Fanconi anemia lymphocytes: effect of DL-alpha-tocopherol (Vitamin E) on chromatid breaks and on G2 repair efficiency

The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells. The effect of 100 microM DL-alpha-tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5 mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample. alpha-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%). The efficiency of the G2 repair process (G2 R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2 R rate, both in FA cells and controls. These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells.     

A comparative study of the potentiating effect of caffeine and poly-D-lysine on chromosome damage induced by X-rays in plant cells.

X-Ray-induced chromosomal aberrations (CA) were potentiated by post-treatments in G2 with either caffeine (caff) or poly-D-lysine (PDL) in root-tip cells of Allium cepa. The enhancement of the yield of CA was concomitant with an increase in the frequency of mitosis. Our results seem to support the idea of a direct relationship between radiation-induced G2 delay and repair of chromosome damage. Here we report on similarities between caffeine and PDL in both decreasing G2 delay and enhancing chromatid aberration yield. The possible molecular mechanism(s) of action responsible for the cytogenetic effects observed are discussed.     

Spectrum of chromosomal aberrations in peripheral lymphocytes of hospital workers occupationally exposed to low doses of ionizing radiation

Chromosome aberrations frequency was estimated in peripheral lymphocytes from hospital workers occupationally exposed to low levels of ionizing radiation and controls. Chromosome aberrations yield was analyzed by considering the effects of dose equivalent of ionizing radiation over time, and of confounding factors, such as age, gender and smoking status. Frequencies of aberrant cells and chromosome breaks were higher in exposed workers than in controls (P = 0.007, and P = 0.001, respectively). Seven dicentric aberrations were detected in the exposed group and only three in controls, but the mean frequencies were not significantly different. The dose equivalent to whole body of ionizing radiation (Hwb) did appear to influence the spectrum of chromosomal aberrations when the exposed workers were subdivided by a cut off at 50 mSv. The frequencies of chromosome breaks in both subgroups of workers were significantly higher than in controls (< or =50 mSv, P = 0.041; >50 mSv, P = 0.018). On the other hand, the frequency of chromatid breaks observed in workers with Hwb >50 mSv was significantly higher than in controls (P = 0.015) or workers with Hwb < or =50 mSv (P = 0.046). Regarding the influence of confounding factors on genetic damage, smoking status and female gender seem to influence the increase in chromosome aberration frequencies in the study population. Overall, these results suggested that chromosome breaks might provide a good marker for assessing genetic damage in populations exposed to low levels of ionizing radiation.     

Chromosomal aberrations in lymphocytes of employees in transformer and generator production exposed to electromagnetic fields and mineral oil

The objective was to study the risk of cytogenetic damage among high voltage laboratory workers exposed to electromagnetic fields and mineral oil. This is a cross sectional study of 24 exposed and 24 matched controls in a Norwegian transformer factory. The exposure group included employees in the high voltage laboratory and in the generator soldering department. Electric and magnetic fields and oil mist and vapor were measured. Blood samples were analyzed for chromosomal aberrations in cultured lymphocytes. In addition to conventional cultures, the lymphocytes were also treated with hydroxyurea and caffeine. This procedure inhibits DNA synthesis and repair in vitro, revealing in vivo genotoxic lesions that are repaired during conventional culturing. In conventional cultures, the exposure group and the controls showed similar values for all cytogenetic parameters. In the DNA synthesis- and repair-inhibited cultures, generator welders showed no differences compared to controls. Among high voltage laboratory testers, compared to the controls, the median number of chromatid breaks was doubled (5 vs. 2.5 per 50 cells; P<0.05) the median number of chromosome breaks was 2 vs. 0.5 (P>0.05) and the median number of aberrant cells was 5 vs. 3.5 (P<0.05). Further analysis of the inhibited culture data from this and a previous study indicated that years of exposure and smoking increase the risk of aberrations. We conclude that there was no increase in cytogenetic damage among exposed workers compared to controls in the conventional lymphocyte assay. In inhibited cultures, however, there were indications that electromagnetic fields in combination with mineral oil exposure may produce chromosomal aberrations.     

