Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

Sevoflurane and isoflurane genotoxicity in kidney cells of mice.

The aim of this study was to evaluate the DNA damage and repair in kidney cells of Swiss albino mice after repeated exposure to sevoflurane and isoflurane and compare their detrimental effects. We used the alkaline comet assay to establish the genetic damage and measured three parameters: tail length, tail moment, and tail intensity of comets. These parameters were measured immediately after exposure to the above mentioned inhalation anaesthetics, two hours, six hours, and 24 hours later and were compared with the control group. Mean values of all three parameters were significantly higher in experimental groups compared to the control group. DNA damage in kidney cells of mice exposed to sevoflurane increased continuously before it reached its peak 24 hours after exposure. Isoflurane induced the highest DNA damage two hours after exposure. Levels of DNA damage recorded 24 h after cessation of exposure to both tested compounds suggest that sevoflurane was slightly more genotoxic than isoflurane to kidney cells of mice. According to these results, the currently used volatile anaesthetics sevoflurane and isoflurane are able to damage DNA in kidney cells of mice. Such findings suggest a possibility for similar outcomes in humans and that fact must be taken into account in everyday clinical practice.

Effects of low doses of glyphosate on DNA damage, cell proliferation and oxidative stress in the HepG2 cell line.

We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be encountered in occupational and residential exposures [the acceptable daily intake (ADI; 0.5 µg/mL), residential exposure level (REL; 2.91 µg/mL) and occupational exposure level (OEL; 3.5 µg/mL)]. The assessments were performed using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell proliferation, alkaline comet assay and cytokinesis-block micronucleus (CBMN) cytome assay. The results obtained indicated effects on cell proliferation, both at 4 and 24 h. The levels of primary DNA damage after 4-h exposure were lower in treated vs. control samples, but were not significantly changed after 24 h. Using the CBMN assay, we found a significantly higher number of MN and nuclear buds at ADI and REL after 4 h and a lower number of MN after 24 h. The obtained results revealed significant oxidative damage. Four-hour exposure resulted in significant decrease at ADI [lipid peroxidation and glutathione peroxidase (GSH-Px)] and OEL [lipid peroxidation and level of total antioxidant capacity (TAC)], and 24-h exposure in significant decrease at OEL (TAC and GSH-Px). No significant effects were observed for the level of reactive oxygen species (ROS) and glutathione (GSH) for both treatment, and for 24 h for lipid peroxidation. Taken together, the elevated levels of cytogenetic damage found by the CBMN assay and the mechanisms of primary DNA damage should be further clarified, considering that the comet assay results indicate possible cross-linking or DNA adduct formation.

Cytotoxic, genotoxic and biochemical markers of insecticide toxicity evaluated in human peripheral blood lymphocytes and an HepG2 cell line.

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future.

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells.

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 µg/mL), residential exposure level (0.002 µg/mL) and acceptable operator exposure level (0.0012 µg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus "cytome" assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.

Polymorphisms in DNA repair genes: link with biomarkers of the CBMN cytome assay in hospital workers chronically exposed to low doses of ionising radiation.

Individual sensitivity to ionising radiation (IR) is the result of interaction between exposure, DNA damage, and its repair, which is why polymorphisms in DNA repair genes could play an important role. We examined the association between DNA damage, expressed as micronuclei (MNi), nuclear buds (NBs), and nucleoplasmic bridges (NPBs) and single nucleotide polymorphisms in selected DNA repair genes (APE1, hOGG1, XRCC1, XRCC3, XPD, PARP1, MGMT genes; representative of the different DNA repair pathways operating in mammals) in 77 hospital workers chronically exposed to low doses of IR, and 70 matched controls. A significantly higher MNi frequency was found in the exposed group (16.2±10.4 vs. 11.5±9.4; P=0.003) and the effect appeared to be independent from the principal confounding factor. Exposed individuals with hOGG1, XRCC1, PARP1, and MGMT wild-type alleles or APEX1, as well as XPD (rs13181) heterozygous showed a significantly higher MNi frequency than controls with the same genotypes. Genetic polymorphism analysis and cytogenetic dosimetry have proven to be a powerful tool complementary to physical dosimetry in regular health surveillance programmes.

