Use of the cytokinesis-block method for the analysis of munuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze munuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for munucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 μg/ml CYB for varying durations (8–48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for munucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of munuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125–1.0 μg/ml) and cyclophosphamide (2–12 μg/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in munucleus frequency over controls at the highest concentration tested (1.0 μg/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in munucleus frequency over controls at the highest tested concentration (12 μg/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze munucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.     

Endogenous sex hormones affect the mutagen-induced chromosome damage by altering a caffeine-sensitive checkpoint

In the present study we analysed the effect of endogenous sex hormones on the SCE frequencies induced in vitro by mitomycin C (MMC), a bifunctional alkylating agent producing high chromosome damage and mitotic arrest. The analysis has been performed on lymphocytes obtained at three different phases of menstrual cycle, from women with regular cycle and hormones dosage. At all phases we further analysed the effect of a post-treatment with caffeine, an agent that it is known to overrride the DNA damage checkpoints.

After MMC, the cultures obtained at ovulation and luteal phases have SCE frequencies statistically higher than the cultures obtained at the progestogenic phase, showing increases of 15 and 25%, respectively. After caffeine, the MMC treated cultures which were set up at the progestogenic phase show a high potentiation of SCE frequencies (28%) whereas the treated cultures set up at ovulatory and luteal phases show little or no potentiation.

These findings demonstrate that the endogenous hormones greatly modulate the SCE frequencies induced by the mutagen; they also indicate that hormones action competes with the caffeine effect. Caffeine acts by abrogating the mitotic arrest produced by DNA damage and induced cells with a higher chromosome damage into a premature mitosis. Our findings suggest that endogenous hormones could overcome the checkpoint controls activated in cells after mutagenic exposure. This action may be an epigenetic mechanism relevant in hormone carcinogenesis.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Rodent species and strain specificities for sister-chromatid exchange induction and gene mutagenesis effects from ethyl carbamated,ethyl N-hydroxycarbamate,and vinyl carbanate

Ethyl carbamate (EC) and two related carcinogens, ethyl N-hydroxycarbamate (ENHC) and vinyl carbamated (VC), caused species-specific increase in sister-chromatid exchange (SCE) formation in the bone marrow cells of rodents. Mice exposed to 400 mg/kg of EC had SCE increases of 6-times-baseline, while rats, Chines hamsters, and golden hamsters showed 3- to 4-times-baseline increases in response to this dose. Lesser, but still significant, differences were found for ENHC and VC; the severest effects consistently occured in mice. Control bone marrow cell-cycle kinetics among the rodent species were similar. Mouse strains A and C57BL/6, which have high and low susceptibilities to EC induction of lung adenomas, respectively, showed nearly identical levels of SCE induction after in vivo exposure to these carbamate. However, testing of VC, a possible metabolite of EC, in vitro revelaed strain-dependent liver enzyme (Aroclor-induced S-9 fraction) capabilities to convert VC to genotoxic products. SCE induction, gene mutation for 6-thioguanine and ouabain resistance, and cytotoxicity in Chinese hamster V79 cells were significantly greater when A strain S-9 enzymes were used as compared with C57BL/6 strain S-9 enzyme preparations. No effect of SCE of reseeding, compared with no reseeding, of VC-treated V79 cells was observed. At a concentration of 25 μg/ml, VC cause 6-times-baselin induction of SCE in the presence of A strain S-9 mix and 4-times-baseline induction in the presence of C57BL/6 strain S-9 mix. These in vitro strain-dependent patterns of response are relevant to the current theory that VC amy be a proximate carcinogenic metabolite of EC.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

Retinol (vitamin A) inhibition of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) induced sister-chromatid exchanges in V79 cells and mutations in Salmonella/microsome assays

When the Chinese hamster cell line V79 and the tester strain of Salmonella typhimurium TA100 were treated with the precarcinogens dimethylnitrosamine (DMN) or diethylnitrosamine (DEN) in the presence of S9 mix, a dose-dependent increase of sister-chromatid exchanges (SCE) in V79 cells and His+ revertants in TA100 resulted. DMN was a far more efficient SCE inducer than DEN, while DEN was a more efficient inducer of His+ revertants than DMN. Retinol (Rol) effectively inhibited DMN and DEN induced SCE in V79 cells and His+ revertants in TA100. Concurrent treatment of V79 cells with Rol at various doses and one dose of DMN or DEN in the presence of S9 mix caused a significant reduction of SCE as compared to SCE induced by DMN or DEN without Rol. Rol inhibition of DMN-induced SCE was dose-dependent. Rol was less efficient in inhibiting DEN-induced SCE, and no consistent dose-dependent inhibition was observed. At all doses, Rol significantly inhibited DMN and DEN induced mutation frequencies in TA100. At the highest dose of Rol (40 micrograms/plate), the inhibition of DMN and DEN induced His+ revertants reached about 90% and 60%, respectively. The possibility that Rol exerts its antimutagenic activities by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of precarcinogens such as DMN and DEN is discussed.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Cytogenetic effects induced by cytochalasin B in Chinese hamster ovary cells

