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 (azg^r) and ouabain-resistant (oua^rr) 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 azg^r and oua^r 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.     

Induction of mitotic gene conversion by 3,3-dimethyl-I-phenyltriazene, I-(3-hydroxyphenyl-)-3,3-dimethyltriazene and by I-(4-hydroxyphenyl)-3,3-dimethyltriazene in Saccharomyces cerevisiae

The induction of mitotic gene conversion by 3,3-dimethyl-1-phenyltriazene (DMPT), 1-(3-hydroxyphenyl)-3,3-dimethyltriazene (3-HO-PDMT) and by 1-(4-hydroxyphenyl)-3,3-dimethyltriazene (4-HO-PDMT) in the diploid strain D4 of Saccharomyces cerevisiae was investigated. The frequencies of the non-reciprocal intragenic recombinations at two unlinked loci ade2 (adenine) and trp5 (tryptophan) were determined. Although all three triazenes showed marked convertogenic activities, significant differences in their genetic effectiveness have been observed. Thus both phenolic triazenes were found to be much stronger convertogens than the unhydroxylated parent compound, DMPT. An attempt is made to account for the established differences in convertogenicity by chemical reactivity that could be expected from the structural features of the tested alkaryltriazenes.     

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

Genetic characterization of ad-3 mutants induced by chemical carcinogens, I-phenyl-3-monomethyltriazene and I-phenyl-3,3-dimethyltriazene, in Neurospora crassa

180 ad-3 mutants of Neurospora crassa induced by 1-phenyl-3-monomethyl-triazene (PMMT) and 56 ad-3 mutants induced by 1-phenyl-3,3-dimethyltriazene (PDMT) were characterized by dikaryon, trikaryon and complementation tests. Results show that the spectrum of genetic alterations induced by PMMT is different from that of PDMT. This suggests that enzymatic dealkylation of PDMT to PMMT does not occur within Neuropsora crassa conidia, and that the mechanism of mutation induction of PDMT in N. crassa is different from that of PMMT. Hydrolytic breakdown products or its intact molecule or some other converted forms might be responsible for the mutagenic activity of PDMT.Mutation induction of PMMT in N. crassa appears to be via alkylation of DNA by carbonium ions produced by this compound, the same mechanism proposed for its carcinogenic activity. The frequencies of leakiness, allelic complementation and nonpolarized complementation patterns among PMMT-induced ad-3 mutants are similar to those of ad-3 mutants induced by other potent chemical carcinogens, such as MNNG and the aflatoxins.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Induction of heavy metal binding groups by dexamethasone and Zn in cultured Chinese hamster ovary cells

Cd binding capacity and pulse polarography were used to study the inducibility of sulfhydryl groups in cultured Chinese hamster ovary cells (wild type and a Cd-resistant mutant) in response to dexamethasone (dex) and Zn. Evidence is presented that both the wild type and the mutant responded to dex and Zn treatment by induction of sulfhydryl groups. In wild type for Zn and dex as well as in the mutant for dex, this induction seems to be in the form of sulfhydryls attached to particulate or membrane fractions in the cells. For Zn in the Cd-resistant mutant the induction was in the form of metallothionein.     

Induction of polyamine limitation in Chinese hamster ovary cells by α-methylornithine

Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of α-methylornithine (αMO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the αMO-treated cells into medium containing the inhibitor resulted in steady-state (exponential) growth at cell densities below 5 × 10³ cells/cm², at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of αMO-treated cultures. Thus, growth rate correlated with polyamine content in the αMO-treated cells. The growth of reseeded. αMO-treated cells became nonexponential at a density (5 × 10³ cells/cm²) far below that at which untreated cells departed from exponential growth (1 × 10⁵ cells/cm²). Medium obtained from high density, αMO-treated cultures inhibited the growth of cells at low density in the presence of αMO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine-limited cells. This is a variable that must be controlled in studies with polyamine-limited animal cells. Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with αMO. The maintenance of mitochondrial integrity in the presence of αMO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. αMO-treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.     

Induction of chromosomal aberrations in cultured Chinese hamster cells in a superoxide-generating system

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.     

Induction of mutations resistant to 6-thioguanine by fast neutrons in cultured Chinese hamster cells

A study was made of induction of mutations, resistant to 6-thioguanine (TGr), and reproductive death of Chinese hamster cells after irradiation by fission-spectrum fast neutrons (mean energy of 0.75 MeV) with doses of 10-130 cGy. A high relative biological effectiveness (RBE) of fast neutrons was shown. The maximum RBE values (13-16) were within the dose range inducing minimum mutagenic and lethal effects. RBE decreased with the dose increase. Inspite of high mutagenic effectiveness of neutrons, estimated according to TGr mutation frequency per cell per dose unit, their relative mutagenic effectiveness, estimated per cell per one lethal event, did not substantially differ from that of X-radiation.     

Mechanisms of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (Dacarbazine) cytotoxicity toward Chinese hamster ovary cells in vitro are dictated by incubation conditions

Decomposition of the antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, Dacarbazine) produces several potentially toxic compounds, the concentration of which depend on incubation parameters such as pH, temperature and illumination. The action of DTIC on chinese hamster ovary (CHO) cell clone formation in the dark (7-8-day incubation) reflects the slow formation of 2-azahypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8)-deficient cells are resistant to DTIC under these conditions, reflecting their inability to utilize 2-azahypoxanthine. The toxicity of DTIC in conventional survival experiments (1-2-h exposure to drug) is dependent upon illumination and is highly influenced by the pH of the medium. Toxicity of DTIC in these experiments appears to reflect rapid accumulation of the immediate photodecomposition product of the drug, 4-diazoimidazole-5-carboxamide (DZC), since HGPRT-deficient cells are not resistant to DTIC under these conditions. The biologically initiated pathway of DTIC action (enzymatic hydroxylation) has little, if any, role in the action of this agent toward cultured CHO cells.     

Genetics of somatic mammalian cells. XVII. Induction and isolation of Chinese hamster cell mutants requiring serine

The induction, isolation and some of the properties of serine-requiring mutants of Chinese hamster ovary cells have been described. These cells promise to be useful in biochemical genetic studies and cancer research.     

Yield of SCEs and translocations produced by 3 aminobenzamide in cultured Chinese hamster cells

Different concentrations of 3-aminobenzamide (3AB), a strong inhibitor of poly(ADP-ribose) polymerase (PARP), were used to study their effect on the BrdU-substituted DNA of the Chinese hamster AA8 cell line. The frequencies of sister chromatid exchanges (SCEs) and translocations were determined using the fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques, respectively. The results indicate that 3AB effectively induced a dose-dependent increase in the frequency of SCEs, but this enhancement in the yield of SCEs was not paralleled by an increase in translocations. These results are discussed in terms of the as yet poorly understood molecular mechanisms of action of the enzyme PARP.     

3-dimensional growth (spheroid formation) of tumor cells in diffusion chambers]

The three-dimension growth -- spheroidoformation of tumor cells of the primary MX-induced sarcoma in mice BALB/c, C57BL, and C3H/Sn, was studied. The tumor cells were cultured on millipore filters of the diffusion chambers implanted into the peritoneal cavity of normal syngeneic mice.