What do human micronuclei contain?

As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.

[Extrachromosomal structures containing small nuclear RNP and coilin in the late vitellogenic oocytes of hibernating grass frogs]. / Ekstrakhromosomnye struktury, soderzhashchie malye iadernye RNP i koilin, v iadrakh pozdnikh vitellogennykh ootsitov zimuiushchikh travianykh liagushek.

We have studied extrachromosomal structures in the germinal vesicle (GV) of the late vitellogenic oocytes of hibernating frogs Rana temporaria. During this period of oogenesis, chromosomes are completely inactivated to be surrounded by a fibrillar karyosphere capsule (Gruzova, Parfenov, 1993). Using immunostaining and in situ nucleic acid hybridization, we have identified three types of extrachromosomal structures: Cajal bodies (CB), nucleoli, and micronucleoli. Immunostaining of GV spreads has revealed that CB and nucleoli contain coilin, a marker protein for CB. The nucleoli were also positively stained with antibodies against Sm-epitope and trimetylguanosine cap of small nuclear (sn) RNP. According to the results of in situ nucleic acid hybridization, the nucleoli contain U6 snRNA. To further investigate a distribution of coilin in GV of the late vitellogenic oocytes of R. temporaria, we injected myc-tagged transcripts of full length human coilin (Wu et al., 1994) into the cytoplasm of oocytes and followed the pathway of the newly translated protein with an antibody specific for the tag. Immunofluorescent staining of spread GV contents demonstrated a specific staining of the nucleoli within 3 h after injection. We suggest that the newly synthesized myc-coilin may be phosphorilated and targeted to the nucleoli.

Micronucleus investigation of alcoholic patients with oral carcinomas.

The micronucleus test (MN) is used as an indicator of genotoxic exposition, since it is associated with chromosome aberrations. An increased mutation rate in oral squamous cells, indicated by an increased MN frequency, is also related to the development of oral carcinomas. We evaluated the frequencies of MN and other metanucleated anomalies in the buccal squamous cells of 30 alcoholics with oral or oropharyngeal carcinomas, and compared them to a control group of abstinent health individuals. Microscopic examination was made of 2000 cells per individual from each of three distinct areas of the mouth: around the lesion (A), opposite to the lesion (B) and in the upper gingival-labial gutter (C); C was used as a control region because of low tumor frequency. There was a seven-fold increase in MN frequency in region B, a three-fold increase in region A and a two-fold, though nonsignificant, increase in C; indicating a gradient of frequencies towards carcinogenesis: C --> A --> B. Comparisons of frequencies of various types of metanucleated cells: binucleated, karyorrhexis (KR), karyolysis (KL) and broken egg (BE) in patients and controls showed, with few exceptions, highly significant differences. This gave us a better understanding of the dynamics of this squamous epithelium, supporting a more efficient biomonitor based on these various metanucleated anomalies: the repair index RI=(KL+KR)/(MN+BE). Also, the apparently contradictory results from regression analysis revealed that the MN frequency decreased with age and alcohol consumption, probably because of slow cell proliferation, and consequently led to a loss of homeostasis due to aging. In addition, in the analysis of nonparametric variables only one CAGE question was significant, confirming the effect of alcohol. In conclusion, the MN test and the repair index could be used for monitoring clinical evolution, by means of intra- and inter-individual cellular comparisons, in subjects with healed or surgically removed tumors or leukoplastic lesions, after chemo- or radiotherapeutic treatments.

Micronuclei assay by laser scanning cytometry.

BACKGROUND: The micronuclei (MN) assay is used to assess the chromosomal/mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were developed: both offer some advantages but also have shortcomings. METHODS: In the present study laser scanning cytometer (LSC), the instrument that combines analytical capabilities of flow and image cytometry, has been adapted for quantitative analysis of MN. The micronucleation of human breast carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in vitro treatment with mitomycin C. Cellular DNA was stained with propidium iodide (PI), protein was counterstained with fluorescein isothiocyanate (FITC). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI and MN were identified on the bivariate PI versus PI/FITC fluorescence distributions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it possible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. RESULTS: The capability of LSC to relocate MN for visual examination was useful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating parameters that excluded objects that were not MN; 93.3+/-3.3 events within the selected gate were MN. It was also possible to successfully apply FISH software to characterize individual cells with respect to quantity of MN residing in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cells (r = 0.87). CONCLUSIONS: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN.

