Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells Characteristics of some recently developed selective systems

Selection conditions have been optimized in the Chinese hamster ovary (CHO) cell system for a number of genetic markers. The genetic systems studied include resistance to the protein-synthesis inhibitors emetine (Emt^r) and diphtheria toxin (Dip^r), resistance to methylglyoxalbisguanylhydrazone (Mbg^r) which affects polyamine transport, resistance to the nucleoside analogs toyocamycin and tubercidin (Toy^r), and resistance to thioguanine (Thg^r) and ouabain (Oua^R). The optimal expression time following mutagenesis for various markers was between 2 and 6 days. A linear dose-response relationship between the concentration of mutagen (ethyl methanesulfonate) and mutation frequency has been observed over the range of 10–700 μm/ml, for all of the above markers except Toy^r. The response of these markers to other mutagens such as tritium (³H) decay and ICR-191 show some specificity. Since the response of a number of genetic markers can be studied simultaneously in the CHO system, it should prove very useful for studies of quantitative mutagenesis and in assay systems for mutagen detection.     

Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod^R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK₂₁) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod^R mutants is not affected by cell density (up to 1 × 10⁶ cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod^R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. The Pod^R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.     

Comparative mutagenicity of alkylsulfate and alkanesulfonate derivatives in Chinese hamster ovary cells.

Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system. Dose--response relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)]. Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose--response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES greater than DMS; EMS greater than MMS greater than iPMS.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

Mutagenic activity of selected aromatic amines and polycyclic hydrocarbons in Drosophila melanogaster

With the use of a series of wild-type and repair-deficient strains and appropriate application procedures, it is possible to demonstrate that carcinogenic aromatic amines and polycyclic hydrocarbons are mutagens in Drosophila. We have shown evidence that AAF, N-OH-AAF, AcO-AAF, BP, DAS and DMBA produce recessive lethals when fed to or injected into adult males. Mutagenic activity was also observed when male larvae were exposed to AAF, BP, DMBA, 3-MC or NA. DA was not mutagenic in the recessive lethal assay under the conditions of the test. DMBA can now be considered as a potent mutagen for Drosophila, although demonstration of its activity depends upon the choice of the treatment procedure and the strain selected. One of the questions concerning the action of aromatic amines and polycyclic hydrocarbons is how their genetic effectiveness in Drosophila can be enhanced. The observation that none of several enzyme inducers (PB, BF, AC, 3-MC) increased their mutagenicity may be interpreted in terms of a more efficient metabolic activation or deactivation. This assumes that active metabolite(s) did not reach the testis in doses sufficient for mutation induction. It also appears that, since the problems pertaining to mutagenicity in Drosophila of aromatic hydrocarbons are obviously a matter of metabolism, the use of repair-deficient strains is no longer an attractive proposal for their elucidation. The present investigation shows that, with weak mutagens, usage of strains mei-9Li or y mei-9a mei-4lD5 does not improve the sensitivity of the recessive lethal method or the test for chromosomal loss. As an alternative, in our opinion more attention should be devoted to possible differences in metabolism between somatic and gonadal tissue. We feel strongly that somatic assay systems might be particularly valuable as a complement to recessive lethal tests on the germ line.     

Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7

2-Aminopurine, 2-amino-N⁶-hydroxyadenine and N⁶-hydroxyaminopurine were compared in suspension test with growing and non-growing cells for their mutagenic and recombinogenic (reciprocal and nonreciprocal) activities in Saccharomyces cerevisiae strain D7. Ethyl methanesulfonate was used as a positive control. No increases above spontaneous frequencies were observed when non- growing cells were treated with the base analogues although EMS induced concentration- dependent responses at all 3 genetic end-points. When growing cells were treated, HAP was recombinogenic and mutagenic and AHA was mutagenic, but only weakly recombinogenic. HAP induced comparable numbers of revertants at much lower concentrations than AHA. 2AP failed to induce any detectable response even at concentrations as high as 2400 μg/ml.     

Mammalian cell transformation and cell-mediated mutagenesis by carcinogenic polycyclic hydrocarbons.