Micronucleus, alkaline, and human 8-oxoguanine glycosylase 1 modified comet assays evaluation of glass-ionomer cements - in vitro.

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10(-2), 10(-3), and 10(-4). Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions.

Genotoxic effect of two bleaching agents on oral mucosa.

AIM: To analyze the genotoxic effect of two hydrogen peroxide-containing bleaching products on oral mucosal cells. MATERIALS AND METHODS: The research was conducted on 22 individuals divided into two groups. Group 1 used ZOOM2 and group 2 the Opalescence BOOST bleaching agent. Specimens of the gingival and the upper lip mucosa were obtained before, immediately after, and 72 h after the bleaching procedure and were analyzed using a micronucleus test. RESULTS: Seventy-two hours after bleaching treatment with BOOST, samples collected from the oral mucosa exhibited a statistically significant increase of all genotoxicity markers, with large effect sizes (Cohen's d>0.8) observed in the total number of micronuclei (MN), number of cells with 3+ MN, karyolysis and bi-nuclear cells. ZOOM2 treatment showed a significant increase, with medium-to-large effect sizes, in the number of cells with 1 MN, karyolysis, nuclear buds and bi-nuclear cells. CONCLUSION: Both preparations demonstrated potential genotoxic effects.

[Normal and cut-off values of the cytokinesis-block micronucleus assay on peripheral blood lymphocytes in the Croatian general population]. / Normalne i granicne vrijednosti mikronukleus-testa na limfocitima periferne krvi u ispitanika opce populacije Republike Hrvatske.

The cytokinesis-block micronucleus (CBMN) assay on peripheral blood lymphocytes is one of the most important methods employed in cytogenetic biomonitoring. For the purposes of biological dosimetry, it is important to know the spontaneous frequency of a biomarker and its normal values in general population. These values are used for population databases, which should be updated regularly. In this study, MN levels were investigated in cytokinesis-blocked lymphocytes of 200 healthy male and female blood donors selected at random from the general population of Croatia. The aim was to assess the variability and determine possible influences of external and/or internal factors on the background levels of MN and to establish the cut-off value for the CBMN assay. The background frequency of MN was (6.90+/-3.32) MN (median 7 MN) and the range was 0 to 18 MN per 1000 binuclear lymphocytes. The cut-off value, which corresponds to 95th percentile of the distribution of 200 individual values, was 12.5 MN. Spontaneous formation of MN was influenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking significantly increased the values of all parameters evaluated by the CBMN assay. Since the existing literature data on smoking-related formation of MN are contradictory, we will continue these investigations to resolve how the number of cigarettes smoked per day and the duration of smoking in years influence the results of the CBMN assay. Our results are consistent with the background MN frequencies reported by other cytogenetic laboratories worldwide. Normal and cut-off values estimated in this study will be used to update the current general population data and as reference for occupationally or accidental exposure.

Correlation between folate and vitamin B12 and markers of DNA stability in healthy men: preliminary results.

The aim of this study was to find correlations between folate and vitamin B12 on baseline damage in white blood cells and their association with smoking, alcohol consumption and ageing. Thirty-six healthy vitamin non-deficient male subjects were selected in a randomized study. Comet assay (SCGE) and micronucleus (MN) assay were used as biomarkers of DNA damage. The amount of DNA damage was correlated with vitamin B12 and folic acid concentration. Positive, but non-significant correlation (canonical R = 0.61; χ²=28.97; P=0.253) was found between micronucleus (MN) frequency or comet assay parameters (SCGE) and five covariates (age, smoking, alcohol consumption, vitamin B12 and folate blood serum concentration). The highest MN frequency was observed in the group with the lowest vitamin B12 concentration (F=3.59; P=0.024). The SCGE assay failed to show significant correlation with vitamin B12 or folic acid concentration. Concentration of vitamin B12 was significantly correlated with incidence of micronuclei. Our results present background data that could be valuable for future genotoxicological monitoring.

Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action.

This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC(50) of BEA (5.0 +/- 0.6) and OTA (15.8 +/- 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 microM) and OTA (1 and 5 microM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 muM of OTA alone and BEA + OTA combinations. Single BEA (0.5 microM) and OTA (1 and 5 microM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans.

Assessment of cyto/genotoxicity of irinotecan in v79 cells using the comet, micronucleus, and chromosome aberration assay.

Irinotecan is a topoisomerase I interactive agent, widely used in the treatment of metastatic colorectal cancer. The genotoxic effects of the maximum single dose (18 microg mL-1), recommended monotherapy dose (9 microg mL-1), and recommended combined therapy dose (4.5 microg mL-1) of irinotecan were studied on V79 cells using the comet assay, chromosome aberration assay, and micronucleus test. The cells were treated with irinotecan for 2 h or 24 h. The statistical significance of the results was determined using the one-way ANOVA test and a nonparametric Mann Whitney U test. The comet assay did not show dose-dependent or time-dependent effects. The chromosome aberration analysis showed large DNA rearrangements, i.e., chromosome exchanges. Although the exposed cultures showed a significant increase in micronucleated cells in respect to control, no dose-dependent relation was established among the treated cultures. Time-dependent effect was also not observed.

[Antineoplastic drugs as a potential risk factor in occupational settings: mechanisms of action at the cell level, genotoxic effects, and their detection using different biomarkers]. / Antineoplasticni Lijekovi Kao Cimbenik Rizika u Radnom Okolisu: Mehanizmi Djelovanja na Razini Stanice i Pregled Metoda za Otkrivanje Njihovih Genotoksicnih Ucinaka.

This article brings an overview of the mechanisms of action of antineoplastic drugs used in the clinical setting. It also describes the genotoxic potentials of the most important classes of antineoplastic drugs involved in standard chemotherapy protocols. Classification of antineoplastic drugs according to the IARC monographs on the evaluation of carcinogenic risks to humans is accompanied by data on their mutagenicity and the most recent updates in the Anatomical Therapeutic Chemical (ATC) Classification System. We report the main findings of biomonitoring studies that were conducted in exposed healthcare workers all over the world between 1980 and 2009 using four biomarkers: sister chromatid exchanges, chromosome aberrations, micronuclei. and the comet assay. The methods are briefly explained and their advantages and disadvantages discussed. Biomarkers provide important information on individual genome sensitivity, which eventually might help to improve current working practices and to manage the risks related with exposure to genotoxic agents. Taking into consideration all known advantages and drawbacks of the existing cytogenetic methods, the micronucleus assay, which is able to detect both clastogenic and aneugenic action, is the most suitable biomarker for assessing harmful effects of antineoplastic drugs currently used in health care.

Evaluation of lead exposure in battery-manufacturing workers with focus on different biomarkers.

The influence of exposure to lead on the frequency of micronuclei (MN), nuclear buds and nucleoplasmatic bridges was investigated in peripheral blood lymphocytes in 15 male battery-manufacturing workers and 15 controls matched for age and smoking habits. In addition to MN test, blood lead (B-Pb) and cadmium (B-Cd), delta aminolevulinic acid dehydratase (ALAD) activity, erythrocyte protoporphyrin (EP), serum vitamin B(12) (S-Vit B(12)) and folate (S-folate) were determined in all subjects. Lead-exposed subjects had significantly higher MN frequency and B-Pb concentrations than controls. In control smokers we found a significant negative correlation between B-Pb concentration and frequency of nucleoplasmatic bridges, and nuclear division index. In control non-smokers a significant positive correlation was observed only between age and nuclear buds frequency, and between S-folate and B-Pb level. In lead exposed smokers, significant positive correlations between MN frequency and S-Vit B(12), S-folate, and nuclear buds frequency were found. A positive correlation in exposed smokers was also found between nuclear buds frequency and S-Vit B(12) concentration. A negative correlation was found between ALAD and EP, and B-Pb in exposed smokers. Exposed non-smokers showed significant negative correlation between MN frequency and B-Cd, and ALAD and EP. The results indicate a genotoxicity of lead, pointing to a micronucleus assay as a relevant test for assessing genotoxic effects resulting from occupational exposure. The other indicators did not necessarily follow the results of THE MN test. Influence of smoking should be further investigated.