Effects of caffeine and cycloheximide during G2 prophase in control and X-ray-irradiated human lymphocytes

The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.     

Adaptive response of human lymphocytes for the repair of radon-induced chromosomal damage

In human lymphocytes low doses of X-rays can decrease the number of chromatid deletions induced by subsequent high doses of sparsely ionizing X-rays. Because of the concern with the carcinogenic effects of low doses of -particles from radon in homes, experiments were carried out to see if low doses of X-rays could also decrease the yield of chromosomal aberrations induced by subsequent exposure to radon. Human peripheral blood lymphocytes were irradiated with low doses of X-rays (2 cGy) at 48 h of culture, exposed to radon at 72 h of culture, and analyzed for the presence of chromatid aberrations at subsequent intervals. The frequency of chromatid aberrations induced by radon alone increased with time after exposure, indicating exaggerated differences in the stage sensitivity of cell cycle stages to high-LET radiation. Furthermore, the numbers of aberrations per cell did not follow a Poisson distribution but were over dispersed, as might be expected since high-LET radiations have a high relative biological effectiveness compared with low-LET radiations. Nevertheless, lymphocytes exposed to 2 cGy of X-rays before radon exposure contained approximately one-half the number of chromatid deletions compared with lymphocytes treated with radon alone and analzed at the same time. Thus, the putative chromosomal repair mechanism induced by low doses of sparsely ionizing radiation is also effective in reducing chromosomal aberrations induced by radon, which hitherto had been thought to be relatively independent of repair processes.     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary 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.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Induction of sister chromatid exchanges by UV light and its inhibition by caffeine

Sister chromatid exchanges in Chinese hamster chromosomes were studied after pulse-labeling cells with ³H-thymidine at various concentrations. Whereas the frequency of chromatid aberrations varied widely, depending upon tritium dose, there was no significant change in the sister chromatid exchange frequency, even with a 40-fold range of variation in the tritium concentration in the medium. When cells were exposed immediately after labeling to UV light at 40 erg/mm² and examined at the second mitosis, the frequency of sister chromatid exchanges was found to be 4 times higher than that of the unirradiated controls. A synchronization treatment utilizing 2 mM thymidine also caused a two-fold rise in the exchange frequency above the control level. Furthermore, when synchronized cells were irradiated with UV light at a dose of 40 erg/mm², the exchange frequency exceeded 5 times that of the untreated controls. However, this effect was detectable only when cells were irradiated at the earlier part of the S phase, while no change was detected when irradiated at the late S or G2 phase. A post-treatment of irradiated cells with caffeine caused a remarkable decrease in the frequency of sister chromatid exchanges. On the other hand, the frequency of chromatid aberrations of the deletion type increased strikingly after the same treatment. The results appear to suggest a certain correlation between the mechanism involved in the induction of sister chromatid exchanges and a post-replication repair of DNA damage.     

Chromosomal effects in lymphocytes of 400 kV-substation workers

Summary In a previous study we found an increased rate of chromosomal aberations in substation workers. To follow up this finding we in this study present data from 38 employees of electric power companies; 19 of the subjects worked with the repair and maintenance of circuit breakers and disconnectors in 400 kV-substations. The other 19 served as controls and were only exposed to normal environmental electromagnetic fields. Coded blood samples were sent to a laboratory for determination of the rate of chromosomal aberrations (CA), sister chromatid exchanges (SCE), and cells with micronuclei (MN). Compared to the control group the exposed men displayed a statistically significant increase in CA and cells with MN. No increase was found in the frequency of SCE. Since in vitro studies of lymphocytes exposed to transient electric currents (spark discharges) produced similar results the increase in chromosomal damage in substation workers may be associated with exposure to transient electric currents during work.     