'LE cells' result from phagocytosis of apoptotic bodies induced by antinuclear antibodies.

Lupus erythematosus (LE) cells are believed to represent phagocytosis by granulocytes of cell nuclei whose DNA has been 'depolymerized' and opsonized by serum factors, most likely antinuclear antibodies and C3b. Since it is known that certain antinuclear antibodies are capable of inducing apoptosis after intracellular penetration; and that the resulting apoptotic bodies can be ingested by non-professional phagocytes, we decided to investigate the possibility that LE cells could result from the phagocytosis of apoptotic bodies induced by antinuclear antibodies. We demonstrate herein, through different methodological approaches, that the ingested material within LE cells corresponds to apoptotic bodies, and that the LE cell phenomenon can be reproduced, in the absence of other serum factors, by penetrating murine monoclonal anti DNA antibodies.

[Studies on micronuclei induced by colchicine and cyclophosphamide using multicolor fluorescence in situ hybridization].

OBJECTIVE: To study chromosomal composition of micronuclei (MN) induced by colchicine (COL) and cyclophosphamide (CP) in mouse bone marrow erythrocytes. METHODS: Multicolor fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes was applied to analyze chromosomal composition of micronuclei induced by COL and CP in mouse bone marrow erythrocytes. RESULTS: About 83.5% of COL-induced MN revealed both centromeric and telomeric signals. Of the CP-induced MN, 74.5% showed telomeric signals only. CONCLUSION: Majority of COL-induced MN contain whole chromosome and that of CP-induced MN mainly contain acentric fragments. Multicolor FISH with centromeric and telomeric DNA probes was a precise technique for analyzing chromosomal composition of MN.

Origin of microcells in the human sarcoma cell line HT-1080.

The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT-1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5-bromo-2'-deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self-renewal of the tumour stem cells is likely based on sporosis.

FISH analysis of 1cen-1q12 breakage, chromosome 1 numerical abnormalities and centromeric content of micronuclei in buccal cells from thyroid cancer and hyperthyroidism patients treated with radioactive iodine.

One of the health consequences of the Chernobyl nuclear power plant accident was a radioactive iodine-related increase in the incidence of thyroid cancer in exposed children. This radioisotope is used in the treatment of thyroid cancer and hyperthyroidism patients providing a convenient opportunity to study cytogenetic damage induced by known doses of radioactive iodine in treated patients. We used pancentromeric FISH on micronuclei and chromosome 1 tandem labelling FISH to monitor overall chromosome breakage and loss, 1q12 breakage and decondensation and chromosome 1 numerical abnormalities in buccal cells from 31 radioactive iodine-exposed hyperthyroidism and thyroid cancer patients. The overall outcome of the study, with 250,000 buccal cells analysed, is that there was no radioactive iodine-related increase in the frequency of micronuclei, 1q12 breakage, 1q12 decondensation or chromosome 1 numerical abnormalities. In addition, neither age nor gender, health status nor radioactive iodine dose modulated the frequency of the above cytogenetic end points. Although several uncertainties of these emerging molecular cytogenetic methodologies will require further experimentation, we conclude that, at the reported exposure levels, radioactive iodine did not induce detectable chromosome damage in buccal cells from treated patients.

Estudo da associaçäo Li2CO3 e adoçante näo calórico, C7H5NO3S, em células de medula óssea de camundongos / A study of Li2CO3 and C7H5NO3 noncaloric sweetener association in mice bone marrow cells

