Benzene-, catechol-, hydroquinone- and phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthesis in Syrian hamster embryo cells.

Benzene is a human carcinogen present naturally in petroleum and gasoline. For the simultaneous assessment of benzene-induced carcinogenicity and mutagenicity, benzene and its principal metabolites, phenol, catechol and hydroquinone were examined for their ability to induce cell transformation and genotoxic effects using the same mammalian cells in culture. Each of the four compounds induced morphological transformation of Syrian hamster embryo (SHE) cells. Catechol was the most potent, inducing transformation at concentrations of 1-30 microM, followed by hydroquinone (3-30 microM), phenol (10-100 microM) and benzene (only at 100 microM). Gene mutations at two loci in SHE cells were induced by all four compounds, with catechol being the most potent; both ouabain-resistant and 6-thioguanine-resistant mutant frequencies were increased. Chromosomal aberrations in SHE cells were especially induced by catechol, lesser by hydroquinone, and to a marginal extent by phenol at only the 100 microM concentration, whereas sister chromatid exchanges in SHE cells occurred with hydroquinone (1-30 microM), catechol (10-30 microM) and phenol (1000-3000 microM). Aneuploidy in the near diploid range of SHE cells was significantly induced by benzene and catechol. All three metabolites induced unscheduled DNA synthesis in SHE cells, whereas benzene did not. This is the first report that the cell transforming activity and mutagenicity of benzene and its metabolites were assessed with the same mammalian cells in culture. The results provide evidence that benzene and several of its metabolites are cell transforming and genotoxic to cultured mammalian cells.

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.

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.

17beta-Estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture.

The ability of 17 beta-estradiol to induce morphological transformation of Syrian hamster embryo cells was examined and dose-dependent increases were observed over the concentration range of 1-10 micrograms/ml. However, treatment of the cells with 17 beta-estradiol failed to induce any detectable increases in gene mutations, chromosome aberrations, sister chromatid exchanges or unscheduled DNA synthesis. In contrast, over the dose range that was effective in inducing cell transformation, 17 beta-estradiol induced numerical chromosome changes (both chromosome gains and losses). These findings are similar to the reported observations with the synthetic estrogen, diethylstilbestrol, and support the hypothesis that aneuploidy induction is important in cell transformation and possibly carcinogenesis induced by estrogens.

Alteration in diethylstilbestrol-induced mutagenicity and cell transformation by exogenous metabolic activation.

It was shown previously that diethylstilbestrol (DES) can induce morphological and neoplastic transformation of Syrian hamster embryo cells in culture in the absence of detectable gene mutations or DNA damage. However, in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant, DES can induce unscheduled DNA synthesis. In this report we have examined whether with exogenous metabolic activation DES can also induce biological effects possibly related to its carcinogenicity, i.e. specific locus mutations in Syrian hamster embryo cells and cell transformation. We observed that DES was mutagenic only in the presence of exogenous metabolic activation. DES induced morphological transformation both in the absence and presence of exogenous metabolic activation. Enhanced cell transformation was observed in the presence of exogenous metabolic activation. These results indicate that two pathways may exist for the induction of cell transformation by DES. One does not apparently involve direct DNA damage, and the other, which requires rat liver post-mitochondrial supernatant-mediated exogenous metabolic activation, is associated with DNA damage and mutagenicity. These results may provide an experimental model to elucidate the biological properties of DES metabolites.

Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts.

The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.

Vincristine sulfate-induced cell transformation, mitotic inhibition and aneuploidy in cultured Syrian hamster embryo cells.

Vincristine, a naturally occurring Vinca alkaloid and widely used anti-neoplastic agent, was examined for its ability to induce cell transformation, inhibition of growth and mitosis, and genetic effects in Syrian hamster embryo cells in culture. Treatment of the cells with doses of less than or equal to 1 ng/ml vincristine sulfate (VCR) had no effect on cell growth, while exposure to greater than or equal to 3 ng/ml reduced the growth rate and treatment with 30 ng/ml resulted in no detectable increase in cell number. At this latter dose the mitotic index of the cells increased significantly suggesting that VCR delayed completion of mitosis. Exposure of the cells to VCR at doses of 1-10 ng/ml for 48 h resulted in morphological transformation of the cells in a doserelated fashion. The vincristine-treated transformed colonies were morphologically indistinguishable from colonies transformed by benzo[a]pyrene or other chemical carcinogens. Morphological transformation was induced by VCR at non-toxic and slightly toxic doses as measured by a reduction in colony-forming ability of the treated cells. Over the dose range which resulted in cell transformation, VCR failed to induce either detectable gene mutations at two genetic loci, unscheduled DNA synthesis, or chromosome aberrations in the Syrian hamster embryo cells. However, a significant dose-dependent increase in aneuploid cells with a near-diploid chromosome number was induced by VCR. Both chromosome losses and gains were induced which is consistent with a non-disjunctional mechanism. These results further support our hypothesis that aneuploidy is one possible mechanism for induction of this early step in the neoplastic transformation of Syrian hamster embryo cells. Furthermore, these findings indicate that VCR may have some carcinogenic potential if exposure to rapidly dividing cells occurs.

