Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe

F.Darroudi 0 2 C.M.Meyers 0 2 V.Hadjidekova 0 2 A.T.Natarajan 0 2 0 Radiation Genetics and Chemical Mutagenesis 1 J.A.Cohen Institute of Radiopaihology and Radiation Protection , Leiden, The Netherlands 2 'MGC, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden , Wassenaarseweg 72, 2333 AL, Leiden - In human hepatoma (Hep G2) cells and peripheral blood lymphocytes (HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization (FISH) assays were applied to study aneugenic and clastogenic potentials of X-rays, directly and indirectly acting chemicals. Induction of MN was studied in vitro following treatment with X-rays, directly acting chemicals, such as methylmethanesulphonate (MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastine sulphate (VBS), and indirectly acting agents, such as cyclophosphamide (CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on the presence of the fluorescent signal in the MN following FISH with a human DNA centromeric probe, MN in the binucleated Hep G2 cells and lymphocytes were scored as centromere-positive or centromere-negative, representing an aneugenic and clastogenic event respectively. In the controls -50% of spontaneously occurring MN were centromere-positive. Treatment of human hepatoma cells and HPBL (in vitro) with potent aneugens such as COL, VCS and VBS increased the number of MN in a dose-dependent manner; of these 75-93% were centromere-positive. X-irradiation induced MN in a dose-related manner in binucleated Hep G2 cells and HPBL, of which 33-40% were centromere-positive, which demonstrates the significant aneugenic potentials of X-rays. Strong clastogenic activity was observed with MMS and frequency of centromerepositive MN was low: -20 and 30% for HPBL and Hep G2 cells respectively. In Hep G2 cells significant aneugenic activity was found with indirectly acting promutagens/ procarcinogens such as HMPA and 2-AAF, in contrast to CP, which came out as a potent clastogen. The noncarcinogen 4-AAF was not able to induce an increase in the frequency of MN in Hep G2 cells. All indirectly acting chemicals tested came out negative when HPBL were used as targets for DNA damage. The results presented correlate positively with data from in vivo assays and indicate that the Hep G2 cell system is a suitable bioactivation system (in vitro) for evaluating the clastogenic and aneugenic potentials of chemicals which require exogenous metabolic activations in order to exert their mutagenic potential. Introduction The need to develop reliable experimental assays to detect aneuploidy has been widely recognized in view of the Aneuploidy can result from malsegregation of chromosomes, disruption of spindle, dissociation of microtubules and inactivation of centrioles. At present different biological end-points are being used to study aneuploidy in vitro and in vivo, such as counting the number of chromosomes (Dulout and Natarajan, 1987; Sbrana et al, 1993) and micronuclei (MN), and MN in binucleated cells (Fenech and Morley, 1985). The micronucleus assay has further been used in combination with CREST staining (Brinkley et al, 1985; Degrassi and Tanzarella, 1988; Thomson and Perry, 1988; Eastmond and Tucker, 1989; Fenech and Morley, 1989; NUsse et al, 1989; Gudi et al, 1990; Miller and Adler, 1991), C-banding (Verschaeve et al, 1988), mouse satellite DNA probes (Miller et al, 1991; Salassidas et al, 1992; Farooqi et al, 1993; Chen et al, 1994; Hayashi et al, 1994) and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes (Raimondi et al, 1989; Eastmond and Pinkel, 1990; Eastmond et al, 1994) to identify the presence of centromere in MN in order to discriminate aneugens from clastogens in vivo and in vitro. However, in vitro tests have the drawback of detecting only the aneugenic and clastogenic potential of mainly directly acting chemicals, and not those of indirectly acting chemicals. The latter may be detected in vitro by the addition of rat liver S9 fractions (Natarajan and van-Kesteren van-Leeuwen, 1981) and the use of metabolically competent genetically engineered cell lines (Crespi et al, 1989). We have used an established human hepatoma cell line (designated as Hep G2) to standardize and validate this assay in mutagenicity testing (Natarajan and Darroudi, 1991; Darroudi and Natarajan, 1993, 1994). The results obtained revealed that human Hep G2 cells are capable of activating different classes of mutagens to form biologically active metabolites, and a positive correlation was found between positive and negative data in human Hep G2 cells (in vitro) and those found in vivo. Two indirectly acting chemicals, hexamethylphosphoramide (HMPA) and safrole, which are known to be carcinogens in vivo (Purchase and Ray, 1981), always came out negative in vitro in mammalian cells F.Darroudi el al (Natarajan and van-Kesteren van Leeuwen, 1981) and in the Salmonella assay (Ashby et al, 1985) in the presence of rat liver S9 fractions. In contrast, positive mutagenic potential for both chemicals was found in human hepatoma cells and Chinese hamster ovary (CHO) cells in vitro in the presence of S9 fractions isolated from Hep G2 cells, using sister-chromatid exchanges, MN in binucleated cells, cytotoxicity and gene mutations assays as biological end-points (Natarajan and Darroudi, 1991; Darroudi and Natarajan, 1993, 1994). In the present study we employed the micronucleus assay in binucleated cells in vitro in combination with a human centromere-specific probe and FISH to study aneuploidy in human Hep G2 and peripheral blood lymphocytes (HPBL). The suitability of these assays was tested using known aneugens and clastogens, and further applied to detect the aneugenic and clastogenic potentials of several indirectly acting carcinogenic and noncarcinogenic chemicals. Materials and methods Cells and culture conditions Human hepatoma cell line (Hep G2). We used a cell line originally established from human liver tumour biopsy designated as Hep G2 (Aden et al., 1979; Natarajan and Darroudi, 1991). The morphological characteristics and epithelial cell shape are compatible with those of liver parenchymal cells (Aden et al., 1979). We applied this cell line in our investigation as the metabolic activation source and the target cells for DNA damage. Hep G2 cells were grown as monolayers in Dulbecco's minimum essential medium (Boehnnger, Germany) supplemented with 15% fetal calf serum (Gibco, UK) and antibiotics. The cultures were incubated at 37C in a 5% CO2 atmosphere. HPBL. Blood was obtained by venipuncture from healthy female volunteers using a hepannized vacutainer and 0.5 ml of whole blood per culture Lymphocytes were stimulated to grow with phytohaemagglutinin (Wellcome) and cultured in medium consisting of F10 (Boehnnger, Germany), 20% heat inactivated (at 56C for 30 mm) fetal calf serum (Gibco, UK), L- (...truncated)