Modified Ames Test Research Articles

Polyamines and their potential to be antimutagens

In order to determine the antimutagenic potential of polyamines, modified Ames tests were performed. Polyamines spermine, spermidine and putrescine all showed antimutagenic potential against EMS-induced reversions. In addition, the polyamines spermidine and putrescine showed potential to reduce the number of spontaneous revertants in modified Ames tests. Since spermidine and putrescine have the potential to reduce spontaneous mutations, we decided to perform DNA fidelity assays. DNA fidelity assays confirmed that putrescine has the potential to reduce the mutation frequency. However, spermidine had no effect. This suggests that putrescine may play a vital role in DNA synthesis and possibly be the active compound that plays a role in affecting EMS-induced mutations in the modified Ames tests. This is possible since all cells have the potential to convert spermine and spermidine to putrescine. However, since the DNA fidelity assay is an in vitro assay, the enzymes required for the conversion of spermine and spermidine to putrescine are absent. The possibility of conversion and the rate of conversion need further study.

Evaluation of the Dermal Carcinogenic Potential of Re-refined Base Stocks Using the Modified Ames Assay, PAC Analysis and the32P-Postlabeling Assay for DNA Adduct Induction

The standard method for assessing the carcinogenicity of lubricating oil base stocks is the mouse skin-painting bioassay. This assay has the advantage of directly measuring the endpoint of interest, dermal carcinogenicity, but has the drawback of being time-consuming and expensive. For this reason, a variety of biological and chemical assays have been developed as predictive alternatives to the in vivo assay. This publication describes the application of three such methods to the assessment of carcinogenic potential of hydrotreated, re-refined oils: the modified Ames test, the analytical determination of 3-7-ring polycyclic aromatic compound content and the 32P-postlabeling assay for DNA adduct induction.

Evaluation of the dermal carcinogenic potential of re-refined base stocks using the modified Ames assay, PAC analysis and the 32P-postlabeling assay for DNA adduct induction.

The standard method for assessing the carcinogenicity of lubricating oil base stocks is the mouse skin-painting bioassay. This assay has the advantage of directly measuring the endpoint of interest, dermal carcinogenicity, but has the drawback of being time-consuming and expensive. For this reason, a variety of biological and chemical assays have been developed as predictive alternatives to the in vivo assay. This publication describes the application of three such methods to the assessment of carcinogenic potential of hydrotreated, re-refined oils: the modified Ames test, the analytical determination of 3-7-ring polycyclic aromatic compound content and the 32P-postlabeling assay for DNA adduct induction.

Comparison of Biological and Chemical Predictors of Dermal Carcinogenicity of Petroleum Oils

Abstract. Various methods have been proposed as a basis for cancer hazard labelling of petroleum products. The Institute of Petroleum analytical method termed IP 346/80 was recently recommended to the European Union as the preferred method for this purpose. In this report we compare IP 346/80 with several other predictors of dermal carcinogenicity, including the Mobil PAC Method, the Modified Ames Test (ASTM Method E 1687-95), and the 32P-postlabelling assay for DNA adducts. Oils for assay were selected from a repository of samples previously subjected to mouse skin-painting bioassay. 120 oils were tested in the Modified Ames Assay, 57 by the Mobil PAC Method, 50 by Method IP 346/80, and 48 by the postlabelling procedure. The ability of each assay to distinguish between carcinogenic and noncarcinogenic oils was examined at various suggested discriminators, e.g. Mutagenicity Indices (MI) of 1.0 and 2.0, 3-7 ring PAC contents of 1% and 2% (w/w), and IP 346/80 DMSO-extractables of 2% and 3% (w/w). Various ad...

Natural antimutagenic agents

Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Mutagenicity and toxicity of water extracts from the Sora river area

The present study was conducted on the waters of the Sora river and effluents entering the river. The samples were extracted with XAD-2 resin at different pH and tested for mutagenicity with the modified Ames test using strains S. typhimurium TA98 and TA100. The majority of the mutagenic activity of the samples was found in the neutral pH fraction of the extracts. Strain TA98 in the presence of metabolic activation was the most sensitive condition of mutagenicity. Of the eleven sample extracts, six were positive; neutral fractions of the effluent from wastewater treatment plant, the water leaching from the municipal dump, the water from the lake lying beneath the dump and the untreated effluent, and acid fractions of two samples taken directly from the river. The water leaching from the municipal dump was also mutagenic and toxic without previous extraction. Mutagenic responses before and after extraction of this sample indicate that components responsible for mutagenicity were partly extracted in the neutral fraction. The toxicity of water samples and extracts was tested with Microtox assay, and acid fractions of the extracts were more toxic than the neutral fractions. Comparing the toxicity to the mutagenicity data indicates that components responsible for toxic and mutagenic response were at least partly separated between acid and neutral fraction respectively.