Induction by modified purines (2-aminopurine and 6-N-hydroxylaminopurine) of chromosome aberrations and aneuploidy in Syrian hamster embryo cells.

The modified purines, 2-aminopurine and 6-N-hydroxylaminopurine, are known point mutagens in prokaryotic organisms. 2-Aminopurine is much less potent than 6-N-hydroxylaminopurine in inducing gene mutation in mammalian cells in culture and this corresponds to the relative activity of these two compounds in inducing tumors in rats and neoplastic transformation of Syrian hamster embryo cells in culture. We report here that these modified purines can induce chromosome aberrations, including chromatid gaps, breaks, and exchanges, as well as numerical chromosome changes in Syrian hamster embryo cells. These chromosome mutations occur over the concentration range of chemical needed to induced morphological transformation of the same cells. It is not known how nucleic base analogs induce chromosome mutations; however, this activity must be considered in attempting to understand the mechanism by which these agents induce neoplastic transformation of cells.

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.

Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S phase).

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1-10 microM), followed by irradiation with near-UV (5-10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival.

Amitrole-induced cell transformation and gene mutations in Syrian hamster embryo cells in culture.

Amitrole, a widely used herbicide, is an animal carcinogen and an inducer of cell transformation. However, it is inactive as a mutagen in bacterial test systems. Thus, it has been suggested that amitrole is a non-mutagenic carcinogen. Over the dose range that induces morphological transformation of Syrian hamster embryo cells in culture, amitrole induced gene mutations at the Na+/K+ ATPase and hypoxanthine phosphoribosyl transferase loci measured concomitantly in the same cells. These results indicate that amitrole may act via a mutational mechanism.

Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride.

The effect of treatment of cultured human oral keratinocytes with sodium fluoride (NaF) has been investigated with respect to induction of unscheduled DNA synthesis (UDS). Oral keratinocytes were isolated from excised buccal mucosa of normal individuals by trypsinization at 4 degrees C overnight, followed by separation of the epithelium of mucosa from lamina propria mucosae with forceps. Isolated cells were cultured in vitro and all experiments were performed with secondary cultures. For detection of UDS, the keratinocytes were cultivated with medium containing 1% fetal calf serum (FCS) for 2 days and then treated with 100-300 micrograms/ml NaF for 4 h in medium containing 1% FCS and 10 mM hydroxyurea (1% FCS-HU medium). Following treatment with NaF, UDS was measured by direct scintillation counting of [3H]thymidine incorporated into DNA of the cells in 1% FCS-HU medium. Significant levels of UDS were induced in a dose-related fashion by NaF treatment. The results suggest that NaF causes DNA damage in cultured human oral keratinocytes.

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.

Dependence on exogenous metabolic activation for induction of unscheduled DNA synthesis in Syrian hamster embryo cells by diethylstilbestrol and related compounds.

Diethylstilbestrol (DES) induces morphological and neoplastic transformation of Syrian hamster embryo cells in vitro in the absence of any measurable induction of gene mutations, which is consistent with the lack of genotoxicity of DES in a number of other assays. However, a few reports of a genotoxic activity of DES in certain systems have been published. In order to understand these differences, we have investigated whether DES induces unscheduled DNA synthesis (UDS) in Syrian hamster embryo cells under the conditions which result in cell transformation and have examined the role of an exogenous metabolic activation system on DES-induced UDS. DES, over a concentration range of 1 to 10 micrograms/ml, failed to induce any detectable UDS in the cells, while other known transforming agents, including UV irradiation (6 to 24 J/sq m), benzo(a)pyrene (0.1 to 1.0 micrograms/ml), and aflatoxin B1 (10 to 100 micrograms/ml), induced significant levels of UDS. In contrast, UDS was induced in a dose-dependent manner by DES (1 to 10 micrograms/ml) after addition of an Aroclor-induced rat liver postmitochondrial supernatant fraction and other cofactors for exogenous metabolic activation. In order to probe the basis for this alteration in UDS induction, the ability of structural analogues and metabolites of DES to induce UDS was examined. In the absence of exogenous activation, the only oxidative metabolite of DES detected in the presence of the cells was cis,cis-dienestrol, which did not induce UDS by itself. In the presence of exogenous activation, cis,cis-dienestrol and its trans,trans-isomer induced UDS but not to a greater extent than DES. With the addition of the exogenous metabolizing system, increased metabolism of DES to cis,cis-dienestrol and additional polar derivatives of DES or dienestrol, possibly hydroxylated derivatives, were observed. With exogenous metabolic activation, tetrafluoro-DES and hexestrol, which differ in their ability to be peroxidatively metabolized to quinone and phenoxyradical intermediates, both induced UDS, although tetrafluorodiethylstilbestrol at 10 micrograms/ml stimulated a higher level of UDS. None of the DES-related compounds examined was active in the UDS assay without exogenous metabolic activation, but all of the compounds can potentially form phenoxyradical intermediates by a peroxidase-mediated reaction. The compounds which can be further oxidized to a quinone were most active in inducing UDS. These results are consistent with the hypothesis that this peroxidase-mediated pathway is important in the induction of UDS, although secondary metabolites may also be involved.(ABSTRACT TRUNCATED AT 400 WORDS)