Makiko M. Hasegawa

Comparative studies on chromosome aberrations induced by polycyclic hydrocarbons in cell-mediated and microsome-mediated assay

Studies were made on chromosome changes in Chinese hamster V79 cells induced by the polycyclic hydrocarbons, benzo[alpha]pyrene (BP), 3-methylchol-anthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA) in the presence and absence of cell-mediated and microsome-mediated activation systems. In cell-mediated assay, BP, MC and DMBA induced concentration-dependent chromosome aberrations in V79 cells, at concentrations of 0.5--1.0 to 10.0 micrograms/ml in the presence of lethally irradiated Syrian hamster embryonal feeder cells at 2.0 X 10(6) cells/60-mm dish. The highest incidences of cells with aberrant chromosomes, observed at concentrations of 20 micrograms/ml, were 24.0% (BP), 23.0% (MC) and 80.0% (DMBA). In the absence of feeder cells, these chemicals did not induce chromosome aberrations in V79 cells, even at the maximum concentrations tested, only background levels being observed. In microsome-mediated assay, BP, MC and DMBA at 20 micrograms/ml also induced chromosome aberrations when combined with microsomes. However, the highest incidences of aberrations were observed with lower amounts of microsomes than usual, the optimal amount of microsomes ranging from 1 to 10%. In this range, the highest incidences of cells with aberrant chromosomes were 10.0% (BP), 9.0% (MC) and 28.0% (DMBA), that is about half to one-third those observed in cell-mediated assay.

Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells

Abstract The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HD) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and morphological transformations is examined. Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to analyze chromosome aberrations and SCEs. 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dose-dependent fashion, with LD being more potent. In the transformation test, LD tar also induced a significant number of Type III foci, while HD was only weakly active. The transformed cells isolated from these Type III foci produced tumors when injected into nude mice. These results show that LD possesses clear clastogenic and transforming capabilities but that HD is weaker in this regard.

Induction of 8-azaguanine or ouabain resistant somatic mutation of Chinese hamster lung cells by treatment with tryptophan pyrolysis products☆

The basic fraction of tryptophan pyrolysis products (TBF) showed strong mutagenic activity on somatic cells of the lung of Chinese hamsters. In this somatic mutation test, TBF was demonstrated to have 5.6 times higher mutagenicity than diethylnitrosamine (DEN) when mutants were selected with 8-azaguanine, and 13.5-fold higher mutagenicity than DEN when mutants were selected with ouabain. From these findings, it is suggested that pyrolyzates of amino acids may have mutagenic actions on somatic cells of animals, as well as carcinogenic actions.

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact. V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (greater than 80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture. The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture. In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive. Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of inocula on plating up to 1-2 X 10(6) cells per 9-cm dish: in liquid culture, even with a lower plating number (2 X 10(5) cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 X 10(6) cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.

Genetic variations in baseline and ultraviolet light-induced sister chromatid exchanges in peritoneal lymphocytes among different mouse strains

The variation in the induction of sister chromatid exchanges (SCEs) in the peritoneal lymphocytes of different mouse strains was investigated. For the baseline SCEs, BALB/c and outbred ICR showed the lowest frequency and DBA/2 and C57BL/6 the highest. BDF1 (C57BL/6 x DBA/2) was ranked among the highest, while CDF1 (BALB/c x DBA/2) was intermediate between the parental strains. Regarding UV-induced SCEs, BALB/c was less susceptible as compared to DBA/2 and C57BL/6. Both BDF1 and CDF1 showed values significantly higher than BALB/c, but not significantly different from DBA/2 or C57BL/6. ICR was ranked in the susceptible group. For the baseline SCEs of bone marrow cells, the overall ranking among strains was essentially the same as that for the baseline, but different from that for the UV-induced, SCEs in peritoneal lymphocytes. The present results can be explained by assuming that the major genetic factor contributing to the strain-dependent difference in the baseline SCEs is due to a codominant trait of a single allele, but that the UV-induced SCEs are complicated by other genetic factor(s).

Induction of 8-azaguanine-resistant mutation and neoplastic transformation of hamster embryonic cells by coadministration of sodium nitrite and aminopyrine

SummaryHamster embryos in utero on the 11th or 12th day of gestation were treated simultaneously with aminopyrine (Ap) and sodium nitrite (NaNO2) by oral administration of the compounds to the mothers by stomach tube. For measurement of induction of 8 AG-resistant mutations, the embryonic cells from treated and control mothers were cultured in MEM plus 10% FBS for 72 h and then selected in medium containing 10 or 20 μg/ml of 8 AG. The number of 8 AG-resistant colonies was markedly increased after co-administration of Ap and NaNO2, and slight induction of mutations was also observed in cells from mothers given NaNO2 alone. This treatment also caused morphological or malignant transformation of cultured cells. About 5-to 6-fold increase in the number of transformed colonies was observed in cells from mothers given Ap plus NaNO2. Cells from the transformed colonies produced tumors when implanted into the cheek pouches of young golden hamsters. These tumors were diagnosed as pleomorphic fibrosarcomas. Similar results were obtained with cells from embryos treated transplacentally with NDMA as positive controls. A single transplacental oral application of Ap at 200 mg/kg or of NaNO2 had only slight biological actions to the cultured embryonic cells. NDMA was produced in the stomach of animals treated simultaneously with Ap and NaNO2. A small amount of NDMA was also detected in the stomach after a single dose of NaNO2.

Increase during passage in culture in susceptibility of syrian hamster embryonic cells to induction of chromosome aberrations by 7,12-dimethylbenz(a)-anthracene

Studies were made on chromosome aberrations in Syrian hamster embryonal fibroblasts (SHEF) at early and late passages in culture. Aberrations were induced with a potent indirect clastogen, 7,12-dimethylbenz(a)anthracene (DMBA). Results showed that cells at late passages were much more susceptible to this chemical than cells at early passages: the frequency of aberrant metaphases was usually less than 20% at the 2nd passage, but it gradually increased with increase in the number of culture passages, reaching 50% or more at the 8th passage in one experiment and 6th-7th passage in another. Induction of chromosome aberrations was also examined in V79 cells co-cultured with hamster cells at early and late passages. V79 cells are known to have no drug-metabolizing enzyme activity. Results demonstrate that the frequency of chromosome aberrations of V79 cells was much greater in the presence of hamster cells at late passages than in the presence of cells at early passages.