A apresentaçäo destes resultados é uma conseqüência da linha de pesquisa desenvolvida com o carbonato de lítio - Li2CO3 (Li) - e adoçantes näo calóricos, em animais de laboratório em fase de estudos, acrescentando-se sacarina (Sc) e utilizando-se camundongos Swiss fêmeas. O objetivo foi avaliar diferenças estatisticamente significantes como indicadoras de mutagenicidade, quando grupos de camundongos receberam os tratamentos. Em nove tabelas, analisou-se o percentual de micronúcleos (MN) calculados em eritrócitos policromáticos (EP) e normocromáticos (EN), isoladamente, e na soma total. Os animais foram tratados por dez dias, v.o. com Li, na dose de 100mg/kg, ou Li associado à sacarina (Sc), nesta mesma diluiçäo. Os resultados obtidos foram comparados com os grupos tratados com ciclofosfamida - Ci. (+) e água - controle (-). A análise de variância seguida do teste de Tukey mostrou diferenças estatisticamente significativas para as médias do percentual de MN em EP, EN e na soma dos totais com valores de F iguais a 16,57**, 9,67** e 15,63**, respectivamente. A comparaçäo dois a dois foi analisada pelos testes "t" de Student (p < 0,05). Diferença significativa só se apresentou para o grupo Ci (+). Considerando-se a metodologia usada em camundongos fêmeas e pela análise estatística estudada, näo foi encontrada indicaçäo de mutagenicidade com a contagem de MN em células de medula óssea para esta espécie de animal.

Co-localization of mitochondrial and double minute DNA in the nuclei of HL-60 cells but not normal cells.

In an attempt to isolate genes located on double minute (Dmin) DNA in HL-60 cells, we prepared DNA probe from purified micronuclei. Micronucleation was induced in HL-60 cells by treatment with hydroxyurea. Screening of a cDNA library unexpectedly produced a number of clones containing mitochondrial DNA (mtDNA) sequences. Here, we show that amplified mtDNA sequences were localized in nuclei and micronuclei of HL-60 and COLO 320DM cells, but not in nuclei of WI-38 normal human fibroblasts or peripheral blood T-cells. To unequivocally demonstrate the presence of mtDNA inside of nuclei and micronuclei, we obtained tomographic fluorescence in situ hybridization (FISH) images of mtDNA by confocal microscopy of consecutive sections of paraformaldehyde (PFA)-fixed material. We also located mtDNA in nuclear buds and purified micronuclei. Dmin DNA and mtDNA were always located at similar sites. The mechanisms of nuclear retention of mtDNA and Dmin DNA and the resulting influence on tumorigenesis are discussed.

Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.

Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG-p53 mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time-weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, peripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN-PCE and MN-NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6-month treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose-dependent increase in the frequency of MN-PCE and MN-NCE and in %PCE. Maximal induction of MN-PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration-dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not exclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN-NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans.

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe.

The DNA content distributions of micronuclei induced in mouse 3T3 cells by ionizing radiation and chemicals was measured by flow cytometry. For a quantitative understanding of these distributions, micronuclei with increasing DNA contents were sorted and analysed for the presence of centromeric signals using fluorescent in situ hybridization (FISH) with a mouse centromeric gamma satellite probe. Radiation-induced micronuclei were found to be produced mainly by chromosome fragments, whereas micronuclei induced by the tear gas chlorobenzylidene malonitrile (CS) were found to be produced mainly by whole chromatids. In contrast, micronuclei induced by vinblastine (VBL) were, according to the shape of their DNA content distributions, produced mainly by whole chromosomes and by combinations of two or more whole chromosomes. With increasing DNA content, micronuclei induced by ionizing radiation also contained one or more whole chromosomes, whereas micronuclei induced by CS or VBL were found to contain several whole chromatids or chromosomes respectively. Computerized random breakage of chromosomes and random combination of chromosome fragments, whole chromatids and whole chromosomes were used according to the FISH results to simulate the measured DNA content distributions of micronuclei. A good agreement was obtained between measured and simulated distributions of micronuclei as well as between results of the measured frequency of micronuclei showing centromeric signals as a function of their DNA content and those predicted by the simulations. These results demonstrate the usefulness of flow cytometry and sorting combined with the FISH technique and computer simulations for producing a more detailed analysis of mechanisms of micronucleus induction.

p53 accumulates in micronuclei after treatment with a DNA breaking chemical, methylnitrosourea, and with the spindle poison, vinblastine.

Micronuclei (MN) induced by N-methyl-N-nitrosourea (MNU) or vinblastine in cultured mammalian cells were analyzed for the accumulation of p53 by immunocytochemical staining with a p53 monoclonal antibody. Our data showed that MN induced by both agents were p53-negative at early post-treatment times, but became positive at late times. Assuming that most MNU-induced micronuclei reflect DNA damage, and most vinblastine-induced micronuclei reflect damage to the mitotic apparatus, we conclude that p53 accumulation in micronuclei is not triggered by DNA damage per se but instead probably stems from DNA degradation occurring during ageing of micronuclei.