Modified ames test with 21 commercial azo dyes metabolic activation by liver S9 from rats and hamsters

Modified ames test with 21 commercial azo dyes metabolic activation by liver S9 from rats and hamsters

Genotoxicity testing of cobalt compounds used in the Danish porcelain industry

Over the past century, cobalt has been an important substance in the blue dye used in the Danish porcelain industry. These blue coloring agents contain about 40% cobalt compounds which are present in the dye either as an insoluble or as a slightly soluble cobalt compound. Previous studies from our Institute have revealed cobalt concentrations in urine from workers using cobalt blue dye at a Danish porcelain factory (Scand J. Work Environ. Health, 14: 378–384, 1988). Also, a comprehensive epidemiological study on the population of employees at this Danish porcelain factory is presently ongoing (F. Tüchsen, M. Jensen, and E. Lynge). The International Agency for Research on Cancer (IARC) recently classified cobalt and cobalt compounds as possibly carcinogenic to humans (Group 2B). Although this classification was based on limited evidence in humans, data from several experimental studies concluded that soluble cobalt(II) compounds are genotoxic to various organisms. However, only a few studies on insoluble cobalt(II) sulfide have been reported. In vitro testing of soluble cobalt(II) compounds in various biological systems may be complicated by the ‘reactivity’ of cobalt itself with a number of substances in the test media. As such, cobalt(II) may precipitate with phosphate in buffers and media yielding it inactive. Also, cobalt(II) may form a chemical complex with various amino acids and other essential biological cofactors rendering it more toxic to the test organism. In the present study, we have explored the potential genotoxic activity of the ‘soluble’ blue coloring agent and cobalt(II) chloride in the Ames test. The test compounds have been studied by using different protocols for the administration of the agents i.e. with the test compound in a complex with different heteroaromatic agents and by using a modified Ames test. Both cobalt compounds were genotoxic to the frame shift strain TA97a. Tnc coloring agent was more genotoxic than equimolar concentrations of cobalt(II) chloride. Adenine reduced the mutagenic effect of cobalt.

簡易気体暴露法による揮発性化合物の変異原性検出の試み テトラクロロエチレンを用いて

The mutagenicity of tetrachloroethylene (PCE) was used to study the advantage of a modified Ames test devised to detect volatile liquid mutagens with bacteria. The test compound, dissolved in DMSO, was spread with filter paper on the lid of a glass plate. These plates were then sealed up with adhesive tape to allow the compound to vapor. A PCE sample containing epoxides was directly mutagenic for Salmonella Typhimurium His- strains, TA100, TA102, TA104 and TA1535. However a purified sample did not increased the number of his+ revertants over the control value when tested with or without CD1 male mice S9 mix. The quantity of the test compound in the soft ager after exposure was achieved by gas chromatography. The quantity of PCE ; 30, 20, 10, 5, 2.5 and 1.25 mg/filter paper in the plate was 4.41, 1.04, 0.49, 0.13, 0.11 and 0.09 mg/soft ager in test plate, respectively. This concentration of PCE may have a great effect on bacteria, and this method may be useful for evaluating the mutagenicity of a volatile liquid. Thus, it is implied that PCE is one of the non-mutagenic carcinogen in Ames test.

