C. Nesti et al., Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C, MUTAGENESIS, 15(2), 2000, pp. 143-147
Primary liver fibroblasts were applied in a cytokinesis-block micronucleus
assay in combination with fluorescence in situ hybridization (FISH) using t
wo 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 standar
d 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 concent
rations tested (7.5 and 15 mu g/ml). At the highest dose (30 mu g/ml) the a
neugen effect was substantially reduced. MN induction obtained with Prot. A
was significantly higher (similar to 3-fold) than with Prot. B at the most
effective concentration. The aneugen effect induced by GF did not change w
hen 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 inc
rease in both protocols. In contrast to GF, the greater clastogenic respons
e induced by MMC in human liver fibroblasts was obtained with Prot. B, simi
lar to 3-fold higher than Prot. A at the most effective concentration and s
imilar to 2-fold with 24 h treatment at 0.17 mu g/ml MMC, With GF, the FISH
data in human liver fibroblasts (80% C + MN) were fairly consistent with t
hose 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 acentri
c fragments rather than whole chromosomes. In conclusion we have demonstrat
ed that chemically induced genetic effects are strongly dependent on the ce
ll culture employed, treatment schedule and intra- and post-treatment exper
imental conditions.