The effects of post-treatments with caffeine during S and G2 on the frequencies of chromosomal aberrations induced by thiotepa in root tips of Vicia faba and in human lymphocytes in vitro

The effects of post-treatments with caffeine on the frequencies of chromosomal aberrations induced by the trifunctional alkylating agent thiotepa were studied in human lymphocytes and in root tips of Vicia faba. In lymphocytes the frequency of aberrations induced in G0 or G1 was most strongly increased when the caffeine post-treatments were given during G2. In Vicia faba, on the other hand, the frequency of aberrations induced in early interphase was unaffected by post-treatments with caffeine during G2, but strongly increased when the root tips were exposed to caffeine during the S phase.     

A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Study on X-ray-induced apoptosis and chromosomal damage in G2 human lymphocytes in the presence of pifithrin-α, an inhibitor of p53

The aim of this study is to investigate the role of the cell-cycle phase in cells exposed to radiation and chemicals in relation to the cellular response. The analysis was focused on the G2 cell-cycle phase, exploring the impact of p53 inhibition in human lymphocytes irradiated with X-rays in the presence or absence of pifithrin-α (PFT-α), a p53-specific inhibitor. Lymphocytes, 44h after stimulation to proliferate, were X-irradiated with 0.5Gy both in the presence or the absence of PFT-α and post-treated with a pulse of 5-bromodeoxyuridine (BrdUrd) to distinguish cells in the S- or G2-phase at the moment of irradiation. At early sampling times after X-ray exposure the following parameters were analysed: cellular proliferation, apoptosis, chromosomal aberrations and p53 expression. The results show an enhancement of apoptotic cells in G2 at early sampling times after irradiation and no differences in terms of chromosomal aberration induction both in cells treated with X-rays alone and in cells treated with X-rays plus PFT-α. Expression of p53 was not detectable at all recovery times. The results suggest a p53-independent apoptotic pathway acting at early times after X-ray exposure in G2 lymphocytes. Furthermore, the same yield of X-ray-induced chromatid breaks was observed both in the presence or absence of PFT-α implying that in G2 X-irradiated lymphocytes this inhibitor of the p53 protein does not affect DNA repair.     

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.     

Effect of caffeine on intrachromosomal distribution of chromatid aberrations induced by chemical mutagens in Crepis capillaris L

The intrachromosomal distribution patterns of chromatid aberrations induced by N-methyl-N-nitrosourethane (MNU), N-ethyl-N-nitrosourethane (ENU) and ethyleneimine (EI) were compared with those induced by combined treatment with the same mutagens and caffeine, the latter being considered as an inhibitor of post-replication repair of DNA.Chromatid aberrations induced by mutagens alone were distributed non-randomly along the chromosomes. In certain regions few aberrations were located; in others pronounced clustering of aberrations was observed and these regions were considered to be hot spots. This refers especially to MNU- and EI-induced aberrations, whereas ENU-induced chromatid aberrations showed a more length-proportional distribution. In ENU experiments, certain chromosomal segments also represented hot spots, but these were less pronounced. The distribution patterns of chromatid aberrations induced by combined treatment with mutagens and caffeine differed significantly from those observed in experiments with the mutagens only. There seemed to be a tendency to approach random distribution here. This was a result both of the decrease in the quantity of the aberrations in the regions, which in the experiments with mutagens only were hot spots, and of its increase in other chromosomal regions. Some of these regions were considered as hot spots but they were less pronounced. These tendencies refer to MNU and EI. Certain differences between the two variants, with the without caffeine, in ENU experiments were observed but these were of lower expressivity.The causes od differential sensivity of chromosomal regions are discussed. The conclusion is drawn that clustering of chromatid aberrations in certain chromosomal regions is due to differences in the repair systems acting in heterochromatic and euchromatic regions.     

Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes.

