Mutants In V79 Cells Research Articles

Rat and hamster hepatocyte-mediated induction of SCEs and mutation in V79 cells and mutation of Salmonella by aminofluorene and dimethylnitrosamine

Aminofluorene (AF) and dimethylnitrosamine (DMN) were examined for their ability to induce multiple genetic endpoints after rat and hamster hepatocyte metabolic activation. The endpoints measured included mutations at the Na +/K +-ATPase (ouabain resistance) and hypoxanthine-guanine-phosphoribosyltransferase (6-thioguanine resistance) loci, and sister-chromatid exchanges (SCEs) in Chinese hamster V79 cells, and mutation of Salmonella typhimurium strains TA98 and TA100. AF, with rat and hamster hepatocyte activation, induced only low levels of mutations at either loci in V79 cells but did induce SCEs. Mutaion of Salmonella by AF after hepatocyte activation also occurred and was a sensitive endpoint for detecting this aromatic amine. DMN induced high levels of mutations at both loci in V79 cells in addition to SCEs in the presence of hepatocytes from both species. DMN was also mutagenic to Salmonella, but only with hamster hepatocytes. Salmonella did not respond as strongly to DMN as the V70 cells. Hamster hepatocytes in activating both carcinogens. The results indicate the variable sensitivity of the genetic endpoints and species differences in activation for two potent chemical carcinogens.

High-density lipoproteins decrease both DNA binding and mutagenicity of r-7, t-80dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene in V79 Chinese hamster cells

The effects of separate lipoproteins or of serum with high or low lipoprotein concentrations on formation of lipophilic carcinogen adducts with DNA and on mutagenicity of the carcinogen was investigated using V79 Chinese hamster lung cells. Binding of r-7, t-8-dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE) to DNA and BPDE induction of 6-thioguanine (6-TG)-resistant mutants in V79 cells was significantly lower after 1 or 4 h when the medium was supplemented with purified HDL. and was lower after 1 h but not 4 h when the medium was supplemented with serum containing a high concentration of mixed lipoproteins (LP). Cells grown in medium without serum or LP supplementation exhibited the highest levels of both BPDE-DNA adduct formation and mutagenesis after 1 h. At 1 h, cells exposed to BPDE in LDL-supplemented medium showed decreased adduct formation and mutagenesis when compared to cells treated with BPDE in PBS-supplemented medium. After 4 h, cells treated with BPDE in LDL-supplemented medium gave the highest levels of adduct formation and the highest mutation frequency. These results suggest that both LDL and HDL effectively decrease the concentration of BPDE available to V79 cells exposed to the mutagen for short periods of time, resulting in decreased interaction of BPDE with DNA and decreased BPDE-associated mutagenesis, but that both BPDE-DNA adduct formation and mutagenesis increased as a function of increased exposure time in the presence of LDL. The results suggest that LDL, but not HDL, uptake by adsorptive endocytosis may be associated with potentiated entry of BPDE into V79 cells as a function of time.

Absence of genotoxic effects in cells exposed to four ketonucleoside derivatives.

The relationship between the structure and genotoxic potential of four new cytostatic compounds--the ketonucleosides KN-35, KN-43, KN-44 and KN-3--was investigated in short-term in vitro assays of hypoxanthine-guanine phosphoribosyl transferase locus mutation in V79 cells, induction of chromosomal aberrations and sister chromatid exchanges on human lymphocytes, induction of chromosomal aberrations and micronuclei in V79 cells, and transformation of Syrian hamster embryo cells. None of the ketonucleosides induced mutagenic effects in any of the assays. Their failure to exhibit significantly genotoxic activity may be ascribed to the probable absence of any reaction between these drugs and the cellular DNA, and indicates that they act by some other mechanism which probably differs from the one observed with alkylating or intercalating antitumoural agents. This suggests that the cytotoxic activity of ketonucleosides cannot be related to genotoxicity.

The reproductive effects assessment group's review of the mutagenicity of vinylidene chloride.

A large number of studies indicate that vinylidene chloride is mutagenic to bacteria and that this activity is largely dependent on microsomal activation. Vinylidene chloride gave positive results for gene reversion and conversion in yeast that was also dependent on metabolic activation, and was positive in tradescantia. In mammalian systems, vinylidene chloride failed to induce gene mutations in V79 cells at two separate loci, failed to induce chromosomal aberrations in mouse bone marrow in vivo, and failed to induce dominant lethals in either mice or rats. Vinylidene chloride was found to alkylate DNA of mice exposed through inhalation and may have caused unscheduled DNA synthesis in kidneys of similarly exposed mice. The studies on the mutagenicity of vinylidene chloride are evaluated in this review.

