Micronucleus Analysis Research Articles

Flavouring Group Evaluation 5, Revision 1 (FGE.05Rev1):Esters of branched‐ and straight‐chain aliphatic saturated primary alcohols and of one secondary alcohol, and branched‐ and straight‐chain unsaturated carboxylic acids from chemical groups 1, 2, and 5 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) ‐ Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with

Flavouring Group Evaluation 5, Revision 1 (FGE.05Rev1):Esters of branched‐ and straight‐chain aliphatic saturated primary alcohols and of one secondary alcohol, and branched‐ and straight‐chain unsaturated carboxylic acids from chemical groups 1, 2, and 5 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) ‐ Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with

Chromosomal Radiosensitivity, DNA repair, SNP and Apoptosis in Lymphocytes of Cervix Cancer Patients

There is considerable evidence that even after similar radiotherapy treatment, patients differ widely in normal tissue reaction. It is believed that the observed differences are related to intrinsic biological factors. Despite the great number of investigations of the relationship between the cellular radiosensitivity and reactions of patients to radiotherapy, there is presently no convincing proof that any of such biological factors alone can be used to predict side effects caused by radiotherapy. Considering the complexity of the mechanisms involved in the maintenance of the cell, tissue and organ function, it seems necessary to analyze the relationship between many different biological factors related to cellular sensitivity to ionizing radiation. The aim of the present study was to examine, in peripheral blood lymphocytes of cervix cancer patients collected before radiotherapy (1) chromosomal radiosensitivity, (2) kinetics of DNA repair, (3) SNP polymorphisms of genes involved in DNA repair and (4) apoptosis. The preliminary study included 30 patients. From each donor, peripheral blood was collected, irradiated in vitro with 2 Gy of gamma rays and the following endpoints were analyzed: Analysis of micronuclei (MN): The frequency of micronuclei, as a measure of chromosomal radiosensitivity, was determined with the micronucleus (MN) assay according to a standard protocol. Analysis of DNA repair: The kinetics of DNA repair was assessed by analyzing the formation and loss of gamma-H2AX foci. After 1 and 24 hours after irradiation, the cells were fixed, incubated with anti-phospho-histone H2AX antibody and analyzed by flow cytometry. Analysis of SNP polymorphism: DNA was isolated from the lymphocytes and RFLP-PCR followed by enzymatic digestion was performed according to the manufacturer’s protocol. The XRCC1-Arg399Gln, XRCC3-IVS5-14.893 and OGG1-Ser326Cys polymorphisms were determined using the restriction enzymes HpaII, PvuII and Fnu4HI, respectively. Analysis of apoptosis: The frequency of spontaneous and radiation-induced apoptotic cells was determined after 1, 6 and 24 hours after irradiation by the annexin method and flow cytometry. The results of the present study have shown a substantial inter-patient variability of lymphocyte chromosomal radiosensitivity, kinetics of DNA repair, SNP polymorphisms and apoptosis. However, no significant relationship was found for any combination of these endpoints. Additional correlation analyses will be performed with the reaction of the healthy tissue of patients to radiotherapy, classified according to the EORTC/RTOG scale.

Micronucleus analysis in patients with colorectal adenocarcinoma and colorectal polyps

To determine, by counting micronucleus (MN) frequencies, whether chromosomal or DNA damage have an effect on the pathogenesis of early colorectal adenocarcinoma (CRC). We analyzed MN frequencies in 21 patients with CRC, 24 patients with colon polyps [10 neoplastic polyps (NP) and 14 non-neoplastic polyps (NNP)] and 20 normal controls. MN frequency was significantly increased in CRC patients and in NP patients compared with controls (3.72 +/- 1.34, 3.58 +/- 1.21 vs 1.97 +/- 0.81, P < 0.001). However, there was no difference in the MN frequency between CRC patients and NP patients (P > 0.05). Similarly, there was no difference in the MN frequency between NNP patients (2.06 +/- 0.85) and controls (P > 0.05). Our results suggest increased chromosome/DNA instabilities may be associated with the pathogenesis of early CRC.

