No abstract

The human in vitro micronucleus (MN) test has become a fast and reliable assay for mutagenicity testing. Currently, this assay is mostly performed with cytochalasin B, which prevents cytokinesis, resulting in polynucleated cells. The number of nuclei per cell indicates the number of nuclear divisions that have occurred since the addition of cytochalasin B. It is recommended that MN are only counted in binucleated lymphocytes, because these cells have finished one nuclear division. Therefore, almost no attention has been paid to MN in mononucleated cells. However, recent studies have indicated that aneugens, but not clastogens, also induce MN in mononucleates. In order to evaluate mononucleates to distinguish between aneugenic and clastogenic effects, we tested some typical aneugens and clastogens in whole blood lymphocyte cultures of four donors with the cytokinesis block micronucleus (CBMN) assay. Results showed that the aneugens diethylstilbestrol (80 microM), griseofulvin (25 microg/ml) and vincristine sulphate (15 microg/ml) increased MN frequencies in mononucleated and binucleated cells, whilst the clastogens mitomycin C (500 ng/ml), bleomycin (6 microg/ml) and doxorubicin (20 microg/ml) increased MN frequency only in binucleates. We also tested the Y heterochromatin decondensing drug berenil (300 microg/ml). Berenil induced an extremely high number of MN in mononucleated as well as in binucleated cells, indicating an aneugenic action. This was confirmed by centromere labelling. The results suggest that MN in mononucleates may be an interesting additional parameter in the CBMN assay. Future studies should clarify whether the micronucleated mononucleate cells have escaped the cytokinesis block and become polyploid.

Tucker, 1989) or by FISH with pan-centromeric DNA probes D-67653 Kaiserslautern, Germany (Becker et al., 1990, Miller et al., 1992. The drugs that have Cultures of human blood lymphocytes from three subjects been studied in most detail for their mechanism of MN were incubated with the clastogen mitomycin C (MMC, induction are the clastogen mitomycin C (MMC) (e.g. Miller 500 ng/ml) and the aneugen diethylstilboestrol (DES, and Adler, 1990;Miller et al., 1991; Rudd et al., 1991; 80 µM) 23 h before harvesting, to induce formation of Surrallés et al., 1995) and the aneugen diethylstilboestrol micronuclei (MN) and numerical and structural alterations (DES) (e.g. Eastmond and Tucker, 1989;Migliore et al., 1996). in metaphase chromosomes. We used fluorescence in situ Many studies have addressed the mechanisms of MN induchybridization (FISH) with painting probes for all human tion (see above), but only a few investigators have examined chromosomes to determine which chromosomes had conthe chromosomal content of MN. The latter studies have shown tributed material to the induced MN. MMC treatment that some mutagens induce preferential occurrence of particular induced an~18-fold increase in MN and led to a significant chromosomes in MN. Chromosomes 1, 9, 15, 16 and Y were increase in hypodiploidy and structural chromosome found in elevated frequencies in 5-azacytidine-induced MN aberrations in metaphase preparations. Undercondensation (Guttenbach and Schmid, 1994; Fauth et al., 1998), chromoof pericentromeric heterochromatin of chromosomes 9 and some 9 in idoxuridine-induced MN (Tommerup, 1984; 1 occurred in 20-75% of metaphases and FISH disclosed Fauth and Zankl, 1999), chromosome 7 in colcemid-induced an abundance of material from these chromosomes in MN (Wuttke et al., 1997) and acrocentric chromosomes in induced MN (62-69% from chromosome 9 and 7-12% from vanadium-and colcemid-induced MN (Migliore et al., 1995; chromosome 1). DES treatment of lymphocytes induced a Caria et al., 1996). seven-fold increase in MN frequency and four-fold increaseUsing FISH analysis with whole chromosome painting in the frequency of numerical aberrations; structural probes (WCPs) for all human chromosomes (Fauth et al., aberrations were not significantly increased. FISH analysis 1998), we investigated which chromosomes had contributed showed that material from all chromosomes was present material to induced MN in MMC-and DES-treated short-term in DES-induced MN, with material from chromosome 1 human lymphocyte cultures. WCPs target the euchromatic present in 16% of MN and material from each other parts of a chromosome and thereby disclose both whole chromosomes being present in 2-10% of MN. Material chromosomes and acentric fragments in MN. However, they from chromosomes 14, 19 and 21 was significantly more fail to distinguish between an entire chromosome or material frequent material from chromosome Y significantly less from a large chromosomal fragment(s) in a particular MN. frequent in DES-treated cells than in controls. The findings...

No abstract

Acrylamide (AA) is a carcinogen as demonstrated in animal experiments, but the relevance for the human situation is still unclear. AA and its metabolite glycidamide (GA) react with nucleophilic regions in biomolecules. However, whereas AA and GA react with proteins, DNA adducts are exclusively formed by GA under conditions simulating in vivo situations. For risk assessment it is of particular interest to elucidate whether AA or GA within the plasma concentration range resulting from food intake are "quenched" by preferential reaction with non-critical blood constituents or whether DNA in lymphocytes is damaged concomitantly under such conditions. To address this question dose- and time-dependent induction of hemoglobin (Hb) adducts as well as genotoxic and mutagenic effects by AA or GA were studied in human blood as a model system.