Nobuko Suzuki

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

Aneuploidy induction in human fibroblasts comparison with results in syrian hamster fibroblasts

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.

Characterization of an unscheduled DNA synthesis assay with Syrian hamster embryo cells

The Syrian hamster embryo cell transformation assay is widely used for studies of carcinogenesis. The characterization of an unscheduled DNA synthesis (UDS) assay for these cells is reported. Benzo[a]pyrene, aflatoxin B1 and UV light induced UDS in the cells in a dose-dependent manner without exogenous metabolic activity. Nitrosopiperidine induced UDS as well as gene mutations and cell transformation only in the presence of an exogenous metabolic activation system. The utility of this UDS assay with these cells is discussed.

Comparison of human versus Syrian hamster cells in culture for induction of mitotic inhibition, binucleation and multinucleation, following treatment with four aneuploidogens.

The responses of human and rodent cells in vitro to aneuploidy-inducing chemicals were compared. Normal human fibroblasts and Syrian hamster embryo fibroblasts were treated with four aneuploidogens; Colcemid, vincristine, and the oestrogens diethylstilboestrol and oestra-1,3,5(10)-triene-3,17beta-diol (17beta-oestradiol). All compounds at a critical dose inhibited cell growth of both cell types. The concentrations of the two oestrogens required to inhibit growth of human and hamster cells were similar, whereas for the two mitotic inhibitors Colcemid and vincristine, the concentrations required for growth-inhibitory effects were lower for human cells than for hamster cells. The growth inhibition was reversible for all treatments except Colcemid. Doses that inhibited cell growth also resulted in large numbers of mitotic cells appearing in a time-dependent manner, indicating that both cell types were arrested in mitosis by all four compounds. The time required for maximum increases in the mitotic indices was greater for human cells, which is consistent with the longer cell cycle of these cells in culture. Few binucleated cells of either type were induced by any treatment except 17beta-oestradiol, which induced a high level of binucleated hamster cells, but not human cells. With time, the mitotic index of all treated cells decreased. For hamster cells, this was always accompanied by a large increase in multinucleated cells. The percentage of multinucleated hamster cells reached 50-60% in the Colcemid- and vincristine-treated cultures and 30-35% in the oestrogen-treated cultures. In contrast, the level of multinucleated human cells was significantly lower for all treatments. Colcemid and vincristine treatments induced 20-25% multinucleated human cells, and the oestrogens induced <5% multinucleated human cells. This latter finding appears to be the most significant difference between the two cell types. These results indicate that human cells respond differently from rodent cells to agents that induce mitotic arrest. This may help in understanding the decreased induction of aneuploidy in human cells by these compounds.