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PubMed1977

In vitro mutagenesis assays as predictors of chemical carcinogenesis in mammals.

In vitro microbial mutagenesis assays coupled with mammalian activation systems offer promising technique to screen chemicals for their potential carcinogenic activity. The correlation between mutagenic and carcinogenic properties for a large array of chemicals is approximately 0.9. The best correlation exists for those carcinogens which are themselves highly electrophilic or produce electrophilic metabolites. Correlation between mutagenicity and carcinogenicity for hormonal, metallic, or physical carcinogens has been disappointing but not unexpected based on their proposed mechanisms of action. In addition to the application of in vitro mutagenesis techniques to screening chemicals for the identification of potential carcinogens, they are useful tools for investigating genetic, biochemical, and pharmacologic properties of different animal species. Studies with the chemical carcinogen dimethylnitrosamine have been conducted and show a functional relationship between mutagenesis and carcinogenesis. The assays can also be conducted using activation systems prepared from the tissues of any mammalian species. This permits a direct assessment of phylogenic extrapolation by comparing the metabolic activation capabilities of tissues from several mammalian species, including human samples. The advantages of mutagenicity testing are the short period of time required for results, the high sensitivity of the assay (microgram of nanogram quantities of chemicals can be used), and the fact that the ultimate agent can be detected biologically without first necessitating chemical identification and isolation. It appears from current studies that in vitro mutagenesis techniques may well open new avenues of investigation into some old toxicologic problems.

Aging↗
PubMed1979

DNA--benzo[a]pyrene adducts formed in a Salmonella typhimurium mutagenesis assay system.

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo[a]pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor I254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was an adduct between the 10 position of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. This adduct is also the major DNA product previously found when human tissues or certain rodent cells were incubated with benzo[a]pyrene. On the other hand, when activation of benzo[a]pyrene was mediated by a phenobarbital-induced B6 mouse-liver enzyme preparation, the extent of binding was quite low and the profile of DNA adducts in S. typhimurium DNA was quite different. Thus, under appropriate conditions, the activation and DNA binding of benzo[a]pyrene inthe microsome mediated S. typhimurium mutagenesis assay generally resembles that seen in intact mammalian cells. Caution must be exercised, however, in the choice of microsome-activation systems.

Animals↗
PubMed1979

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.

Aflatoxins↗
PubMed1976

A comparative study of different experimental protocols for mutagenesis assays with the 9-azaguanine resistance system in cultured Chinese hamster cells.

Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in whcih the cell density was maintained below critical values both before and during mutant selection. The identification of such a plateau allows, besides more reliable and reproducible estimates of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.

Azaguanine↗
PubMed1979

Intrahepatic mutagenesis assay: a sensitive method for detecting N-nitrosomorpholine and in vivo nitrosation of morpholine.

An intrahepatic host-mediated mutagenicity assay capable of detecting low levels of N-nitrosomorpholine (NMOR) is described. The indicator organism was Salmonella typhimurium TA1530 which had been injected intravenously 10 min prior to the administration of the test compound. The bacteria were subsequently recovered from the liver and scored for revertants by standard methods. The lower limit of detectibility of this system for intubated NMOR was 0.2 microgram/g body weight. This assay was then used to study the formation of NMOR in vivo from morpholine and nitrite which had been sequentially gavaged to mice. Under acidic conditions (pH 3.4) 12--19% of the administered morpholine was converted to NMOR in the presence of excess nitrite. This nitrosation, and the subsequent uptake and activation of the NMOR, took place so rapidly that most of the total mutagenic response was complete within 15 min. This response was inhibited by prior intubation of ascorbic acid, a known inhibitor of nitrosation, and enhanced by sodium thiocyanate, a nitrosation catalyst.

Animals↗
PubMed1979

Absence of noxious effects of selected neuroleptics in dominant-lethal mutagenesis assay.

In a dominant-lethal assay in mice the following tricyclic neuroleptics were tested: prothiaden, imipramine, oxyprothepin decanoate and docloxythepin. No dominant-lethal effect was induced by these neuroleptics, even when administered at doses many times as great as clinical doses. The reduced percentages of pregnancies, in females who had copulated with males receiving docloxythepin, observed during and immediately after its administration, were directly connected with marked sedation induced in the males by this neuroleptic.

Animals↗
PubMed1977

Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol colon carcinogenesis: correlative studies on selenium effects on the mutagenicity and sister chromatid exchange rates of selected carcinogens.