We determined the kinetics of the induction of chromosomal aberrations and micronuclei (MN) by mitomycin C (MMC, 0.1 µg/ml) in Chinese hamster ovary (CHO) cells treated with cytochalasin B (Cyt-B, 3 µg/ml). In cells treated with Cyt-B as well as with Cyt-B plus MMC the highest yield of binucleated cells was obtained 24 h after treatment. After 40 h of treatment with Cyt-B the frequency of MN in binucleated cells was significantly higher than that observed at previous times in the same cultures as well as in controls. In cultures treated with MMC the frequency of MN increased with time, reaching the highest value at 24 h. The frequency of chromosomal aberrations was also significantly higher in cells treated both with Cyt-B and Cyt-B plus MMC than in controls and exceeded that of MN in parallel cultures. These data confirm the capacity of MMC to induce chromosomal alterations in mammalian cells; in particular they indicate that Cyt-B is able to induce cytogenetic effects in CHO cells. Using immunofluorescence microscopy, after reaction with CREST antikinetochore antibodies, we found that in cells treated with Cyt-B or Cyt-B plus MMC the frequency of MN without kinetochore was, respectively, about 70 and 85%, indicating that under our experimental conditions MN originate mainly from acentric chromatid fragments. Present data suggest that the method based on the blockage of cytokinesis by Cyt-B normally used in the MN assay should be reconsidered.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Sister-chromatid exchanges and thioguanine resistance in V79 Chinese hamster cells after treatment with the aneuploidy-inducing agent carbaryl +/- S9 mix

Carbaryl induced sister-chromatid exchanges (SCEs) but no thioguanine resistance in V79 Chinese hamster cells. Addition of S9 from Aroclor-pretreated rats, or glutathione, reduced the toxic effects of carbaryl. Glutathione or S9 mix reduced the effect of carbaryl on SCE. However, the latter result indicates that carbaryl's effect may be enhanced at a certain compound/S9 ratio. Since treatment with microsomes alone, but not S9 mix, was clastogenic it cannot be excluded that this enhancement of SCE was due to perturbations in the S9 mix by carbaryl rather than to formation of some particular SCE-inducing metabolite from the compound. The effects of carbaryl on chromosomes and chromosomal distribution are comparable to those sometimes reported for TPA. This, in conjunction with the weak indications on carcinogenic activity of carbaryl, makes it of interest that the compound be tested for promotion or co-carcinogenicity in vivo.     

Induction of 8-azaguanine- and ouabain-resistant mutants by cyclophosphamide and 1-(pyridyl-3)-3,3-dimethyltriazene in Chinese hamster cells cultured in diffusion chambers in mice.

A host-mediated assay is described for induction of 8-azaguanine-resistant (azgr) and ouabain-resistant (ouar) mutants in Chinese hamster V79 cells cultured in diffusion chambers (DC) in C3H mice. Injection of the hosts with the indirect mutagen/carcinogen cyclophosphamide (CPP) or 1-(pyridyl-3)-3,3-dimethyltriazene (PyDT) caused a dose-dependent increase in mutation frequency at the loci of azgr and ouar in the V79 target cells. Plating efficiency of V79 cells in DC in mice was decreased depending upon the dose of CPP or PyDT given to the hosts. In addition, the relationship between expression time and mutation frequency was examined and discussed. The data support the use of this system as an effective screening procedure for suspected environmental mutagens or carcinogens, especially those that need to be metabolically activated in vivo.     

Ultraviolet-light exposure induces a heritable sensitivity to the induction of SCE by mitomycin-C

The dose-response relationship for mitomycin-C (MMC)-induced sister-chromatid exchange (SCE) has been determined in the progeny of Chinese hamster lung fibroblasts (V79) exposed to 5.0 J/m2 ultraviolet light-C (UVC, 254 nm) and in the progeny of non-UVC-irradiated controls. Progeny of UVC-irradiated cultures exhibited sensitivity to MMC-induced SCE at doses of MMC that were not detectably lethal. This sensitivity was manifest as an increase in SCE per cell in a large proportion of the cells derived from UVC-exposed cultures and thus appears not to result from the expression of a rare event such as mutation.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6

The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species.     

A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate

6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10³ – 10⁴ cells/0.2 ml growth medium/0.4 cm²) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 10⁵ colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 10⁵ colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.