Francesca Degrassi

Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy.

The immunofluorescent staining of kinetochores in micronuclei with antikinetochore antibodies was used to develop an in vitro assay for aneuploidy-inducing agents. The results show that about 80% of micronuclei induced by either colchicine or chloral hydrate contained kinetochores; only 9% of X-ray-induced micronuclei reacted positively to the antibody. These findings indicate that the in vitro micronucleus assay coupled with immunofluorescent staining of kinetochores can be a useful method for assessing the ability of chemicals to induce aneuploidy and/or chromosome aberrations.

Induction of chromosomal aberrations and SCE by camptothecin, and inhibitor of mammalian topoisomerase I

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with camptothecin (CM), an inhibitor of mammalian topoisomerase I. While no chromosome-type aberrations were found in G1-treated cells, instead there was a dose-dependent induction of chromatid-type aberrations. These types of chromosomal alteration were not induced during the treatment itself but during the S phase, as CM is not efficiently removed with the normal washing procedure after treatment.

Chromosomal aberrations induced by restriction endonucleases

Restriction endonucleases (REs) are able to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells. The G1 phase of the cell cycle seems to be especially sensitive for the induction of chromosomal aberrations by REs. The different capacities of REs to induce chromosomal aberrations are probably correlated with the number of recognition sites in the genome.

Micronucleus test in Vicia faba root tips to detect mutagen damage in fresh-water pollution

Abstract We have developed a micronucleus test in the root tips of Vicia faba, as a first alarm system for mutagen pollution of fresh water. The test can also give analytical results in the laboratory. Secondary root tips of Vicia faba were treated with mutagens, chromatoclastic and mitoclastic ones, which are known to have different modes of action (X-rays, methyl methanesulfonate, mitomycin C, ethidium bromide and colchicine). A number of variables (dose, exposure time, interval between treatment and fixing) were considered. The patterns of the micronucleus frequencies and of the ‘generating events’ (chromosome aberrations and mitotic anomalies) were compared. The micronucleus test in Vicia faba root tips showed a broad spectrum of mutagen damage, had low control levels, a good correlation between micronuclei and generating events and a good dose-effect relationship for both. This test has the sensitivity, reliability, low cost and manageability necessary for our purposes.

Cytokinesis-block micronucleus assay with kinetochore detection in colchicine-treated human fibroblasts

A modified micronucleus assay using antikinetochore antibody has been developed in cytokinesis-blocked human fibroblasts as a simple method to identify aneuploidy-inducing agents. Different protocols for inducing binucleated cells by cytochalasin B in colchicine-treated human fibroblasts were investigated. A dose-related increase in kinetochore-positive micronuclei was obtained when cytochalasin B was given subsequent to colchicine treatment. No induction of micronuclei was observed in combined treatments of the two substances. These results indicate that the detection of kinetochores in micronucleated cytokinesis-blocked human fibroblasts can be effectively applied to the identification of environmental agents with aneuploidy-inducing potential. However, in testing such compounds particular attention should be paid to the protocol used for inducing cytokinesis-blocked cells.

The production of chromosomal alterations by β-lapachone an activator of topoisomerase I

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) after exposure to beta-lapachone, an activator of mammalian topoisomerase I, were studied in Chinese hamster cells. A dose-dependent increase in the frequencies of SCE was observed in continuous treatments with beta-lapachone. Chromatid-type aberrations were obtained in cells exposed to beta-lapachone for one cell cycle but also in cells exposed during the G2 phase of the cell cycle, with a marked induction of exchange-type aberrations for both treatment schedules. We therefore propose that activation of topoisomerase I by beta-lapachone results in the production of chromosomal alterations. The cell cycle dependence of beta-lapachone clastogenic effects strongly suggests a mechanism for the formation of chromosomal aberrations after this drug closely resembling the one observed for the topoisomerase I inhibitor, camptothecin.

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes “lost” as micro-nuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.

Genotoxic activity of nitrilotriacetic acid in Chinese hamster cells.

Nitrilotriacetic acid (NTA), a chelating agent, was tested for its ability to induce chromosomal damage in Chinese hamster cells. The chemical was shown to exert a weak genotoxic activity increasing the frequency of micronuclei after prolonged treatments. The analysis of kinetochore containing-micronuclei showed that NTA prevailingly induces chromosomal aberrations as compared to chromosome loss in hamster cells. Furthermore, immunostaining with an alpha-tubulin antibody showed clear alterations in the interphase microtubule network of cells treated for 24 h with 3 mM NTA. The microtubule effects of the chemical may be partly responsible for its cytotoxic effects.