The introduction of a polycyclic hydrocarbon such as benzo(alpha)pyrene (BP) into normal golden hamster embryo cell cultures results, in addition to cytotoxicity, in malignant cell transformation. Studies on the effect of different doses of BP on the normal cells showed that the frequency of transformed colonies was directly related to the dose of the carcinogen. Analysis of this dose-response curve suggests a one-event ("one-hit") response for transformation by this carcinogen. The one-event response for transformation by carcinogenic polycyclic hydrocarbons and the fact that these carcinogens bind to DNA in susceptible cells suggests that transformation can involve a single alteration in the genetic constitution of the treated cells. Carcinogens may, therefore, produce somatic mutations, some of which may involve the genes that control malignancy. Recently, considerable progress has been made in developing models for the study of chemical mutagenesis in mammalian cells. Using resistance to 8-azaguanine as a marker, positive correlations between mutagenicity and transformation were obtained with chemically reactive carcinogens such as N-acetoxy-N-2-fluorenyl-acetamide, N-methyl-N'-nitro-N-nitrosoguanidine and K-region epoxides of polycyclic hydrocarbons. However, no such correlations were obtained with the carcinogenic polycyclic hydrocarbons themselves, since the cell lines used in chemical mutagenesis do not metabolize these carcinogens. In order to obtain better correlations, we have developed a cell-mediated mutagenic assay with carcinogenic hydrocarbons in which Chinese hamster cells, which are susceptible for mutagenesis, were co-cultivated with lethally irradiated rodent cells that can metabolize these compounds. Using this cell mediated assay, we obtained mutagenesis with the carcinogenic hydrocarbons 7,12-dimethylbenz(alpha)anthracene (DMBA), BP, 3-methylcholanthrene and 7-methylbenz(alpha)anthracene; the most potent carcinogen, DMBA, gave the highest frequency of mutations. The polycyclic hydrocarbons, pyrene and benz(alpha)anthracene, which are not carcinogenic were also not mutagenic. We have therefore demonstrated a relationship between the carcinogenecity of polycyclic hydrocarbons and their mutagenicity in mammalian cells, without having to isolate their reative metabolic intermediates. It should be possible to use in this system human cells from different organs and individuals to screen for environmental chemicals hazardous to humans which have to be metabolically activated.     

The use of different test systems with yeasts for the evaluation of chemically induced gene conversions and gene mutations

The wide variety of genetic alterations that can be induced in human populations when exposed to chemical genotoxic substances present in our environment may be predictable using laboratory organisms such as yeasts.In the present paper methodologies are described for analysing the genetic effects induced by a well known chemical mutagen, ethyl methanesulfonate (EMS). Haploid or diploid yeast cells have been treated in vitro, in buffer or in the presence of mouse liver microsomes, and in vivo, in the peritoneum of the mouse (host-mediated assay).With these different methods of assaying the genetic activity of a compound, its metabolic activation occuring in the mammalian body is taken into account: this might lead to a more reliable extrapolation of data from laboratory experiments to man.The relationships between doses and frequencies of the induced genetic effects are described by equations obtained after regression analysis of the data, thus allowing a quantitative comparison among different methodologies and different genetic systems.One genetic system analyzed is represented by forward-mutations scored phenotypically on a non selective medium. Mutations induced in five loci with different sensitivity and average data of mutation-induction per locus have been derived. The second genetic system was provided by scoring on a selective medium mitotic gene-conversions induced in two loci with different kinetics.Haploid cells of Schizosaccharomyces pombe and diploid cells of Saccharomyces cerevisiae were submitted to analysis for the evaluation of gene-mutations and gene-conversions respectively.     

Mutagenesis by nitrosoguanidine, ethyl methanesulfonate, and mutator gene mutH in continuous cultures of Escherichia coli.

Induction of T5-R mutations by alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) was examined in glucose limited chemostat cultures of non-mutator and mutator (mutH) bacteria. In agreement with the proposal that NTG mutagenizes DNA at the replication fork, this mutagen (6.8 X 10-minus 6 M) showed replication-dependent mutagenesis in continuous culture. EMS (5-10-minus M)) induced mutagenesis could not be correlated with growth rate, which probably means that induction of mutagenic lesions (promutations) by this mutagen does not involve replicating genes. A large synergic response was found for the mutH gene in combination with NTG, supporting the hypothesis that the mutH gene product acts during DNA replication.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Synergistic effect of an Escherichia coli mutator gene on mutagenesis by ultraviolet radiation and by alkylating agents

E. coli strains differing in a gene responsible for high spontaneous mutability (mut HI) were compared for their mutability by UV radiation and by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate. All three exogenous mutagenic agents induced significantly higher frequencies of mutants with impaired carbohydrate-fermenting ability when the mutator allele rather than the wild-type allele was present. Thus the mut HI gene product possibly increases the probability of replication error due to alterations in the structure of the template strand of DNA. An attempt to detect an synergistic effect for UV-induced suppressor mutations was unsuccessful. The failure may have been due to the particular method used for scoring this type of mutation.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

Conversion of environmental pollutant to mutagens by visible light.

