Induction of chromosomal aberrations (unstable and stable) by inhibitors of topoisomerase II, m-AMSA and VP16, using conventional Giemsa staining and chromosome painting techniques

Mutagenesis vol.13 no.l pp.39-43, 1998 Induction of chromosomal aberrations (unstable and stable) by inhibitors of topoisomerase II, m-AMSA and VP16, using conventional Giemsa staining and chromosome painting techniques P.Mosesso1, F.Darroudi2*3, M.van den Berg2, S.Vermeulen2, F.Palitti1 and A.T.Natarajan2'3'4 'Dipartimento di Agrobiologia e Agrochirruca University degli studi dclla Tuscia, Via San Camillo de Lellis, Viterbo 1-01100, Italy, 2MGC, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands and 3 J.A.Cohen Institute of Radiopathology and Radiation Protection, Interuniversity Institute, Leiden, The Netherlands Frequencies of symmetrical and asymmetrical exchange aberrations induced by two inhibitors of topoisomerase II, namely, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) and etoposide (VP16), were estimated in human peripheral blood lymphocytes. The aberrations were scored using conventional Giemsa staining and fluorescence in situ hybridization (FISH) techniques, using chromosomespecific DNA libraries. Stable aberrations (translocations) were detected using two cocktails of DNA libraries specific for three chromosomes, namely 1, 3 and X and 2, 4 and 8, representing ~40% of the whole human genome. The frequencies of dicentrics and translocations increased in a dose-dependent manner, however, m-AMSA was found to be a more potent inducer of chromosomal aberrations in comparison with VP16 (at concentrations at which comparable frequencies of aberrations were induced) by 20- to 30-fold. When corrected for DNA content of chromosomes in each cocktail, a higher frequency of translocations with the cocktail consisting of chromosomes 2, 4 and 8 in comparison with 1, 3 and X was evident The genomic translocation frequency calculated from chromosome painting analysis for m-AMSA exceeded that estimated for dicentrics by ~2-fold. However, for VP16 almost equal frequencies of both types of chromosome exchange were found. Introduction Environmental and occupational exposure of man to genotoxic agents is a major risk factor in carcinogenesis. Estimation of the level of exposure is a important parameter for the determination of possible deleterious health effects. In most studies with human peripheral blood lymphocytes the frequency of chromosomal aberrations, especially dicentrics, is used as the biological end point. For ionizing radiation this has been used since the 1960s to estimate exposure dose (Bender and Gooch, 1966; Buckton et ai, 1967). Following exposure to genotoxic chemicals such as vinyl chloride, styrene or benzene, quantification of chromosomal aberrations has also been used as a biomarker. Evidence that elevated frequencies of chromosomal aberrations in peripheral blood lymphocytes can predict an increased cancer risk is provided by the reports of the Nordic cohort study of cancer incidence and its correlation with chromosomal aberrations, sister chromatid exchange and micronuclei frequencies. The chromosomal aberration frequencies were found to be most closely correlated with cancer incidence in comparison with other two assays (Hagmar et ai, 1994). Generally, the majority of observed chromosomal aberrations in Giemsa stained preparations are dicentrics, rings and acentric fragments which are unstable aberrations and will lead to cell death during proliferation. Therefore, cells containing these types of aberrations decrease with post-exposure time. Balanced symmetrical chromosome exchanges (translocations) may persist for a longer time and therefore can provide an estimate of cumulative frequencies of chromosome aberrations. From the human point of view, stable aberrations such as translocations are very important, since they are associated with congenital abnormalities in newborns as well as in neoplasms of humans (Mitelman etal., 1991). The introduction of the fluorescent in situ hybridization (FISH) technique with chromosome-specific composite DNA probes (Pinkel et ai, 1986; Lichter et ai, 1988; Natarajan et ai, 1991, 1992) has opened up the possibility of accurately and rapidly detecting numerical and structural chromosome aberrations. When human lymphocytes are irradiated in vitro with Xrays and scored for the frequencies of dicentrics and (reciprocal, terminal and interstitial) translocations using FISH and chromosome-specific DNA probes, it has been found that the frequency of translocations is 1.5-3 times greater than dicentrics (Cremer et ai, 1990; Natarajan et ai, 1992; Schmid et ai, 1992; Bauchinger et ai, 1993; Tucker et ai, 1993). For the radiomimetic and chemotherapeutic agent bleomycin the frequency of symmetrical exchanges was also higher (~2.5-fold) than asymmetrical ones (Hoffmann et ai, 1994; Ellard, et ai, 1995). At present, for screening and evaluation Giemsa stained preparations are used to detect potential carcinogens, by analysing predominantly chromatid exchanges and breaks, because most of the chemicals act in a S-dependent way. The two inhibitors of topoisomerase II 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) and etoposide (VP16) are reported to act like S-independent agents (i.e. ionizing radiation), leading to induction of chromatid and chromosometype aberrations following treatment in Gj and G\ respectively in Chinese hamster ovary cells (CHO wild type and xrs mutant) and human lymphocytes (Andersson and Kihlman, 1989; Darroudi and Natarajan, 1989). Furthermore, it appears that anti-topoisomerase-induced DNA strand breaks are not resealed by the normal repair pathways (Zwelling and Mattem, 1982; Zwelling et ai, 1982). While arabinofuranosylcytosine (ara-C) post-treatment strongly enhances the frequency of chromosome-type aberrations induced by X-rays (Preston, 1980), it does not affect the frequency in m-AMSA-treated Go or Gi lymphocytes (Andersson and Kihlman, 1989), pointing to a difference in the processing of induced strand breaks by these two agents. *To whom correspondence should be addressed at: MGC, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands. Tel: +31 71 5276168; Fax: +31 71 5221615; Email: © UK Environmental Mutagen Society/Oxford University Press 1998 39 P.Mosesso et aL In the present paper the relative frequencies of symmetrical and asymmetrical chromosome exchanges induced in GQ human peripheral lymphocytes treated in vitro with two chemotherapeutic drugs, m-AMSA and VP-16, inhibitors of DNA topoisomerase II, were estimated. The frequencies of chromosome exchanges, i.e. dicentrics and translocations, following Giemsa staining and chromosome painting techniques respectively were determined. A comparison was made between different types of aberrations induced by these two chemicals. Materials and methods Cell culture and treatment conditions A whole blood sample was collected from a healthy male donor in a hepariniz (...truncated)