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Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase δ and ϵ, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step. [ABSTRACT FROM AUTHOR]

Werner syndrome (WS) is a rare autosomal disorder characterized by premature aging exhibiting chromosome instability and predisposition to cancer. Cells derived from WS patients show a variety of constitutionally stable chromosomal aberrations as detected by conventional chromosome banding techniques. We have employed the fluorescence in situ hybridization (FISH) technique using painting probes for 12 different chromosomes to detect stable chromosome exchanges in three WS cell lines and three control cell lines. WS cell lines showed increased frequencies of both stable and unstable chromosome aberrations detected by FISH and Giemsa staining, respectively. One WS lymphoblastoid cell line (KO375) had a 5/12 translocation in all the cells and ~60% of the cells had an additional translocated chromosome 12. A high frequency of aneuploid cells was found in all the WS cell lines studied. Though WS cells are known to be chromosomally unstable, unlike other chromosome instability syndromes they are not sensitive to mutagenic agents. We studied the frequencies of X-ray-induced chromosomal aberrations in two WS cell lines and found an ~60% increase in the frequencies of fragments and no consistent increase in the frequencies of exchanges. [ABSTRACT FROM PUBLISHER]

We have tried to characterize the nucleotide excision repair (NER) events associated with the nuclear skeleton in both repair-proficient and repair-deficient human cell lines following UV irradiation. The repair patches were labelled with biotin-16-dUTP and the repair sites were visualized by fluorescence microscopy using fluorescence-conjugated antibodies to biotin. The intensities of repair labelling measured for the three human cell lines of normal, xeroderma pigmentosum group C (XP-C) and Cockayne syndrome group B (CS-B) are in good agreement with their known repair capabilities. Digestion of nuclei with DNase I markedly solubilized the repair patches in normal (3-fold reduction after 1 h post-UV incubation) and transcription-coupled repair (TCR)-defective Cockayne syndrome cells (6-fold reduction after 1 h post-UV incubation). The intensity of repair labelling remained the same in TCR-proficient XP-C cells after DNase I digestion, indicating that the repair events mediated by the TCR pathway are tightly associated with the nuclear skeleton. Treatment with ammonium sulphate after DNase I digestion further reduced the intensity of repair patches in both normal and Cockayne syndrome cells, but not in XP-C cells. The tight association of repair patches generated by the TCR pathway with the nucleoskeleton in XP-C cells reinforces the concept of functional compartmentalization of the nucleus, where NER is highly heterogeneous. [ABSTRACT FROM PUBLISHER]

Chinese hamster primary embryonic cells (at G1 phase) were treated with 1.0 or 3.0 μg/ml bleomycin and chromosomal aberrations in first division metaphases were analysed by fluorescence in situ hybridization (FISH) using arm-specific painting probes for chromosomes 3, 4, 8 and 9. We observed that bleomycin induced all classes of chromosome-type aberrations very efficiently. The interesting findings were: (i) the frequency of induced interstitial translocations (i.e. insertions) was approximately equal to that of reciprocal translocations; (ii) the frequency of induced pericentric inversions was higher than that of centric rings. In our earlier studies, we found that X-rays induced a low frequency of interstitial translocations in comparison with reciprocal translocations and equal frequencies of centric rings and pericentric inversions. These data suggest that bleomycin differs from X-rays with respect to the induction of some specific types of aberrations. The results of a χ2 test examining the hypothesis that formed aberrations among the chromosomes or chromosome arms are randomly distributed on the basis of their relative lengths revealed a differential involvement of these chromosomes in the aberrations following exposure to bleomycin. In general, chromosome 8 was found to be more involved in induced aberrations than expected, chromosome 4 was randomly involved, whereas chromosomes 3 and 9 were less involved. This study demonstrates the utility of arm-specific painting probes for efficient detection of a large variety of chromosomal aberrations induced by bleomycin. [ABSTRACT FROM PUBLISHER]

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]