Evaluation of the Relationship between PAH Content and Mutagenic Activity of Fumes from Roofing and Paving Asphalts and Coal Tar Pitch

Evalution of the Relationship between PAH Content Mutagenic Acitivity of Fumes from Roofing and Paving Asphalts and Coal Tar Pitch. MACHADO, M. L., BEATTY, P. W., FETZER, J. C., GLICKMAN, A. H., AND McGINNIS, E. L. (1993). Fundam. Appl. Toxicol. 21, 492–499. Fume condensates from asphalt and coal tar pitch were evaluated to determine if polycyclic aromatic hydrocarbon (PAH) composition, crude oil source, or temperature at which the fume was generated correlated with mutagenic activity. The fume condensates were tested for mutagenic activity using a modified Ames Test. Benzo[a]pyrene (BP) and other PAHs were detected in all samples. The concentration of BP in coal tar pitch was 18, 100 ppm while the concentration in asphalt was less than 6 ppm. Coal tar fumes contained between two and three orders of magnitude more BP, as well as other PAH species, than asphalt fumes. Coal tar fume condensates were also approximately 100 times more mutagenic than those of asphalt. Generation temperature, crude oil source, and/or process conditions affected the PAH concentrations but not the mutagenicity in roofing asphalt fume condensates. With paving asphalt fumes, PAH content and mutagenicity varied with crude oil source but not with processing conditions; due to limited data, it was not possible to determine the effect of generation temperature. Coal tar pitch fumes generated at 316°C contained significantly higher concentrations of PAHs than those generated at 232°C and the mutagenic activity generally paralleled the PAH content. A subset of the paving asphalts demonstrated good correlation between mutagenicity and three- to seven-ring PAH content. These results indicate that asphalt fumes are far less mutagenic than coal tar fumes. Asphalt fumes differ in their ability to induce mutagenic activity, and, most likely, in their potential carcinogenicity.

Evaluation of the Relationship between PAH Content and Mutagenic Activity of Fumes from Roofing and Paving Asphalts and Coal Tar Pitch

Evaluation of the Relationship between PAH Content and Mutagenic Activity of Fumes from Roofing and Paving Asphalts and Coal Tar Pitch. Machado, M. L., Beatty, P. W., Fetzer, J. C., Glickman, A. H., and McGinnis, E. L. (1993). Fundam. Appl. Toxicol. 21, 492-499.Fume condensates from asphalt and coal tar pitch were evaluated to determine if polycyclic aromatic hydrocarbon (PAH) composition, crude oil source, or temperature at which the fume was generated correlated with mutagenic activity. The fume condensates were tested for mutagenic activity using a modified Ames Test. Benzo[a]pyrene (BP) and other PAHs were detected in all samples. The concentration of BP in coal tar pitch was 18,100 ppm while the concentration in asphalt was less than 6 ppm. Coal tar fumes contained between two and tree orders of magnitude more BP, as well as other PAH species, than asphalt fumes. Coal tar fume condensates were also approximately 100 times more mutagenic than those of asphalt. Generation temperature, crude oil source, and/or process conditions affected the PAH concentrations but not the mutagenicity in roofing asphalt fume condensates. With paving asphalt fumes, PAH content and mutagenicity varied with crude oil source but not with processing conditions; due to limited data, it was not possible to determine the effect of generation temperature. Coal tar pitch fumes generated at 316°C contained significantly higher concentrations of PAHs than those generated at 232°C and the mutagenic activity generally paralleled the PAH content. A subset of the paving asphalts demonstrated good correlation between mutagenicity and three- to seven-ring PAH content. These results indicate that asphalt fumes are far less mutagenic than coal tar fumes. Asphalt fumes differ in their ability to induce mutagenic activity, and, most likely, in their potential carcinogenicity.

Evaluation of the dermal carcinogenicity of lubricant base oils by the mouse skin painting bioassay and other proposed methods.

Lubricant base oils are petroleum products that are predominantly derived from the vacuum distillation of crude oil. Various types of refinement can be employed during the manufacturing process, and evidence suggests that certain of the associated process streams produce skin cancer. Polycyclic aromatic compounds (PACs), some of which are considered as the causative agents, are removed, concentrated or chemically converted during the refinement process. In order to understand the effects of various types of refinement processes on carcinogenic potential, 94 oils were evaluated in the mouse epidermal cancer bioassay. This Exxon database is unique, because of the wide range of crude oils and processing histories represented. Seven processing history classifications are described, and conclusions concerning the impacts of each refinement process on dermal carcinogenicity are discussed. This research also included an evaluation of selected biological and chemical test methods for predicting carcinogenic potential. These included a modified version of the Ames test for mutagenicity, as well as analytical characterizations of the polycyclic aromatic structures in the oils. For classification purposes, a sample was considered to be carcinogenic if it resulted in the production of two or more tumor-bearing animals (in test groups of either 40 or 50 animals). The modified Ames test was considered to be positive if the mutagenicity index was > or = 2.0, and PAC analyses were similarly designated as positive or negative according to proposed guidelines. All of the alternative test methods showed similar agreement with dermal carcinogenicity bioassay data; concordance values were > or = 80%. However, each test was incorrect in ca. 10%-20% of the cases evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicological and chemical characterization of organic micropollutants in river po waters (Italy)