HGPRT- mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations.

In order to determine the mutagenic specificity of mutagenic and carcinogenic agents in mammalian cells, a reversion system capable of distinguishing between frameshift mutations and various kinds of base pair substitutions would be useful. We report here a method for the isolation and characterization of HGPRT- Chinese hamster V79 cell mutants that might form the basis for such a system. Two mutants of different specificity have been partially characterized. DEW-1, isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, is revertible by the base pair substitution mutagens MNNG and ethyl methanesulfonate (EMS), but not by frameshift mutagens. DSW-3, isolated following ICR-191 treatment, is specifically reverted by frameshift mutagens, but not by EMS or MNNG. With the further characterization of these and other mutants, it should be feasible to determine not only whether an agent is mutagenic in V79 cells, but also to determine the type(s) of mutation(s) it produces.

A mutagenic metabolite produced by Fusarium moniliforme isolated from Linxian county, China.

Fusarin C, a fungal metabolite, was recently isolated and identified from corn meal inoculated with Fusarium moniliforme which was one of the most common fungi associated with corn in Linxian county, a high-incidence area of esophageal cancer. In the presence of S-9 mix, fusarin C significantly increased the number of revertants in Salmonella typhimurium TA 100, and induced SCE, micronuclei, chromosome aberrations and 6-TG resistant mutants in V79 cells. The toxic action of fusarin C on V79 cells was much stronger in the absence of S-9 mix. However, fusarin C did not show, at the largest concentration used, any significant mutagenic or clastogenic effect on the cells without the addition of S-9 mix. The possible relationship between the consumption of corn contaminated with F. moniliforme and the cause of esophageal cancer was discussed.

Inhibition of metabolic cooperation by phorbol esters in a cell culture system based on adenosine kinase deficient mutants of V79 cells.

In Chinese hamster V79 cells, stable mutants which are greater than 1000-fold resistant to the adenosine analog, tubercidin (Tubr mutants), and which exhibit high degree of cross-resistance to various other adenosine analogs, viz. toyocamycin, formycin A, 6-methylmercaptopurine riboside and 8-azaadenosine, have been isolated. The inability of the mutant cells to phosphorylate [3H]tubercidin and lack of adenosine kinase activity (AK- phenotype) in their cell extracts provide evidence that the mutant cells are unable to convert adenosine analogs into their toxic phosphorylated derivatives. When AK- cells are co-cultured with increasing numbers of parental V79 (AK+) cells in medium containing tubercidin, then due to metabolic cooperation between AK+ and AK- cells, a cell density-dependent decline in recovery of the resistant cells is observed. However, diphtheria toxin resistant (Dipr) mutants of V79 cells, which are altered in elongation factor-2, showed no similar cell density effect. Addition of 0.01 microgram/ml of phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to growth medium in these experiments markedly enhanced the recovery of the Tubr mutants, but it had no effect on the recovery of Dipr mutants, which suggests that TPA was enhancing recovery of AK- mutants by inhibiting metabolic cooperation between AK- and AK+ cells. Maximum effect of TPA on the recovery of AK- mutants (3- to 5-fold enhancement) was observed at a density of 6 X 10(5) V79 cells/60 mm diameter dish and it was independent of the particular adenosine analog that was used as selective agent. Studies with a number of different phorbol derivatives show that only those phorbol esters which show tumor promoting activity in the mouse skin system inhibited metabolic cooperation between AK+/AK- cells in a dose-dependent manner. An excellent correlation was observed in these studies between the relative tumor promoting activity of various phorbol esters, their relative binding affinities to the cell surface receptors, and the concentrations at which they inhibited metabolic cooperation in the AK-/AK+ cell system. The AK-/AK+ cell system thus provides a new system for examining the effect of tumor promoters on metabolic cooperation between cells.

Chinese hamster cell mutants with defective endocytosis of low-density lipoprotein contain altered fatty acid composition in the membrane

Chinese hamster V79 cell mutants resistant to compactin (ML236B), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, are defective in endocytosis of low-density lipoprotein (1). Two resistant clones, MF-2 and MF-3, differ in lipid composition from the parental V79 strain. In both the total cells and membrane fraction, the ratio of palmitoleic acid (16:1)/palmitic acid (16:0) is 0.4-0.5 in MF-2 and 1.7-1.8 in MF-3 while that in V79 is 0.2-0.3. By contrast, a hybrid clone between V79 and MF-3 shows a ratio of palmitoleic acid to palmitic acid very similar to that of V79. The synthesis of palmitoleic acid from acetate in the resistant clone is higher than in V79.