Selenium (Se) inhibition of either the activation of test compounds and/or mutagenic events elicited by activated compounds is suggested by experimental rat assays, mutagenesis assays, and assays with human lymphocytes in culture. The colon tumor incidence in 1,2-dimethylhydrazine (DMH)-treated rats was reduced from 87% to 40% by 4 ppm Se supplements in the drinking water. Supplemental Se decreased the total number of colon tumors induced by DMH more than three-fold and by methylazoxymethanol (MAM) almost two-fold. Coexposure of Salmonella typhimurium TA 1538 to an effective molar ratio of Se/2-acetylaminofluorene=10, Se/N-OH-acetylaminofluorene=10 and SE/N-OH-aminofluorene=300 reduced the mutagenicity to 65, 68, and 61% of their respective controls with mutagen alone. With a molar ratio of Se/N-OH-AAF=100, Se reduced the activity to 28% of the mutagenicity of N-OH-AAF alone. Preliminary data indicating MAM is mutagenic in S. typhimurium TA 1535 and His G 46(6837) are presented. In toxicity studies exposure of human lymphocyte cultures to 1.3 X 10(-9) to 1.6 X 10(-5) M Se yielded sister chromatid exchange (SCE) rates equivalent to background levels of 6--7 SCE per cell. The SCE frequencies of lymphocytes cultured with Se and selected carcinogens are discussed.

Animals↗
PubMed1978

Screening of safrole, eugenol, their ninhydrin positive metabolites and selected secondary amines for potential mutagenicity.

The mutagenicity of safrole, eugenol, the secondary amines, with which they combine during metabolism, and the ninhydrin positive urinary metabolites of safrole and eugenol was tested. The panel of tests included the direct bacterial assay, a microsomal mutagenesis assay and a host-mediated assay. With the direct bacterial assay employing four mutant strains of Salmonella typhimurium (TA1530, TA1531, TA1532, TA1964), all the compounds gave negative results. In the microsomal mutagenesis assay, employing the same four mutant strains, safrole and safrole metabolite II were mutagenic with strains TA1530 and TA1532. Dimethylamine was also found to be a weak mutagen in the microsomal mutagenesis assay with strain TA1530. Safrole and safrole metabolite II were also mutagenic in the host-mediated assay with strains TA1950 and TA1952. Negative results were observed for safrole metabolites I and III, eugenol, eugenol metabolites I and II, piperidine, pipecolic acid, proline, and pyrrolidine in all three assay systems.

Animals↗
PubMed1976

Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential sample mammalian assay for mutagenesis.

Since mutagenic substances induce structural changes in DNA, studies were undertaken to determine whether substances would modify DNA replicative activity. DNA synthesis was quantitated 3.5 h after drug administration as uptake into DNA of a 30-min pulse of 10 muCi of [3H] thymidine. Combinations of methylurea (20000 mg/kg) and sodium nitrite (150 mg/kg) administered p.o. resulted in gastric synthesis of nitrosomethylurea and inhibited testicular DNA synthesis by 83%. Combinations of methylurea and sodium nitrite of 1000 and 100 mg/kg respectively inhibited DNA synthesis by 75%. With dimethylamine and sodium nitrite, a combination which results in gastric synthesis of dimethylnitrosamine, inhibitions of 65 and 57% were observed at 2000 mg/kg together with 150 mg/kg and 1000 mg/kg in combination with 100 mg/kg, respectively. In separate experiments, dimethylnitrosamine (50 mg/kg, p.o.) and diethylnitrosamine (100 mg/kg, i.p.) inhibited thymidine uptake by 30 and 89%, respectively. The mutagenic polynucelar hydrocarbon 3-methylcholanthrene (15 mg/kg) inhibited DNA synthesis by 95% and safrole (640 mg/kg), a mutagenic methylenedioxybenzene derivative, inhibited by 60%. Cadmium chloride (10 mg/kg), acetylaminofluorene (160 mg/kg) and dibutylnitrosamine (500 mg/kg) also induced statistically significant effects. Noncarcinogenic analogues of these substances (anthracene, 125 mg/kg; diphenyl-nitrosamine, 500 mg/kg; piperonyl butoxide, 640 mg/kg, and methylurea, 2000 mg/kg in combination with sodium nitrate) were inactive. Highly toxic substances (potassium cyanide, 2.5 mg/kg; 2,4-dinitrophenol, 20 mg/kg; and lead acetate, 150 mg/kg) were also inactive. This index of mutagenicity appears to have considerable sensitivity and therefore may have potential in drug evaluation.

Animals↗
PubMed1979

Selection of carcinogens and related compounds tested for mutagenic activity.

A list of 102 chemicals was prepared for subsequent mutagenesis assays in a National Cancer Institute program to determine the extent of correlation between carcinogenesis and mutagenesis in standardized assays. The chemicals were divided into five major categories: 37 aromatic amines, 11 polycyclic aromatic hydrocarbons, 8 nitrosamines and nitrosamides, 16 alkylating agents, and a miscellaneous category consisting of 11 heterocyclic compounds, 7 amides, ureas and acylating agents, 5 antimetabolites, 4 inorganic chemicals, and 3 promoters. The chemicals were further described as procarcinogens (requiring metabolic activation to exert their biologic activities), ultimate carcinogens (direct-acting chemicals not requiring metabolic activation), and noncarcinogens (compounds shown to be inactive in one or more adequate carcinogenicity tests). An extensive bibliography documents the selection and categorization of the compounds.

