title: Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C.
abstract: Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 microg/ml). At the highest dose (30 microg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher ( approximately 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, approximately 3-fold higher than Prot. A at the most effective concentration and approximately 2-fold with 24 h treatment at 0.17 microg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.
authors.affiliation: Dipartimento Uomo e Ambiente, Universita' di Pisa, Via S.Giuseppe 22, and Istituto di Mutagenesi e Differenziamento del CNR, Via Svezia 10, 56100 Pisa, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Nesti
authors.forename: F
authors.initials: F
authors.lastname: Trippi
authors.forename: R
authors.initials: R
authors.lastname: Scarpato
authors.forename: L
authors.initials: L
authors.lastname: Migliore
authors.forename: G
authors.initials: G
authors.lastname: Turchi
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D002478:Cells, Cultured; D002875:Chromosomes / Q000187:drug effects; D004249:DNA Damage; D001728:Dicumarol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects*; D006118:Griseofulvin / Q000494:pharmacology*; D006801:Humans; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D008099:Liver / Q000187:drug effects*; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects*; D015162:Micronucleus Tests / Q000379:methods*; D016685:Mitomycin / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D006118:Griseofulvin; D016685:Mitomycin; D001728:Dicumarol