Evidence for mutagenic repair in V79 cell mutant with aphidicolin-resistant DNA polymerase-alpha.

An aphidicolin-resistant ( aphr ) mutant of Chinese hamster V79 cells, aphr -4-2, is shown to be slow-growing, sensitive to ultraviolet (UV) irradiation, hypermutable for spontaneous and UV-induced mutations, and known to contain an aphr mutant DNA polymerase-alpha, with a 10-fold reduction in the Km for dCTP but not for dATP. We show here that the mutant had a normal repair replication measured by unscheduled DNA synthesis assay. The mutant was specifically sensitive and hypermutable to UV and N-methyl-N'-nitro-N-nitrosoguanidine, but it had normal sensitivity to ionizing radiation and dimethyl sulfate. Unlike the V79 (wt) cells, the mutant exhibited further enhancement in the already elevated mutability following UV and conditioned medium treatment. The mutant characteristic is explained by the presence of an error-prone long-patch excision repair synthesis. The association in the mutant properties--an aphr DNA polymerase-alpha, UV sensitivity, and hypermutability to UV-induced mutation--provides the genetic evidence that DNA polymerase-alpha is likely to be involved in UV-induced DNA repair synthesis.

On the nature of the mutations induced by the diolepoxide of benzo[a]pyrene in mammalian cells

We have previously described the induction by r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 8-azaguanine resistant (AGr) Chinese hamster V79 cell mutants, 40% of which were found to contain material which cross-reacted (CRM) with antiserum to hypoxanthine-guanine phosphoribosyltransferase (HPRT) and whose AGr phenotype we ascribed to missense mutation (Brookes et al., 1982). We now report that we have been unable to demonstrate by Southern blotting any change in the HPRT gene in 11 CRM-negative mutants. We have, moreover, found HPRT mRNA of normal size and amount in most of these mutants. Examination of the revertants of one mutant indicates the probable occurrence of changes within an amino acid codon in the genesis of mutant and revertant. Our results suggest that BPDE functions primarily as a point mutagen.

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.

Mutagenicity of cyclophosphamide in mammalian cells treated in vitro and in utero.

Chinese hamster V79 cells were treated with cyclophosphamide (CP) and various concentrations of S-15 mix in vitro, and then examined for chromosome aberrations, sister chromatid exchange (SCE) and induction of 8-azaguanine (8AG)-resistant mutations. Cells from hamster embryos treated with CP in utero by intraperitoneal injection were tested for drug-resistant mutation. CP caused a marked dose-dependent increase of chromosome aberrations, SCE and drug resistant mutations in V79 cells in the presence of lower concentrations of S-15. The inductions of 8AG-resistant mutations in V79 cells by CP with 1% S-15 were 7.4 times higher than those in the control. Trans- placentral treatment with CP also caused a marked increase in 8AG-resistant mutations of hamster embryonic cells.

Ultrastructural changes during the enhancement of cellular 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase in a Chinese hamster cell mutant resistant to compactin (ML 236B).

Chinese hamster V79 cell mutants resistant to compactin (ML236B) were isolated. A resistant clone, MF-2, grown in the presence of 2 micrograms/ml of ML236B for 1 week showed a 30-fold increase in 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-Co A) reductase activity compared to MF-2 grown in the absence of ML236B, and cells grown for 4 weeks showed a 53-fold increase. Apparent ultrastructural changes in thin sections of the MF-2 cells were observed after growth in ML236B: dilated cisternae in the rough endoplasmic reticulum had or did not have flocculated contents; there was significant distension of perinuclear space; and vesicular inclusion bodies were present in nuclei.

Higher efficiency of hamster hepatocytes than rat hepatocytes for detecting dimethylnitrosamine and diethylnitrosamine in hepatocyte-mediated Chinese hamster V79 cell mutagenesis assay

The mutagenecities of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on Chinese hamster V79 cells were examined by cocultivation of the cells with primary cultures of rat hepatocytes or hamster hepatocytes. Mutations in V79 cells were selected by resistance to ouabain. The mutation frequencies induced by all doses of DMN (1, 5, 10 and 50 μg/ml) and DEN (10, 50, 100 and 500 g/ml) were substantially higher on cocultivation with hamster hepatocytes than with rat hepatocytes. Thus it is proposed that hamster hepatocytes are useful for detecting mutagenesis in V79 cells induced by nitrosamines.

