytes of both the exposed groups. Within the two exposed groups As induced higher incidences of CA in the symptomatic than the asymptomatic individuals. These results suggest that asymptomatic individuals have relatively lower sensitivity and susceptibility to induction of genetic damage by As compared with the symptomatic individuals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

It has been shown repeatedly that following irradiation of human lymphocytes in the G0 stage, more translocations are induced than dicentrics. To check the role of DNA double-strand breaks (DSB) alone for the induction of symmetrical and asymmetrical chromosome aberrations, the frequencies of induced exchange aberrations by the restriction enzyme AluI were analyzed. The enzyme was introduced into cells using the pellet pipetting technique. Frequencies of induced translocations and dicentrics were determined using a chromosome painting assay with chromosome-specific DNA libraries for chromosomes 1, 4 and X (representing 16.8% of the human genome). The number of translocations detected was ~3-fold higher than the number of dicentrics, indicating that the increased frequency of translocations compared with dicentrics found in irradiated human lymphocytes does not result from DNA lesions other than DSB but from differential processing of DSB. [ABSTRACT FROM PUBLISHER]