On April 6, 1993, near the town of Tomsk (Russia) there was an accident at the Siberian Chemical Plant (SCP) which resulted in extensive contamination of an area of 250 km(2) to the north of SCP with long-lived radionuclides such as (239)Pu, (137)Cs and (90)Sr. Cytogenetic methods and electron spin resonance (ESR) spectrometry of tooth enamel were used to estimate the radiation doses received by the population. The ESR signal intensity and the chromosomal aberration frequency in lymphocytes of the tooth donors showed a good correlation. The data showed that 15% of the inhabitants of the Samus settlement received a radiation dose >90 cGy. The exceptions were results of an examination of fishermen, where ESR gave high values (80-210 cGy) but both the chromosome assay and the cytokinesis block micronucleus method gave lower ones (8-52 cGy). A large increase in chromosome damage was observed in people born between 1961 and 1969. It was found that during these years several serious accidents at the Siberian Chemical Plant had occurred causing radiation pollution of the area. The number of cells with chromosome aberrations was significantly less among the people arriving in Samus after 1980. We found good correlations between the level of carotene consumption and a decrease in frequency of both micronuclei in binucleated lymphocytes (r = 0.68, P < 0.01) and chromatid aberrations (r = 0.61, P < 0.01) among the inhabitants. We also examined the inhabitants of Samus for opisthorchis infection, which was present in 30% of the population. The Samus inhabitants affected by Opisthorchis felineus showed significantly increased levels of micronuclei in binucleated lymphocytes and chromatid aberrations as compared with the controls.

The distribution of camptothecin (CPT)-induced break points in late S or G2 phase of the cell cycle observed in Chinese hamster chromosomes was analysed in 400 metaphases. Contrary to expectation, they were not localized in the heterochromatic regions, suggesting that these chromatid-type aberrations arise by a mechanism which does not involve collision of the CPT-trapped 'cleavable complex' with the replication fork. Since many break points mapped more frequently to light bands (DAPI negative) than dark bands (DAPI positive) with a frequency of 73 and 15% respectively, it could be argued that the presence of the CPT-trapped 'cleavable complex' probably interferes with chromatin condensation. In fact, the euchromatic regions, which are expected to be more actively condensed in G2 phase, were more involved in chromosomal damage. These results do not completely confirm the idea that some residual DNA synthesis occurring in G2 is responsible for the G2 clastogenic effects of CPT as the heterochromatic regions should, in this case, be more involved.

Molecular characterization of some cytologically apparent terminal deletions in human tumours revealed that these were subtelomeric cryptic translocations undetectable by classical cytogenetic methods. To determine whether subtelomeric cryptic translocations occur following exposure of mammalian cells to ionizing radiation we used four cell lines exhibiting variable telomere lengths. Our G1 and G2 radiation experiments revealed that a small percentage of broken chromosomes exhibited telomeric signals. This occurred exclusively in cell lines exhibiting FISH-detectable telomeres, suggesting that telomeric signals at radiation-induced chromosome breaks were the result of subtelomeric cryptic translocations. In addition, telomeric signals at G2 chromatid breaks were usually paired with telomeres of intact sister chromatids, further supporting the notion that subtelomeric cryptic translocations are responsible for the presence of telomeric sequences at radiation-induced chromosome breaks. In one of the cell lines we identified what looked like de novo telomeric signals at derived chromatid breaks observed 20 h following irradiation. Our previous study suggested that these signals may be the result of amplification of interstitial telomeric sites in the first cell cycle and spontaneous breakage of interstitial telomeric sites in subsequent cell cycles. Taken together our results suggest that a small percentage of radiation-induced chromosome/chromatid breaks may be modified by subtelomeric cryptic translocations and that interstitial telomeric sites may be involved in radiation-induced chromosome instability.

Frequencies of symmetrical and asymmetrical exchange aberrations induced by two inhibitors of topoisomerase II, namely, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) and etoposide (VP16), were estimated in human peripheral blood lymphocytes. The aberrations were scored using conventional Giemsa staining and fluorescence in situ hybridization (FISH) techniques, using chromosome-specific DNA libraries. Stable aberrations (translocations) were detected using two cocktails of DNA libraries specific for three chromosomes, namely 1, 3 and X and 2, 4 and 8, representing approximately 40% of the whole human genome. The frequencies of dicentrics and translocations increased in a dose-dependent manner, however, m-AMSA was found to be a more potent inducer of chromosomal aberrations in comparison with VP16 (at concentrations at which comparable frequencies of aberrations were induced) by 20- to 30-fold. When corrected for DNA content of chromosomes in each cocktail, a higher frequency of translocations with the cocktail consisting of chromosomes 2, 4 and 8 in comparison with 1, 3 and X was evident. The genomic translocation frequency calculated from chromosome painting analysis for m-AMSA exceeded that estimated for dicentrics by approximately 2-fold. However, for VP16 almost equal frequencies of both types of chromosome exchange were found.