Illumination of DMBA, 3-methylcholanthrene, 2-aminoanthracene and chrysene with visible light resulted in the formation of direct-acting chemicals endowed with genotoxic and frameshift mutagenic activities. These findings may be of relevance in assessing the potential health hazards inherent in the planned conversion to diesel fuels which is expected to result in increased atmospheric levels of polycyclic aromatic hydrocarbons.     

Chemical induction of streptomycin-resistant mutations in Escherichia coli. Dose and mutagenic effects of dichlorvos and methyl methanesulfonate

A procedure for the quantitative determination of induced streptomycin-resistant mutants in E. coli was applied to study and compare mutation induction by the organophosphate dichlorvos and by methyl methanesulfonate (MMS). Both compounds increased the frequency of mutants even under conditions where no inactivation of cell was observed. Mutation induction by these agents as a function of both concentration and exposure time was measured. The dose-response curves found with both mutagens were non-linear; atp higher doses more mutants were induced per unit dose than at lower doses. Possible relationships between dose-effect curves and the chemical nature of alkylating mutagenic agents are discussed.     

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.     

UV-light-induced mutations in synchronous CHO cells

Asynchronous and synchronous CHO cells were irradiated with germicidal UV light to determine the fluence response curve for cell killing, and the induction of resistance to 6-thioguanine, ouabain, and diphtheria toxin. For asynchronous populations the data show a sigmoidal response for induced reproductive death, as has been seen by other, with a D₀ of 6 J/m² and an extrapolation number of 2.5. The induction of mutations appears to be a linear function for all three mutagenic markers up to a dose of 17 J/m².Reproductive death induced in the synchronous populations is a function of the time at which exposure occurs in the cell cycle, with late G₁ and early S being the sensitive stages. The induction of resistance to 6TG, ouabain, and diphtheria toxin (DT) all seem to depend on the time of exposure in the cell cycle. As in the case of UV-induced reproductive death, the more sensitive periods for mutation induction appear also to be the G₁ and early S period of the cell cycle, with the largest cyclic variation occurring for induced DT resistance.A comparison of the results reported here for the UV exposure with exposures of synchronous CHO cells to X-rays and ethylnitrosourea suggests that there are different age-specific responses to mutation induction for each agent, and that there are often different age responses for different mutagenic end- points with the same mutagen.     

5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides

The induction of 5-methyltryptophan (5-MT) resistance mutations was assayed as a test system for mutagenic chemicals in Escherichia coli. It is assumed that different premutational alterations in several genes of the Escherichia coli chromosome will lead to 5-MT-resistant mutants. The chemicals used were three monofunctional alkylating agents as reference compounds, namely β-propiolactone (β-PL), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methyl methanesulfonate (MMS), which are all mutagenic in the 5-MT system; of the eight organophosphorus insecticides tested, four have definite mutagenic activity (Dichlorvos, Oxydemetonmethyl, Dimethoate, and Bidrin), one is probably mutagenic (Methylparathion) and the remaining three (Parathion, Malathion and Diazinon) do not induce 5-MT resistance mutations in the conditions used here (< 30% survival). The relative mutagenic activity after a treatment time of 60 min is (in decreasing order) MNNG > MMS > Dichlorvos > Oxydemetonmethyl, Dimethoate and Bidrin. The concentration-dependent mutagenic activity of all mutagenic compounds is nearly linear when plotted on a log-log scale (with slopes varying from 1.0 to 1.5) and could be taken as an indication that one premutational reaction will be sufficient for the induction of one 5-MT-resistant mutant.     

Metabolic activation of selected aromatic amines and polycyclic hydrocarbons by isolated subcellular fractions of Drosophila melanogaster

The applicability of microsomal preparations from Drosophilamelanogaster as the metabolic factor in the Salmonella mutagenicity assay with strains TA98 and TA100 was evaluated. Isolated cellular fractions (S27) from PB-pretreated flies activated N-acetyl-2-aminofluorene (2-AAF), N-hydroxy-N-aceyl-2-aminofluorene (N-OH-AAF), benzo[a]pyrene (BP), 9,10-dimethylanthracene (DA) and 2 -naphythylamine (NA)_into mutagenic metabolites, 7,-12-Dimethylbenz[a]-anthracene (DMBA) was ineffective under the conditions of the test.This study was performed in an effort to determine optimal conditions for activating, by Drosophila enzymes, aromatic amines and polycyclic hydrocarbons, with 2-AAF and BP as model mutagens. The following alterations improved the sensitivity of this combined Salmonella/Drosophila assay. (1) Incubation of the plates at 25°C for 1 night instead of permanent exposure at 37°C. (2) Isolation of S27 fractions instead of the conventional S9, because 9000 × g was not sufficient tio spin down Drosphila mitochondria.