Alkylating Agents↗
PubMed1978

The inhibitory effect of cysteine on the mutagenic activities of several carcinogens.

The Salmonella/microsome mutagenesis assay was used to determine the effect of cysteine (alpha-amino-beta-mercaptopropionic acid) on the mutagenic actions of several carcinogens: N-methyl-N'-nitro-N-nitrosoguanidine. N-acetoxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, 4-nitroquinoline-1-oxide, methyl methanesulfonate, 5-nitro-2-furaldehyde semicarbazone, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, aflatoxin B1 and the nitrosation products of methylurea and methylguanidine. Cysteine, at non-toxic concentrations, significantly decreased the frequency of reversion to histidine prototrophy when it was added to treatment mixtures. The extent of the inhibition of mutagenic action by cysteine depended on the carcinogen studied as well as the doses of cysteine and carcinogen employed. Cysteine (2.5--10 mM) completely inhibited the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine and methylguanidine nitrosation products while only partially preventing the mutagenic effects of the other carcinogens assayed. Inhibition of 5-nitro-2-furaldehyde semicarbazone-induced mutagenesis occurred only with higher cysteine concentrations (20--200 mM).

2-Acetylaminofluorene↗
PubMed1978

Mutagenesis by N-acetoxy-2-acetyl aminofluorene of chinese hamster V79 cells is unaffected by caffeine.

8-Azaguanine (AZG)- and 6-thioguanine (TG)-resistant cells (mutants) were induced in Chinese hamster V79-4 cells by 0.1--2.5 microgram/ml N-acetoxy-2-acetyle aminofluorine (AcAAF) treatments in the presence of 5% fetal bovine serum (FBS). The frequency of resistant colonies increased from 1 to 47 per 10(5) survivors. The effect of caffeine (50--200 microgram/ml) during the mutagenesis expression period was determined by adding caffeine 1--24 h after AcAAF. The medium was replaced after 48 h exposures so that caffeine was absent during subsequent selection with AZG or TG. No significant change in the AcAAF-induced mutant frequency occurred with any treatment combination although caffeine greatly enhanced the lethality associated with AcAAF treatments. Thus, caffeine interferes with postreplication repair in V79-4 cells without affecting the probability of error of the repair process. These results were obtained with a quantitative mutagenesis assay in which the cells were reseeded prior to selection to achieve maximum expression without interference from metabolic crossfeeding. In contrast, the commonly used in situ assay is subject to serious interference from crossfeeding and yields an artifactual enhancement of AcAAF mutagenesis by caffeine.

Acetoxyacetylaminofluorene↗
PubMed1979

The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test.

Evaluation of 6 carcinogenic nitrosamines in the hepatocyte primary culture/DNA repair test revealed that all were active. Three non-carcinogenic analogs were negative. This sensitivity to nitrosamines, together with considerations concerning the metabolic capability and end point of this assay, indicates that it will be a useful addition to screening batteries that include bacterial mutagenesis assays.

Animals↗
PubMed1976

Mutagenic activity of tryptophan metabolites produced by rat intestinal microflora.

The catabolism of tryptophan by rat intestinal microflora was studied for the production of mutagenic metabolites that might be involved in the etiology of colon cancer. Various tryptophan metabolites were assayed for mutagenic and comutagenic activity in the Ames bacterial test system. These included metabolites that were identified by thin-layer chromatography in cultures of rat fecal bacteria, other compounds structurally related to tryptophan, whole unfractionated mixed fecal bacteria culture filtrates, and concentrated solvent extracts. A total of 27 materials were tested with 5 Salmonella strains in the mutagenesis assay. Most substances were inactive, and only one compound, o-aminoacetophenone, which was unlikely to be produced in the intestine, showed weak comutagenic activity. Our results did not support the hypothesis that tryptophan metabolites produced by intestinal microflora are major etiologic factors in cancer of the colon.

Acetophenones↗
PubMed1977

In vitro studies of chemical mutagens and carcinogens. I. Stability studies in cell culture medium.

A quantitative microbial assay was used to study the stability of known mutagenic and carcinogenic compounds in cell culture medium. Ten direct-acting carcinogens, when incubated in culture medium with 15% fetal bovine serum at pH 7.2-7.4 and 37 degrees C, became inactive at varying rates. Biologic half-lives of the test compounds ranged from 8 minutes to 67 hours. In contrast, six procarcinogens showed no significant inactivation after 3 weeks' incubation. The biologic half-lives of each compound were presented, and the significance of these findings as they relate to cell culture carcinogenesis and mutagenesis assays was discussed.

Carcinogens↗
PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