River Po waters were collected at the end of the drainage basin, extracted by means of XAD-2 resins and tested for toxicity on aquatic organisms ( Daphnia magna and Selenastrum capricornutum) and for mutagenicity with a modified Ames test. The extracts were analyzed by HRGC/FID and selective detectors for the determination of the most common classes of toxic compounds. The relationships between toxicological responses and analytical results are discussed.

The mutagenic activity and polycyclic aromatic hydrocarbon content of mineral oils

Naphthenic distillates (raw or acid-treated) and motor and emulsifiable aluminium rolling oils were tested for mutagenicity in the Salmonella/microsome assay using the TA98 and TA100 strains. The polycyclic aromatic hydrocarbon (PAH) content of oil samples was also determined in parallel. In the presence of metabolic activation, both untreated and acid-treated naphthenic distillates were found to be mutagenic on a modified Ames test. One untreated sample showed the highest value of mutagenic potency (50 net revertants/mg oil for strain TA98). The PAH content of naphthenic distillates was about 10% (w/w) and was slightly reduced by sulfuric acid/earth treatment (1%). Non-mutagenic paraffin- and solvent-extracted crankcase oils became active, both with and without enzyme activation, after long use as gasoline engine lubricants, whereby their PAH content doubled (from 1.5% to 3%, w/w). A refined emulsifiable mineral oil also became directly mutagenic in both Salmonella strains after prolonged use in an aluminium hot-rolling mill. As the PAH levels found in used rolling oils was very low, we cannot explain their mutagenic activity. Mutagenicity was greatly reduced following careful cleaning of the oil bath and of the entire rolling machine. The present data reveal both the potential risk of occupational exposure to unrefined or mildly treated oils and the formation of mutagens in highly refined oils if the latter are used at high temperatures. The formation of mutagens in oils used in the metal-working industry can be prevented by careful industrial hygiene measures.

Consideration of Variables in Evaluation of Toxicity to Skin

Certain systems for estimation of toxicity to skin are discussed, in which fuller consideration of variables led to improved study design and better prediction of human hazard. Three areas are considered: skin irritation; dermal carcinogenesis; prediction of carcinogenic potential (in vitro). (1) Variation in the guidelines of various U.S. and foreign agencies for evaluation of skin irritation and corrosiveness of chemicals was addressed by use of multiple-site patching in the rabbit. Data obtained from this “composite assay,” as well as from other published studies, indicated that no single set of guidelines was adequate for proper human hazard assessment. (2) Practical questions are raised about latency time and incidence of skin tumors from limited exposure to chemical carcinogens. In benzo[a]pyrene (BaP) carcinogenesis in mice, the duration of treatment is a key factor in tumor onset and frequency, with a “saturation level” resulting from interplay of duration and dose level. Age at inception of treatment can also be a factor in susceptibility to BaP-induced skin cancer, depending on conditions of exposure. (3) The question of carcinogenic potential of oil industry raw materials requires short-term answers, such as might be provided by an in vitro mutagenicity test. However, such tests are not appropriate for these water-immiscible mixtures. Studies on bioavailability of oil-related materials enabled modification of the Ames bacterial test, vastly improving sensitivity. With this modified Ames test, high correlation is seen among mutagenicity, content of polycyclic aromatics, and dermal carcinogenicity.

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes I. Mutagenicity in Salmonella typhimurium

Ptaquiloside, a potent carcinogen of an illudane-type sesquiterpene glycoside isolated from Pteridium aquilium, and its related compounds, hypolosides having the same nucleus isolated from the Pteridaceae, exhited marked mutagenicity in the modified Ames test with Salmonella typhimurian TA98 and A100 using a preincubation at pH 8.5 Illudins M and S, sesquiterpenes of the same illudane type from basidiomycetes, also exhibited mutagenicity. The structural requirements for mutagenicity are discussed.

The use of rat urine extracted on XAD-2 resin and assayed in a microsuspension-modified Ames test as an in vivo indicator of genotoxic exposure

The measurement of urinary mutagenicity is a non-invasive monitoring tool which often reflects an animal's recent exposure to genotoxic agents. Although studies in man are indispensable for monitoring industrial and/or environmental exposure to genotoxins, a sensitive laboratory animal model is necessary for mechanistic studies on the role of specific chemical exposure in altering urinary mutagenicity. The objective of this study was to enhance the sensitivity of the methodology used for detecting urinary mutagenicity in rats by using XAD-2 resin to extract and concentrate the urine and a microsuspension modified Ames test to quantify mutagenicity. The polycyclic aromatic hydrocarbon benzo[ a]pyrene (BP) and the aromatic amine 2-acetylaminofluorene (AAF) were used as test compounds. Under the conditions of our study, AAF administered to rats by gavage at doses of 1 mg/kg or higher induced a dose-dependent increase in urine mutagenicity. The greatest mutagenic response was seen when S9 was present during the microsuspension-modified Ames test and β-glucuronidase (BG) was not included. Similarly, BP administered to rats by gavage at doses of 10 mg/kg or higher induced a dose-dependent increase in urinary mutagenicity. The relative importance of BG and S9 were quite different with BP than with AAF. With BP, mutagenicity was greatest when both S9 and BG were present during the microsuspension-modified Ames test, and least with S9 and without BG. In both AAF- and BP-treated animals, extraction of the urine on XAD-2 resin markedly enhanced the mutagenic response compared to neat urine, but partitioning of the XAD-2 eluate into methylene chloride always diminished the mutagenicity of the urine extract. The results demonstrate the sensitivity and reproducibility of rodent urinary mutagenicity assays when XAD-2 resin is used to extract and concentrate the urine and a microsuspension-modified Ames test is used to quantify mutagenicity. This sensitive method should facilitate mechanistic studies on the roles of specific environmental agents in affecting urinary mutagenicity and, in addition may be used during acute, subchronic and chronic rodent bioassays as a non-invasive in vivo indicator of genotoxic exposure.

Mutagenesis of Escherichia coli (CSH50) by asbestos.

The mutagenic character of richterite asbestos was detected in Escherichia coli strain CSH50, using a modified Ames test. Two sets of experimental plates which contained naturally occurring asbestos were used, to one set of which S-9 rat liver homogenate was added. Control plates contained no asbestos. Mutant colonies appeared in significantly greater frequency in both experimental sets as compared with controls, and experimental plates with rat liver homogenate contained the greatest number of mutant colonies. It is postulated that enzymes contained in rat liver metabolize some unknown mutagen introduced with the natural asbestos sample other than the inert richterite fibers themselves. This finding, together with the unlikelihood that bacterial cell walls could be easily penetrated by asbestos fibers, tends to negate fiber penetration as the mechanism of mutagenesis. The mutagenic mechanism operative in E. coli (CSH50) may be applicable to animal cell systems.

The effects of cis-platinum(II)diamminodichloride, UV light and N-methyl- N′-nitro- N-nitrosoguanidine on Escherichia coli: Plasmid mediated resistance to mutagens

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride ( cis-PDD). Survival after treatment determined as colony forming ability was greater in rec + strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl- N′-nitro- N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA + and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.

Superoxide dismutase protects against paraquat-mediated dioxygen toxicity and mutagenicity: studies in Salmonella typhimurium.

Paraquat is univalently reduced to the relatively stable, but oxygen-sensitive, paraquat radical (PQ.+). This PQ.+ can react with dioxygen to generate the superoxide radical, which can further generate other more deleterious species of oxygen free radicals (i.e., hydroxyl radical, OH.). These oxygen free radicals are known to cause chromosomal breaks; therefore, it was logical to postulate that paraquat is a mutagen. This proved to be the case when tested in a modified Ames test using a liquid incubation assay. Salmonella typhimurium strains TA98 and TA100 were grown in the presence of various concentrations of PQ, as well as in the presence of known mutagenic compounds: mitomycin C, azide, and proflavine. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium and these toxic and mutagenic effects were oxygen dependent. Furthermore, cells containing high levels of superoxide dismutase were more resistant to the toxic and mutagenic effects of paraquat than were cells containing a normal level of this enzyme.