The genotoxic risk in health care workers occupationally exposed to cytotoxic drugs--a comprehensive evaluation by the SCE assay.

Present study aimed at an integral assessment of sister chromatid exchange (SCE) frequencies in the health care workers occupationally exposed to cytostatics. The results of 500 individual analyses were evaluated. Drug handling practice was investigated in parallel and the results showed that cytostatics are mostly prepared outside hospital pharmacy (98%) and mainly handled by nurses (96%). Mean frequency of SCE was 5.63 +/- 2.28, while HFC represented 9.65% of the cells analysed. Both values were higher compared to previously established control values for Croatian population. The duration of exposure, profession, age, gender, smoking habit, medical exposures, and simultaneous exposure to other occupational mutagens significantly contributed to SCE and HFC values. The usefulness both biomarkers in the assessment of cytogenetic damage is confirmed. Since current practice in Croatian hospitals does not include regular monitoring of workplaces, to ensure maximal occupational safety, a surveillance on exposed health care workers, including periodic biomonitoring, is recommended.

Assessment of genotoxic risks in Croatian health care workers occupationally exposed to cytotoxic drugs: a multi-biomarker approach.

The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes of Croatian health care workers occupationally exposed to cytotoxic drugs. A comprehensive multi-biomarker approach using the alkaline comet assay and cytogenetic endpoints (analysis of structural chromosome aberrations, SCE assay, lymphocyte proliferation kinetics and cytokinesis-block micronucleus assay) was employed. The study included two populations of subjects: 50 health care workers occupationally exposed to cytotoxic drugs and 50 control subjects matched in age, gender and smoking habit. An investigation regarding the handling practice with cytotoxic drugs was conducted in parallel. Results obtained indicate high exposure levels at workplace that should be reduced. The values recorded among the occupationally exposed subjects were as follows: mean comet tail length: 17.46+/-0.08 microm; the incidence of long-tailed nuclei: 54.68+/-3.93%; 4.48+/-0.33 structural chromosome aberrations per 200 cells; 5.81+/-0.04 SCE per 50 cells; 29.28+/-2.21% of high-frequency cells; proliferation rate index: 1.97+/-0.12; and 16.32+/-0.85 micronuclei per 1000 binuclear cells. All these values indicated higher levels of DNA and cytogenetic damage compared to the general population. Obtained results also confirmed that the frequency of long-tailed nuclei in the alkaline comet assay represents a helpful complement to other well-established comet parameters. The age of subjects and smoking habit significantly influenced the values of both comet and cytogenetic endpoints. Overall results of this study confirmed that handling cytotoxic drugs without appropriate safety precautions involves a potential genotoxic risk for exposed subjects. Before a strict monitoring of exposure levels on each workplace becomes a standard practice in Croatian hospitals, cytogenetic surveillance of exposed workers is also recommended, at least in cases of accidental exposure.

Genotoxic effects of anaesthetics in operating theatre personnel evaluated by the comet assay and micronucleus test.

Genetic damage induced by anaesthetic gases in occupationally exposed populations was investigated using the comet assay and micronucleus test. The study included two groups of subjects: 50 operating theatre medical workers (anaesthesiologists, technicians and nurses) and 50 control subjects corresponding in sex, age and smoking habit. The exposed group revealed an increase in genome damage in both tests. In the comet assay, exposure to anaesthetics was a highly significant predictor of the tail length for technicians, while sex proved to be significant predictor of tail moment for women in exposed group. Micronucleus frequency increased significantly, showing threefold increase in exposed groups (RR>3.029). Univariate analysis showed significant influence of duration of exposure, while multivariate analysis showed age to be significant predictor of micronucleus frequency. The obtained results call for further, targeted investigation of exposure risk.

Chromosome aberrations in peripheral blood lymphocytes of Croatian hospital staff occupationally exposed to low levels of ionising radiation.

Medical staff is an occupational group exposed to different agents suspected to induce genetic damage. Among them ionising radiation is the most studied. Cytogenetic analysis of human chromosomes in peripheral lymphocytes allows direct detection of mutation in somatic cells. This study investigated the cytogenetic effects of low-level ionising x-radiation in 48-hour peripheral blood lymphocyte cultures sampled from 765 hospital staff occupationally exposed to several agents known or suspected to induce chromosome damage and compared them with 200 control subjects. The exposed subjects were divided in eight (8) groups according to their specialties and job titles. The exposed groups manifested an increase in all types of chromosome aberrations. Acentric fragments were the most frequent chromosome-type aberration. Dicentric chromosomes were statistically significant only in urologists/gynaecologists. Age and smoking significantly influenced the incidence of dicentrics in the exposed groups. The frequency of ring chromosomes was low in all exposed groups (range: 0-2), and none were found in the control group. These findings indicate the importance of periodic medical checkups of hospital staff occupationally exposed to low doses of ionising radiation. The purpose is to create an individual cytogenetic register, where changes could evidence individual risks.

Chromosome damage in workers in cigarette manufacturing industry.

To investigate whether occupational exposure to tobacco dust is genotoxic, a group of employees in a tobacco factory was tested for structural chromosome aberrations (CA), cytokinesis-block micronucleus assay (CBMN) and sister chromatid exchanges (SCE) that are well established as indicators of early biological effects. The study group consisted of 40 tobacco workers and an equal number of matched controls. The results obtained in the exposed group showed a significant increase in chromosome aberrations (R=0.26), micronucleus frequency (R=0.56) and in sister chromatid exchanges (R=0.75), which was additionally influenced by smoking. A significant increase in high frequency cells (HFC) in the exposed group was also observed. Like the SCE frequency, the HFC frequency increased significantly in smokers of the control and exposed smokers. The study indicates that occupational exposure to tobacco dust induces genome damage. A higher risk was observed in women. The micronucleus frequency and sister chromatid exchange tests seem to be more reliable indicators of genome damage than chromosome aberrations in monitoring chronically exposed subjects.

Stable and unstable chromosome aberrations measured after occupational exposure to ionizing radiation and ultrasound.

AIM: To evaluate chromosome aberration and fluorescent in situ hybridization (FISH) assays as a method to estimate of health risk, we monitored 9 male subjects occupationally exposed to low doses of both ionizing radiation and ultrasound during a period of over 3 years. METHODS: Sampling was performed at 6-month intervals during a three-year period. First we used conventional chromosomal aberrations analysis. When the aberration frequency for a particular subject reached the background, we measured translocations in the final sample, using fluorescence in situ hybridization. Chromosome painting probes for chromosomes 1, 2, and 4 were used simultaneously. RESULTS: Dicentric and ring chromosomes were eliminated within a year. Translocations persisted and deviated from control values in all examinees. Translocations were detected long after unstable aberrations decreased to the background level. CONCLUSION: Fluorescence in situ hybridization-based translocation detection was a reliable method for monitoring chronic occupational clastogen exposure. Chromosome aberration assay correlated with translocation frequency. Stable chromosomal aberrations reflected cumulative genome damage during job exposure.