Isolation of micronuclei: high yield by sucrose gradient versus maximum purity by cell sorting.

Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.

Flow cytometric detection of micronuclei by combined staining of DNA and membranes.

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB and DPH were comparable to the results obtained with the combination of EB and HO.

Genotoxic effects of the carbamate insecticide methomyl. I. In vitro studies with pure compound and the technical formulation "Lannate 25".

The carbamate insecticide methomyl and the methomyl-containing technical formulation "Lannate 25" were tested on whole blood human lymphocyte cultures. Both products induced dose-dependent increases in chromosome aberrations and micronuclei. Lannate 25 induced DNA damage as measured by the alkaline elution assay and hydroxylation of guanine at the C8 position. Sister chromatid exchanges were not increased significantly with either product. Overall, the technical formulation was more active than the pure compound, when compared at similar concentrations of active principle. Moreover, a different ratio of CREST-positive/CREST-negative micronuclei was observed with the two products, pure methomyl being relatively more active than Lannate 25 in the induction of CREST-positive micronuclei. On the basis of these results, previous evaluations of methomyl as a nongenotoxic compound should be reconsidered.

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei.

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine (BrdU) on cell cycle progression and micronucleus induction were studied in different mammalian cell cultures. Simultaneous flow cytometric measurements of DNA content and side scatter of nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependent temporary block in the G2/M phase of the first cell cycle. NIH 3T3 cells and human amniotic fluid fibroblast-like cells, on the contrary, did not show any cell cycle disturbances in the presence of BrdU. Micronucleus frequency increased as soon as CHE cells started to divide and reached a plateau when all cells have divided. The height of this plateau was almost equal for 60 and 100 microM BrdU. This saturation of micronucleus induction was due to a saturation of BrdU incorporation into DNA already at a doses of 60 microM as shown by the BrdU/Hoechst quenching technique. Indirect immunofluorescent staining of kinetochores with CREST antibodies revealed that nearly all BrdU-induced micronuclei were kinetochore-negative suggesting the presence of acentric chromosome fragments in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed several peaks representing micronuclei which contain DNA fragments of defined sizes induced by non-random breakage of chromosomes 1 and X as verified by flow karyotyping and C-banding.

An investigation of micronucleus and mutation induction by oxazepam in mammalian cells.

The benzodiazepines are a class of drugs that are widely used in the treatment of various psychiatric disorders. One member of this class, oxazepam, is also a common metabolite of several other benzodiazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is inconsistent, we investigated the oxazepam-induced formation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and L5178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all three cell lines. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were formed in the first mitosis after treatment. Kinetochore staining (CREST-antiserum) revealed the presence of kinetochores in approximately 50% of the micronuclei in all three cell types. This result was further confirmed by in situ hybridization in L5178Y cells and indicates the presence of whole chromosomes or centric fragments as well as acentric fragments in the oxazepam-induced micronuclei. The L5178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.

A comparison of the 8-hydroxydeoxyguanosine and micronucleus techniques for the assessment of X-ray and UV induced genotoxicity.

A comparison was made of the relative sensitivities of the cytokinesis-block micronucleus and the 8-hydroxydeoxyguanosine assays for the assessment of X-ray and UV-induced genotoxicity in mouse splenocytes in vitro. A detectable (p < 0.05) increase in micronuclei occurred at about 1/5 the X-ray exposure required to induce a significant increase in the level of 8-hydroxydeoxyguanosine. With UV radiation a significant (p < 0.05) rise in micronuclei was achieved at about 1/10th the dose needed to produce a detectable increase in 8-hydroxydeoxyguanosine levels. The data confirm the value of the cytokinesis-block micronucleus technique for the detection of genotoxicity at low-level X-irradiation, and indicate that it is more sensitive for that purpose than the 8-hydroxydeoxyguanosine assay. The data also demonstrate, for the first time, the production of 8-hydroxydeoxyguanosine by UVA/B-irradiation of intact cells, but point again to a more sensitive assessment of UV-related genotoxicity by the cytokinesis-block micronucleus method.