A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

The frequency of induced premature centromere division in human populations occupationally exposed to genotoxic chemicals.

Premature (early) centromere division (PCD, i.e., the separation of centromeres during the prometaphase/metaphase of the mitotic cycle) seems to be a possible manifestation of chromosome instability in human chromosome-breakage syndromes. Chromosome instability also frequently occurs in the peripheral blood lymphocytes (PBL) of humans occupationally exposed to clastogenic agents, and is considered an etiologic factor of neoplastic diseases. In order to investigate the importance of PCD in cancer risk assessment, we studied the frequency of PCDs in PBL of 400 Hungarian subjects. The various groups comprised 188 control donors and 212 subjects occupationally exposed to different genotoxic chemicals, such as acrylonitrile (ACN) and/or dimethylformamide (DMF), benzene, cytostatic drugs, ethylene oxide (ETO), mixed exposure in the rubber industry, mixed organic solvents including CCl4, hot oil-mist, bitumen, and polychlorinated biphenyls (PCB). Data were compared with chromosomal aberration frequencies determined in the same samples. PCD yields are significantly higher in populations exposed to mixed chemicals, crude oil and cytostatic drugs, compared with controls. PCDs involving more than three chromosomes are also more frequent in ETO- and oil mist-exposed groups than in the others. The results indicate that the induction of PCDs is neither incidental nor artificial. As a consequence, we suggest that PCD can be developed into a new, exposure-related cytogenetic biomarker for a more adequate occupational cancer risk assessment. A further, follow-up epidemiological and cytogenetic investigation of PCD is in progress.

The centromere as a target for the induction of chromosome damage in resting and proliferating mammalian cells: assessment of mitomycin C-induced genetic damage at kinetochores and centromeres by a micronucleus test in mouse splenocytes.

The cytokinesis-block micronucleus assay (MN) on murine splenocytes was used for the estimation of chromosome damage in a resting cell population in vivo that can be induced to proliferate in vitro. Mitomycin C at different doses (10(-8), 6 x 10(-8), 10(-7), 6 x 10(-7) and 10(-6)M) was used to induce cytogenetic damage in resting and cycling splenocytes. Antikinetochore antibodies (CREST) and two-colour fluorescence in situ hybridization (FISH) with minor and major satellite DNA were applied. These approaches allowed the detailed characterization of the mechanisms by which MN originates, since it was possible to identify breaks induced in pericentric heterochromatic (resulting in MN containing the major but not the minor satellite DNA) or detachment/disruption of kinetochore (resulting in different frequencies of MN containing kinetochore or both probes). Based on the evidence that resting and cycling mouse splenocytes are characterized by different spatial distribution of centromeric regions, the hypothesis was tested that the damage induced by mutagens at centromeres is influenced by the phase of the cell cycle in which the cells are treated. Data presented here show that resting and cycling splenocytes are both sensitive to mitomycin C action, and indicate that this compound has an aneugenic potential, besides its strong clastogenic activity. In particular, results obtained after CREST and FISH characterization of MN differed when cells were treated during proliferation, suggesting a disruption/detachment of kinetochores induced by mitomycin C at this cell stage. Furthermore, under the same treatment condition the proportion of MN containing the major satellite DNA only was greater than expected on the basis of random breakage at this site. Treatment of resting cells produced aneugenic damage, but without evidence of disruption/detachment of kinetochores or preferential breakage at the centromere. These results indicate that the amount and type of chromosome damage induced by mitomycin C in mouse splenocytes differ in relation to the proliferative status of treated cells.