Transgenic mice with foreign DNA inserted into three pairs of chromosomes were exposed to 2 Gy X-rays in order to study the induction and persistence of chromosome malsegregation and aneuploidy up to 28 days after exposure. By tracing the marker chromosomes in cytokinesis-blocked binucleated splenocytes using fluorescence in situ hybridization (FISH), reciprocal products of chromosome malsegregation in the daughter nuclei were analysed. FISH with murine minor satellite DNA was employed to detect chromosome loss (MN with a centromere) in binucleated splenocytes. In addition to its clastogenic effects, X-irradiation also showed aneugenic activity, which was observed as centromere positive micronuclei (C + MN) and malsegregated marker chromosomes detected by FISH. The initial frequency of micronuclei (MN) analysed by Acridine Orange staining immediately after X-ray exposure was found to be 42.3 per 100 binucleated cells. The MN frequency declined in an exponential manner and at day 14, reached about half the value observed immediately after irradiation and 14% MN were detected at day 28. Of these MN, 25% were centromere positive at day 0 as detected by minor satellite signal after FISH. The percentage C + MN increased further at day 3 and declined at day 14 to the level observed at day 0. There were 7.6% malsegregated cells immediately after X-irradiation as analysed by two colour FISH. This value increased further during later intervals and remained stable until day 28. A combination of the Acridine Orange staining and FISH with minor satellite DNA and marker DNA to detect aneuploidy and chromosome malsegregation, was utilized in the present study to demonstrate the induction and persistence of aneugenic and clastogenic damage in transgenic mice irradiated in vivo.

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.

X-ray induced chromosomal translocations were studied in mouse spermatogonial stem cells by meiotic analysis at the spermatocyte stage many cell generations after induction. Using a composite DNA probe for mouse chromosomes 1, 11 and 13, in combination with fluorescence in situ hybridization, the involvement of these three chromosomes in translocation formation was recorded. The obtained results at various sampling times ranging from 75 to 320 days after irradiation show no significant differences in the participation pattern of the painted chromosomes in multivalent formation with increasing sampling time. Pooling of the data at the different dose levels of 5 and 7 Gy indicated significant overrepresentation of the specifically stained bivalents in translocation formation. This suggests that clonal proliferation cannot be held responsible for the observed non-randomness. Experiments with the DNA-repair inhibitor 3-aminobenzamide showed no change in the recorded overrepresentation, indicating that it is probably not the repair of lesions that is causing this phenomenon but rather non-random induction.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.

The cytogenetic effects of mimosine, a naturally occurring plant amino acid known to arrest cell-cycle progression at the G1-S border in cultured cells, have been studied. It was found that mimosine inhibits the cell-cycle progression in a dose-dependent manner in primary and transformed Chinese hamster fibroblasts as well as primary lymphocytes and transformed lymphoblastoid cells of human origin. In the Chinese hamster fibroblast cells, the first division metaphases analysed were found to be highly damaged or pulverized. The damaged cells which could pass through the next cell division, showed very high frequencies of sister chromatid exchanges (SCEs) compared with untreated second division cells. No such cytogenetic alterations could be detected in the human cells. The absence of clastogenic effect in cells of lymphoid origin appears to be related to the known capacity of these cells to undergo apoptosis, thereby efficiently eliminating cells with high frequencies of chromosomal aberrations. Our study demonstrates the clastogenic potency of mimosine and suggests the need for a careful interpretation of the results while using mimosine for cellular or molecular studies pertaining to cell cycle events.

Normal Chinese hamster primary fibroblasts were exposed to methotrexate, an antifolate agent known to induce fragile sites. In a total of 156 banded metaphases prepared from methotrexate-treated cells, 93 breaks and gaps were observed and mapped to the individual hamster chromosomes and their bands. About two-thirds (69%) of all breaks and gaps mapped to the band Xq21, which is significantly more than expected. Some other bands exhibited fragility but the effect was not significant. In 6% of cells, homozygous fragility of Xq21 was observed, suggesting that this is a common fragile site. It has been reported previously that various chemicals induce preferential localized fragility of Xq in Chinese hamster ovary (CHO) and V79 cell lines. Our results suggest that preferential fragility of Xq in the presence of chemical compounds represents examples of fragile-site induction. A tendency of the in vitro chromosomal aberration test to yield positive results in the absence of positive data in other test systems, such as the Ames test and the mouse bone marrow micronucleus test, might be due to fragile-site expression induced by chemical compounds under study.