We have studied the induction of chromosomal aberrations in human lymphocytes exposed in G0 to X rays or carbon ions. Aberrations were analyzed in G0, G1, G2 or M phase. Analysis during the interphase was performed by chemically induced premature chromosome condensation, which allows scoring of aberrations in G1, G2 and M phase; fusion-induced premature chromosome condensation was used to analyze the damage in G0 cells after incubation for repair; M-phase cells were obtained by conventional Colcemid block. Aberrations were scored by Giemsa staining or fluorescence in situ hybridization (chromosomes 2 and 4). Similar yields of fragments were observed in G1 and G2 phase, but lower yields were scored in metaphase. The frequency of chromosomal exchanges was similar in G0 (after repair), G2 and M phase for cells exposed to X rays, while a lower frequency of exchanges was observed in M phase when lymphocytes were irradiated with high-LET carbon ions. The results suggest that radiation-induced G2-phase block is associated with unrejoined chromosome fragments induced by radiation exposure during G0.     

Effects of sodium butyrate on X-ray and bleomycin-induced chromosome aberrations in human peripheral blood lymphocytes.

Peripheral blood lymphocytes from normal human volunteers or from Down syndrome patients were pre-treated with sodium butyrate (a compound which is known to induce structural modifications in the chromatin through hyperacetylation of nucleosomal core histones) and exposed to X-irradiation or treated with bleomycin in vitro in the G0 and/or G1 stage(s) of the cell cycle. The frequencies of chromosomal aberrations in the first mitosis after treatment were scored. The results show an enhancement in the yield of aberrations in the butyrate pre-treated groups. However, the absolute frequencies of chromosomal aberrations as well as the relative increases with butyrate pre-treatment varied between blood samples from different donors suggesting the existence of inter-individual variations. There is a parallelism between the effects of X-irradiation or of combined treatments in G0 and G1 stages and between effects observed in the X-ray and bleomycin series. The increase in the yields of chromosomal aberrations in butyrate-treated and X-irradiated lymphocytes (relative to those which received X-irradiation alone) is interpreted as a consequence of the inhibition of repair of DNA damage by butyrate.     

Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the Comet assay with cytogenetic methods and immunology

Several substances used in rubber processing are known to be genotoxic. Workers in a rubber tyre factory, exposed to a broad spectrum of contaminants such as benzo[a]pyrene, benzo-fluoranthene, naphthalene, acetonaphthene, alkenes and 1,3-butadiene have been regularly examined for several years: chromosomal aberrations in lymphocytes, mutagenicity of urine (by use of the Ames test) and various parameters of blood and urine were assessed. An elevated level of mercapturic acid derivatives was found in the urine of employees, which is indicative of environmental exposure to toxicants with alkylating activity. We have now extended this study by examining genotoxicity with the modified Comet assay in parallel with chromosomal aberrations and micronucleus formation as well as immunological endpoints. Twenty-nine exposed workers from this factory were compared with 22 non-exposed administrative staff working in the same factory, as well as with 22 laboratory workers. The absolute numbers of peripheral leukocytes were significantly higher in the exposed group than in either of the control groups (p < 0.001). The erythrocyte mean cell volume was significantly higher in exposed workers in comparison with laboratory controls (p < 0.05). Percentages of lymphocytes, polymorphonuclear leukocytes, monocytes and eosinophils were not altered. The proliferative response of T- and B-cells to mitogen treatment when calculated per number of lymphocytes and adjusted for smoking, age and years of exposure did not differ between exposed and control groups. Endogenous strand breaks (including alkali-labile sites) and altered bases (formamidopyrimidine glycosylase- and endonuclease III-sensitive sites) were measured by the Comet assay in lymphocyte DNA. Exposed workers had significantly elevated levels of DNA breaks compared with office workers (p < 0.00001) or with laboratory controls (p < 0.00001). Micronuclei occurred at significantly higher frequencies in the exposed group than in controls (p < 0.00001), though the frequencies were all within the normal range. Significant correlations were seen between individual values of strand breaks, micronuclei and chromatid/chromosome breaks and certain immunological parameters.