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

Abstract

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 methylmeth-anesulphonate (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 approximately 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 centromere-positive MN was low: approximately 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 non-carcinogen 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.

Validation of the micronucleus-centromere assay for biological dosimetry

“…Similar results were observed by others, not only in human lymphocytes (Huber et al, 1996;Darroudi et al, 1996;Vral et al, 1997) but also in other cell lines (Darroudi et al, 1996;Salassidis et al, 1992;Weissenborn and Streffer, 1991). Due to a relatively low degree of interindividual variability, a statistically significant difference in the frequency of MNC+ was observed between 0 and 0.25 Gy.…”

Khat (Catha edulis) consumption causes genotoxic effects in humans

“…To distinguish between clastogenic and aneugenic effects, additional experiments with a pancentromeric probe were carried out. 19 Because many khat chewers consume alcohol and tobacco in parallel, we compared for possible synergistic or additive effects in a second study. In both experimental series, data on daily consumption were based on information from the participants.…”

“…FISH with a pancentromeric probe was carried out as described earlier 19 and 1,000 -2,000 cells per individual were analyzed for the presence or absence of a centromeric signal.…”

Next generation platforms for high-throughput biodosimetry

“…Fluorescent in situ hybridization improves the detection of different types of aberrations and can be used in all cytogenetic methods recommended by the IAEA for radiation dose estimations: translocation assay (1) , dicentric assay (27) , micronucleus assay (28) and PCC assay (29) . Moreover, FISH can be used for the automation of the scoring process, because it facilitates the development of fast automated image systems (30,31) .…”