Alkylating properties and genetic activity of 4-vinylcyclohexene metabolites and structurally related expoxides

The mutagenicity of the epoxides 4-vinyl-1,2-epoxycyclohexane, 4-epoxy-ethyl-1,2-epoxycyclohexane, 4-epoxyethyl-1,2-dihydroxycyclohexane, 1,2-epoxycyclohexane and styrene oxide was assayed on the TA100 strain of S. typhimurium and V79 Chinese hamster cells. In the latter cell system, both point mutation (6-thioguanine resistance) and chromosomal damage (anaphase bridges and micronuclei) were scored. Genetic effects were related to the alkylating properties of the epoxides. For this purpose, alkylation of 4-( p-nitrobenzyl)pyridine (NPB) and sodium- p-nitrothiophenolate (NTP) was measured and values for the substrate constant ( s) were calculated. 4-Epoxyethyl-1,2-epoxycyclohexane, 1,2-epoxycyclohexane and styrene oxide, characterized by the highest reactivity toward NBP and by an s value in the vicinity of 1, were mutagenic in all test systems. 4-Vinyl-1,2-epoxycyclohexane and 4-epoxyethyl-1,2-dihydroxycyclohexane, characterized by lower NBP reactivity and higher s values (1.30–1.38), did not induce reversion in S. typhimurium or 6-thioguanine-resistant mutants in V79 cells, but were as effective as the 3 other compounds in the induction of chromosomal damage.

Chromosomal aberrations induced by hydrogen peroxide in cultured mammalian cells.

Chromosomal aberrations were observed in cultured mammalian cells (CHO-K1 Chinese hamster cells, V79 Chinese hamster cells, Syrian hamster cells, and BALB/c mouse cells) after treatment with hydrogen peroxide (H2O2; 0.1-0.5mM or 0.01-0.1mM) for 3h. The cytotoxic and clastogenic effects of H2O2 were clearly reduced by the addition of catalase. In contrast to the clastogenic potential, H2O2 did not enhance the frequency of mutation in V79 cells, whether the marker for mutation was 8-azaguanine resistance or ouabain resistance.

Excess thymidine is not mutagenic in Chinese hamster V79 fibroblasts

The ability of thymidine to enhance the frequency of HGPRT −, 6-thioguanine resistant V79 cell mutants has been investigated as a function of post treatment growth interval. We have also studied the relative sensitivities towards exogenous thymidine of wild type and mutant cell lines. Our data imply that increases in mutant frequency following exposure of V79 cells to thymidine can be explained on the basis of a greater sensitivity of wild type cells compared with HGPRT deficient cells.

Chromosomal aberrations and mutation in cultured mammalian cells induced by pyrrolizidine alkaloids

4 pyrrolizidine alkaloids, heliotrine, lasiocarpine, petasitenine, and senkirkine, were tested for their effects on V79 Chinese hamster cells. All the alkaloids produced cytoplasmic vacuolization, and caused cellular and nuclear enlargement. Chromosomal aberrations were induced in cells treated with the alkaloids. Heliotrine and petasitenine induced interchromosomal exchanges, and lasiocarpine and senkirkine caused chromatid gaps. All the alkaloids induced an 8-azaguanine-resistant mutation in V79 cells by direct treatment for 48 h. Mutation was also induced by treatment with the alkaloids for 1 h in the presence of a metabolic activation system.

Mutagenic, transforming and promoting effect of pickled vegetables from Linxian county, China.

A recent epidemiological survey in China showed that there is a regional distribution of esophageal cancer and a correlation between mortality for this cancer and environmental factors, especially the consumption of pickled vegetables. A series of experimental studies with pickled vegetable extract were done using different in vitro biological systems. The results showed that pickled vegetable extract induced 6-thioguanine-resistant mutants in V79 cells and increased sister chromatid exchanges in the same cells and in Syrian hamster embryo cells. Pickled vegetables extract induced transformed foci in Syrian hamster embryo cells and in 3-methylcholanthrene initiated C3H/10T1/2 cells. The mutagenic, transforming and promoting activities of pickled vegetable extract seen in vitro conform with in vivo results and provide evidence for the presence of a mutagen and/or a carcinogen in pickled vegetable extract. A possible role of pickled vegetables consumption in the etiology of esophageal cancer is discussed.

Cis-platinum(II) diamine dichloride causes mutation, transformation, and sister-chromatid exchanges in cultured mammalian cells

The anti-tumor agent cis-platinum (II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose—response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. These findings suggest that this chemical is a potential human carcinogen.