A pilot study of evaluation of the antioxidative activity of resveratrol and its analogue in a 6-month feeding test in young adult mice

Resveratrol, a polyphenolic phytoalexin, has free-radical scavenging activity and we found that it induces chromosomal aberrations, micronuclei, and sister chromatid exchanges in vitro. We synthesized its analogue 4-hydroxy- trans-stilbene (4-OH) and found that it has the same in vitro clastogenic activities as resveratrol, suggesting that the 4′ hydroxy group of resveratrol is responsible for the effect. We fed resveratrol and 4-OH to young adult ICR mice at 0, 0.2, 2, or 20 ppm in their standard powder diet for 6 months and investigated the antioxidative effects. Half of each group was given 3000 ppm potassium bromate (KBrO 3) in water for the last week to cause oxidative damage. Body weight gain tended to increase in males at 0.2 ppm resveratrol or 4-OH, and in females at 2 ppm 4-OH. Micronucleus (MN) analysis in bone marrow erythrocytes showed that the KBrO 3 tendency to induce MN was not prevented by the dietary resveratrol or 4-OH, which themselves did not induce MN under the present conditions. In this pilot study, resveratrol and 4-OH showed no obvious effect, either beneficial or adverse, at doses that are feasible in daily life for humans.

Genetic Toxicity Test of Methylcarbamate by Ames, Micronucleus, Comet Assays and Microarray Analysis

Carbamates have excellent insecticidal activities against a broad spectrum of insects. They possess knocking-down, fast-killing, and systemic effects, however, they are toxic to mammals. In this study, we have carried out in vitro genetic toxicity test of methylcarbamate and microarray analysis of differentially expressed genes in response to methylcarbamate. Methylcarbamate did not show mutations in base substitution strain TA1535 both with and without exogenous metabolic activation. Methylcarbamate did not show mutations in frame shift TA98 both with and without exogenous metabolic activation. Methylcarbamate showed DNA damage based on single cell gel/comet assay in L5178Y cells both with and without exogenous metabolic activation. Methylcarbamate did not increase micronuclei in CHO cells both with and without exogenous metabolic activation. Microarray analysis of gene expression profiles in L5178Y cells in response to methylcarbamate selected differentially expressed 132 genes that could be candidate biomarkers of genetic toxic action of methylcarbamate.

Genetic Toxicity Test of Emodin by Ames, Micronucleus, Comet Assays and Microarray Analysis Showing Differential Result

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a major constituent of rhubarb. Although it has been claimed to have a wild spectrum of therapeutic value, its side effects, especially in human kidney cells have not been well characterized. In this study, we have carried out in vitro genetic toxicity test of emodin and microarray analysis of differentially expressed genes in response to emodin. The result of Ames test showed mutations with emodin treatment in base substitution strain TA1535 both with and without exogenous metabolic activation. Likewise, emodin showed mutations in frame shift TA98 both with and without exogenous metabolic activation. The result of COMET assay in L5178Y cells with emodin treatment showed DNA damage both with and without exogenous metabolic activation. Emodin did not increase micronuclei in CHO cells both with and without exogenous metabolic activation. 150 Genes were selected as differentially expressed genes in response to emodin by microarray analysis and these genes would be candidate biomarkers of genetic toxic action of emodin.

Different behavior of SCE‐eliciting lesions induced by low and high doses of busulfan

Our previous studies suggested a dose-dependent transition in the types of DNA lesions induced by busulfan, as measured using the comet assay and by micronuclei analyses. The aim of the present study was to investigate the dose-dependent induction of different sister-chromatid exchange-eliciting lesions; lesions were distinguished by their efficiency in producing sister-chromatid exchange (SCE), and by their reparability during G1. Synchronously dividing murine salivary gland cells were assayed in vivo. Groups of mice were intraperitoneally injected with either 30 or 80 micromol busulfan/kg body weight solution at early or late G1. The rate of SCE/micromol busulfan/kg body weight obtained by exposure at late G1 with the high dose was twice that of the low dose. SCE induction during early G1 was higher than at late G1 with both doses; only the low-dose response was statistically significant. The frequency distribution of SCEs per cell demonstrated that cells exposed at the late G1 phase showed typical profiles that closely fit a Gaussian curve. However, an irregular profile was obtained for cells treated during early G1, which showed some cells with high-SCE frequency. Cells treated in early G1 have more time to repair lesions before DNA synthesis; therefore, the results suggest that instead of repair, secondary SCE-eliciting lesions during G1 were produced, especially at the lower dose. The results obtained in this study indicate that there are dose-dependent differences in the types of SCE-eliciting lesions induced by busulfan.