The effect of beta-carotene on the frequencies of micronuclei (MN) induced in cytochalasin blocked binucleated Chinese hamster ovary cells (CHO) by a bifunctional alkylating agent mitomycin C (MMC), a monofunctional alkylating agent methyl methanesulphonate (MMS) and a radio-mimetic agent bleomycin (BLEO) was investigated. Four different modes of application of the combination of clastogens and beta-carotene were examined (pre-treatment, simultaneous treatment, pre- +simultaneous treatment and post-treatment). The results obtained showed no effect of beta-carotene on the frequencies of MN induced by MMS, a slight but not statistically significant reduction of MMC-induced MN only when beta-carotene was used in low concentrations (0.25 and 0.5 microM) and a potentiation of the clastogenicity of bleomycin by beta-carotene in three of the treatment regimes utilized, post-treatment being ineffective. On the basis of these results it can be concluded that the effect of beta-carotene on clastogenesis induced by chemicals depends on the type and mechanism of action of the clastogen used as well as the treatment protocol employed.

In the mouse splenocyte assay cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization techniques were applied to study aneuploidy following in vitro/in vivo treatments. MN can be formed by lagging acentric chromosome fragments or whole chromosomes. In order to discriminate MN produced by agents causing chromosome breakage (clastogens) from those arising following treatment with agents causing spindle malfunctioning (aneugens) the mouse centromere satellite DNA probe was used in combination with the fluorescent in situ hybridization technique. MN in mouse splenocytes were induced in vitro by colchicine, vincristine sulphate and vinblastine. For in vivo treatment, mice were injected intraperitoneally with diethylstilbestrol (DES) and hexamethylphosphoramide (HMPA) or whole body X-irradiated. Depending on the presence of the fluorescent signal in the MN following in situ hybridization with centromere-specific probe MN in the binucleated splenocytes were scored as centromere-positive (C+) or centromere-negative (C-). Treatment of mouse splenocytes (in vitro) with potent aneugens such as colchicine, vincristine and vinblastine increased significantly the number of centromere-positive MN (P < 0.001). Following in vivo treatment and in vitro culturing of mouse splenocytes significant (P < 0.001) aneugenic activities were observed with indirectly-acting chemicals such as DES and HMPA. X-irradiation caused a slight increase in the frequency of centromere-positive MN (approximately 20%) in binucleated mouse splenocytes. In addition the Giemsa C-banding technique was used to detect C+ MN and a comparison was made between the mouse centromere specific probe and the conventional C-banding technique to detect centromeres in MN.

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

Recombinant lambda phage DNA containing segments of human O6-methylguanine-DNA transferase gene was employed to localize this gene among the human chromosomes using non-radioactive in situ hybridization technique. This gene was found to be present at the telomeric end, 26q, of the long arm of the chromosome 10.

An established human hepatoma cell strain (Hep G2) was used in micronuclei (MN) and sister chromatid exchange (SCE) assays to evaluate the clastogenic potential of several indirectly-acting mutagenic carcinogens. Benzo[a]pyrene, cyclophosphamide, dimethyl nitrosamine, hexamethylphosphoramide, pyrene and safrole were selected for this study based on the positive and negative results reported with conventional in vitro assays employing rat liver S9 fraction for metabolic activation. Two directly-acting mutagens, methyl methanesulphonate and mitomycin C, were also included in this study. In this system, the human hepatoma cells act as the metabolic activation source as well as the target cell for DNA damage. The results obtained demonstrate that the Hep G2 cells are metabolically competent to activate different classes of mutagens into biologically active metabolites. The non-carcinogen pyrene did not induce any increase in the frequencies of MN and SCE in Hep G2 cells. Furthermore, a good correlation was found between positive and negative data obtained for the tested chemicals in this in vitro assay with literature data obtained in in vivo tests using rodents.

Although aneuploidy is a serious human health problem, the experimental methodology devised until now to study the mechanisms involved in the induction of aneuploidy and for the screening of aneuploidy-inducing agents has not been so much employed to have the necessary validation. A procedure using primary cell cultures of Chinese hamster embryo cells grown on cover glasses is described. To avoid the excessive scattering and subsequent loss of chromosomes, a hypotonic treatment with a 0.17% sodium chloride solution, at room temperature, followed by in situ fixation has been standardized. This procedure improves the method through the reduction of the spontaneous frequency of aneuploid cells. Experiments carried out with cells treated with X-rays, X-rays plus caffeine, and the synthetic estrogen diethylstilbestrol (DES) demonstrated the accuracy of the system since the average chromosome number remained constant in spite of the induction of high frequencies of aneuploid cells. Moreover, the method allows for the analysis of other cytogenetic endpoints such as anaphase-telophase alterations, structural chromosome aberrations or sister chromatid exchanges.