Genotoxicity assessment in fish peripheral blood: a method for a more efficient analysis of micronuclei

The authors describe a method for analysis of micronuclei using a nucleic acid‐specific fluorochrome, acridine orange, and ultraviolet microscopy in order to establish a simple and reliable technique for routine genotoxicity assessment in fish peripheral erythrocytes.

Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method

In order to assess the applicability of the cytokinesis-block micronucleus assay to frozen cells in human biomonitoring and in vitro radiosensitivity studies, basal and radiation-induced micronuclei and nucleoplasmic bridges (NPBs) were analysed in 28 lymphocyte samples stored frozen from 18 to 123 weeks. All samples successfully proliferated and produced a sufficient number of binucleated cells to be analysed. The length of the cryopreservation period did not influence cell proliferation, nor the incidence of micronuclei and NPBs, both in untreated and irradiated cells. Spontaneous levels of micronuclei were modulated by age (P=0.007) and by gender (P=0.024), as previously shown for cultures set up using fresh cell samples. Irradiation with 2 Gy gamma-rays significantly increased both micronuclei and NPBs, which were significantly correlated to each other (P=0.004). Radiation-induced micronuclei significantly increased with the age of donors (P=0.035), confirming previous findings obtained with fresh cell samples. The spontaneous incidences of micronuclei observed in cultures set up with frozen lymphocytes were compared with data recorded from the same subjects 5 years before using fresh blood samples. A high correlation was observed between the two data sets (P=0.004 after removing age and gender effects), highlighting the stability during the time of micronuclei as a biomarker of genomic stability, and supporting the suitability of frozen cells for cytogenetic analyses in biomonitoring and susceptibility studies.

Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay

Glyphosate is a widely used broad-spectrum weed control agent. In the present study, an in vivo study on the genotoxic effects of a technical herbicide (Roundup) containing isopropylamine salt of glyphosate was carried out on freshwater goldfish Carassius auratus. The fish were exposed to three doses of glyphosate formulation (5, 10 and 15 ppm). Cyclophosphamide at a single dose of 5 mg/l was used as positive control. Analysis of micronuclei, nuclear abnormalities and DNA damage were performed on peripheral erythrocytes sampled at intervals of 48, 96 and 144 h posttreatment. Our results revealed significant dose-dependent increases in the frequencies of micronuclei, nuclear abnormalities as well as DNA strand breaks. Our findings also confirmed that the alkaline comet assay and nuclear deformations in addition to micronucleus test on fish erythrocytes in vivo are useful tools in determining the potential genotoxicity of commercial herbicides.

Evaluation of genotoxicity of oral exposure to tetravalent vanadium in vivo

The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic potential in vivo is not elucidated. In this study, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO 4), a tetravalent vanadium salt, were investigated. To this aim male CD1 mice were administered with VOSO 4 in drinking water over the dose range 2–1000 mg/l for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment, micronuclei in both blood reticulocytes and bone marrow polychromatic erythocytes were determined; in addition, DNA lesions detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined by atomic absorption spectrometry. Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups. The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and lack of significant genotoxic potential in vivo.

Micronucleus analysis in a Portuguese population exposed to pesticides: Preliminary survey

The general population is exposed in their everyday life to different chemicals namely to pesticides. Many of these compounds are capable of inducing mutations in DNA and lead to several diseases including cancer. With this study we intended to evaluate DNA damage inflicted by pesticide exposure in a population occupationally exposed to those chemicals by means of the micronucleus (MN) test. The obtained results showed a significant increase in MN frequency in occupationally exposed individuals ( p<0.001) compared with the control group. Higher frequencies of MN were associated with a specific workplace (greenhouses) and the lack of protective measures (gloves) during labour activities. These results reinforce that conditions in workplace should be improved to minimize exposure to these chemicals. This study also emphasizes the need to reinforce the good practices campaigns in order to enlighten those who work with pesticides on the potential hazard of occupational exposure and the importance of using protective measures.

Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part IV:Recommendation of a Working Group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a Simple and Straightforward Approach to Genotoxicity Testing

Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.

Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa Stainings in Preimplantation Mouse Embryos Micronucleus Assay Including a Triple Dose Study

Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.

Evaluation of the genotoxic effects of thyroxine using in vivo cytogenetic test on Swiss albino mice

Thyroid hormones enhance aerobic metabolism favoring oxidative stress which may lead to covalent damage of various molecules including DNA. Previous investigations revealed that thyroid hormones induce DNA damage on human lymphocytes and sperm in the in vitro Comet assay. However, cytogenetic evaluation of genotoxic effects of thyroxine gave equivocal results: increase of sister chromatid exchanges, and no incerase of micronuclei in cultured human lymphocytes. Therefore, the aim of the present study was to further evaluate the possible genotoxic effects of thyroxine using in vivo cytogenetic test on Swiss albino mice. Three experimental concentrations of thyroxine were used (0.1 mg/kg, 0.5 mg/kg and 2.5 mg/kg). The mice were divided into several groups depending on the duration of the treatment with thyroxine. Thus, we treated mice for 1, 3, 7 and 10 days. Positive (Nmethyl- N'-nitro-N-nitrosoguanidine) and negative controls were also formed for the same time periods. Cytogenetic endpoinds (numerical and structural aberrations, chormosome gaps and breaks) were analysed in bone marrow cells from femures. The results obtained in this investigation showed that thyroxine has not induced chromosome damage or aberrations. This is in agreement with our previous analysis of micronuclei in human peripheral blood lymophocytes treated with thyroxine. On the other hand, we observed a decrease of mitotic index especially in animals treated for a longer period of time with the highest dose of thyroxine. Therefore, it can be concluded that thyroxine does not induce genotoxic effects which could be detected by cytogenetic analysis.

Scanning aneugen and clastogen by micronuclei analysis using flow cytometry

To explore a flow cytometry (FCM)-based method for discriminating aneugen- or clastogen-induced micronuclei. Cells were stained with anti-CD71-FITC and PI, and the PI fluorescent signal intensity of micronucleated reticulocyte (MN-RET) in the peripheral blood of NIH mouse treated with COL or CP was detected by flow cytometry. The ratio of the median of the intensity of MN-RET fluorescent signals to that of nucleated cell was low in the cyclophosphamide treated mouse, while the median was high in the colchicine treated mouse. The flow cytometry-based micronucleus assay can be used to discriminate primarily smaller MN induced by the clastogen exposure from the larger MN induced by an aneugen.

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.

The Histone Deacetylase Inhibitor Trichostatin A Has Genotoxic Effects in Human Lymphoblasts In Vitro

Histone deacetylase inhibitors (HDACi) are a class of putative chemotherapeutic agents for which the mechanism of toxicity has not been fully identified. To explore the possibility that HDACi are genotoxic, human TK6 lymphoblastoid cells were exposed to trichostatin A (TSA) and genetic damage was measured. TSA caused a dose-dependent increase of G1-arrested cells at 24 h that correlated with increasing levels of p21 and apoptosis. Significantly elevated frequencies of structural chromosomal aberrations in cells exposed to TSA were observed using both the kinetochore-antibody micronucleus assay and nonbanding metaphase chromosome analysis. Increased tail intensities, indicative of elevated levels of DNA damage, were observed using the alkaline comet assay. Elevated levels of phosphorylated histone gammaH2AX protein were observed as early as 3 h following TSA exposure and peaked at 12 h for 200nM TSA. Significant levels of aneuploidy at the 200nM TSA dose were observed using metaphase analysis, but interestingly, kinetochore-positive micronuclei were not detected at any dose using the kinetochore micronucleus assay, suggesting that TSA induces aneuploidy via a nondisjunction event rather than chromosome lagging. Increases in chromosomal loss and breakage were observed using simultaneous FISH metaphase analysis of chromosomes 5, 7, 8, and 21, consistent with data obtained from the micronucleus and metaphase chromosome analyses. We conclude that TSA is both a clastogen and aneugen in the TK6 cell line and propose that the observed cytostatic and apoptotic properties of TSA may partially be due to this genotoxicity.

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm™)

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm™ engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm™ model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30–50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine “ in vivo-like” assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods; the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed a genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer.