Showing papers on "Ames test published in 2006"

High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay.

Abstract: The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity assay (Ames test) is offset by the low specificity of the established mammalian cell assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and non-genotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability.

Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial

Abstract: Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.

DNA damage and mutagenicity induced by endosulfan and its metabolites.

Abstract: Endosulfan is a widely used broad-spectrum organochlorine pesticide, which acts as a contact and stomach poison. Nontarget species, such as cattle, fish, birds, and even humans, are also affected. Studies on the genotoxicity and mutagenicity of endosulfan have been inconsistent and nothing is known about the genotoxicity of its metabolites. In the present study, endosulfan (as a commercial isomeric mixture and as the alpha- and beta-isomers), and metabolites of endosulfan (the sulfate, lactone, ether, hydroxyether, and diol derivatives) were assayed for their ability to induce DNA damage in Chinese hamster ovary (CHO) cells and human lymphocytes using the Comet assay and were assayed for their mutagenicity using the Salmonella reversion assay (Ames test with TA98, TA97a, TA102, TA104, and TA100, with and without S9 activation). The compounds produced statistically significant (P < 0.01), concentration-dependent (0.25-10 microM) increases in DNA damage in both CHO cells and human lymphocytes. Endosulfan lactone caused the most DNA damage in CHO cells, while the isomeric mixture of endosulfan produced the greatest response in lymphocytes. The test compounds also were mutagenic in Salmonella strains at concentrations of 1-20 mug/plate (P < 0.05), with TA98 being the most sensitive strain and the diol and hydroxyether metabolites producing the highest responses. The results indicate that exposure to sublethal doses of endosulfan and its metabolites induces DNA damage and mutation. The contribution of the metabolites to the genotoxicity of the parent compound in Salmonella and mammalian cells, however, is unclear, and the pathways leading to bacterial mutation and mammalian cell DNA damage appear to differ.

Electrophilic Intermediates Produced by Bioactivation of Furan

Abstract: The industrial and environmental chemical, furan, is a liver toxicant and carcinogen in laboratory animals. It has been classified as a possible human carcinogen. The mechanism of tumor induction is unknown. However, toxicity is initiated by cytochrome P450 catalyzed oxidation of furan to an α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial. This metabolite reacts readily with protein and DNA nucleophiles and is a bacterial mutagen in Ames assay strain TA104. Metabolism studies indicate that this reactive metabolite is formed in vivo. It is also an intermediate leading to other metabolites whose role in furan-derived toxicities has yet to be explored.

Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro.

Abstract: Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.

Current trends and perspectives in nutrition and cancer prevention.

Abstract: There is an increasing evidence that dietary phytochemicals may play important roles as chemopreventive or chemotherapeutic agents in prevention of many diseases, including tumors. The purpose of this study was to examine antimutagenic effects and effect on the immune response of representative series of substances which commonly occur in human diet. Using the Ames bacterial mutagenicity test and in vivo chemiluminescence test, we investigated antigenotoxic and immunomodulatory effects of juices and vegetable homogenates (carrot + cauliflower, cauliflower, red cabbage, broccoli, onion, garlic) on the genotoxicity of AFB1 and pyrolysates of aminoacids. Using the Ames test and in vivo micronucleus, the chemiluminescence test, the blastic transformation test and the comet assay we examined antimutagenic effects of chemically identified chemoprotective substances in the pure form (resveratrol, diallylsulphide, phenethyl isothiocyanate, ellagic acid, epigallocatechin gallate, genistein and curcumin) on mutagenicity induced by three reference mutagens: aflatoxin B1 (AFB1), 2-amino-3-metylimidazo[4,5,-f] chinolin (IQ) and N-nitroso- N-metylurea (MNU) and effect of phytochemicals on the immunosuppression caused by these mutagens. All complete vegetable homogenates and substances of plant origin tested, showed a clear antimutagenic and immunomodulatory activities on mutagenicity and immunosuppression induced by reference mutagens. Only in the Ames test the effect of some phytochemicals against direct mutagen MNU was lower compared to indirect mutagens AFB1 and IQ. Similarly, resveratrol and epigallocatechin gallate had no inhibitory effect on mutagenicity MNU in the Ames test.

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N′-diacetyl metabolites

Abstract: para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.

Determination of antimutagenic properties of acetone extracted fermented milks and changes in their total fatty acid profiles including conjugated linoleic acids

Abstract: Kefir has a unique microbial content that results in complex fermented milk drink. In this study, the antimutagenic power of acetone-extracted kefir was determined and compared to acetone-extracted yogurt and milk using different chemical mutagens in the Ames Salmonella microsomal test. Acetone extracts were fat portions of the kefir, yogurt and milk samples. The direct-acting mutagenic agents methylmethanosulphate (MMS) and sodium azide (SA), and the indirect-acting mutagenic agents aflatoxin B1 (AFB1), 2-aminoanthracene (2AA), and 3,2-dimethyl-4-aminobiphenyl (DMAB) were used in Ames test with TA 100 Salmonella typhimurium. Acetone-extracted fermented milk samples significantly inhibited mutagenicity for different percentages of MMS, SA, AFB1 and 2-AA. Results indicate that milk also had a lower antimutagenic effect. Additionally, the fatty acid profiles of milk, yogurt and kefir were determined. A two-step methylation method followed by gas chromatography was used to identify conjugated linoleic acids (CLA). Fermentation slightly affected fatty acid composition. Three isomers of CLA (c9, t11; t10, c12; t9, t11), butyric, palmitic, palmitoleic, oleic acids, which have been proven as antimutagenic components of milk fat, were in higher concentrations in kefir.

Genotoxicity of Microbial Volatile Organic Compounds

Abstract: Luminescent and light absorption umu tests were used to investigate the genotoxicity of microbial volatile organic compounds (MVOCs), which have been reported to occur in conjunction with the growth of filamentous fungi. Investigation of 20 types of MVOC samples confirmed the SOS-inducing activity of 1-octen-3-ol, 2-methyl1-propanol, 2-heptanone, 3-octanol, 1-pentanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-methyl-2butanol, 3-octanone, 2-hexanone, 2-butanone, 3-methyl-2-butanone, 2-pentanol, ethyl isobutyrate, and terpinen-4ol. Of these materials, 3-methyl-2-butanone and 3-methyl-2-butanol, which were positive in both the luminescent and light absorption umu tests, were clearly shown also to be mutagenic based on the results of the Ames test. Each of these 20 MVOCs is known to be produced by microorganisms commonly detected in indoor environments, and long-term exposure could be a health hazard.

Mutagenicity and Acute Toxicity Evaluation of 2-Dodecylcyclobutanone

Abstract: Mutagenicity and acute toxicity of 2-dodecylcyclobutanone (2-DCB), a unique radiolytic product, were evaluated. Mutagenicity was evaluated by the Ames assay using 5 standard Salmonella tester strains with S9 enzyme activation and 5 concentrations of 2-DCB. Sodium azide (NaN 3 ), fenaminosulf, and 2-aminofluorene (2-AF) served as positive controls. The Ames assay showed no difference between the 5 concentrations of 2-DCB and the controls, including samples incubated with S9. The results indicate that 2-DCB does not produce point or frameshift mutations in Salmonella and is not activated by S9. Acute toxicity of 2-DCB was evaluated by the Microtox acute toxicity system and compared with cyclohexanone and 2-nonenal (both GRAS additives). The effective concentrations that caused a 50% reduction in light emission by Vibrio fischeri cells (EC 50 ) were; 21.72 6 14.57 ppm for 2-DCB, 37.40 6 0.45 ppm for cyclohexanone, and 1.65 6 0.26 ppm for 2-nonenal. The maximum number of cells affected by 2-DCB was 65% 6 4%, while it reached 90% to 100% for the other 2 compounds. Our results suggest that even though the EC 50 for 2-DCB is lower than that for cyclohexanone, it was not toxic enough to decrease light emission of V. fischeri beyond 60% to 70%. These results indicate that the potential risk from 2-DCB, if any, is very low.

Genetic toxicity testing of 3-methyl-2H-furo[2,3-c]pyran-2-one, an important biologically active compound from plant-derived smoke

Abstract: Aerosol smoke, aqueous smoke solutions and 3-methyl-2H-furo[2,3-c]pyran-2-one isolated from plant-derived smoke stimulate seed germination and enhance seedling vigour of many species. Consequently, smoke technology has important potential applications in agriculture, horticulture and other environmental sectors. However, use of 3-methyl-2H-furo[2,3-c]pyran-2-one as a germination promoter and/or growth stimulant is only possible provided it can be shown that it is not hazardous, i.e. not toxic or genotoxic. We therefore performed an investigation to evaluate the genotoxic effects of this compound in five strains of Salmonella typhimurium in the Ames test (1.5 μg/plate to 7.5 ng/plate), and in the VITOTOX® test (1500–0.045 ppb). In all tests performed, no mutagenic activity was induced by 3-methyl-2H-furo[2,3-c]pyran-2-one, with or without S9 metabolic activation. Due to the potential wide use of this smoke-derived compound, it is of importance that no toxic and genotoxic effects were observed at the concentrations tested.

In vitro anti-mutagenic effect of magnolol against direct and indirect mutagens

Abstract: Magnolol, a component of the bark of Magnolia obovata, has been reported to possess various biological activities, such as anti-carcinogenicity, anti-promotion activity and anti-oxidative activity. These findings suggest potential for this compound in cancer chemoprevention. Interestingly, there have been no reports to date on the potential anti-mutagenic activity of magnolol, involving inhibition of initiation processes of the primary stage of carcinogenicity. In this study, anti-mutagenic activity of magnolol against mutagenicity induced by direct mutagens [1-nitropyrene (1-NP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)] and indirect mutagens [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), benzo(a)pyrene (B(a)P), 2-aminoanthracene (2-AA) and 7,12-dimethylbenz[a]anthracene (DMBA)] were investigated using the bacterial mutagenicity test (Ames test). Results show that magnolol strongly inhibits mutagenicity induced by indirect mutagens, but does not affect direct mutagens. To elucidate the mechanism of this effect against indirect mutagens, effect of magnolol on CYP1A1- and CYP1A2-related enzyme activities of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) were investigated. Magnolol strongly and competitively suppressed these enzyme activities, suggesting it inhibited mutation induced by indirect mutagens through suppression of CYP1A1 and CYP1A2 activity.

The mutagenic potential of madder root in dyeing processes in the textile industry.

Abstract: The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated.

Inhibitory effects of beer on mutation in the Ames test and DNA adduct formation in mouse organs induced by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

Abstract: An evaluation of the antigenotoxic potential of beer components against carcinogens contained in the human diet, namely heterocyclic amines (HCAs) including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was determined. The protective mechanism involved was also investigated. Beer samples were found to inhibit the mutagenicity of HCAs in the Ames test. Beer solution, consisting of a freeze-dried and dissolved sample, given as drink-water significantly reduced the formation of PhIP-DNA adducts in mouse colon and lung compared to control mice fed with PhIP in the absence of beer solution. Furthermore, beer solution added in the diet as a food additive mimic significantly reduced the amount of DNA adducts present in the liver and lung of mice fed with PhIP. In an effort to investigate the mechanism responsible for the observed protective effect, the effect of beer solutions on HCA metabolizing enzymes was investigated. Beer solutions inhibited the activity of CYP1A1 and CYP1A2, as determined from deethylation and demethylation assays using 7-ethoxy- and 7-methoxyresolufin, respectively. Considering the overall suppression of PhIP genotoxicity by beer, this study confirmed that beer components can interfere with the enzyme activity involved in the metabolism of HCAs and subsequently suppress the observed genotoxicity. The results of this study showed that beer components act in a protective capacity against the genotoxic effects of heterocyclic amines in vivo.

Activity related to the carcinogenicity of plastic additives in the benzophenone group.

Abstract: This study examines the activities relating to the carcinogenicity of six types of benzophenone derivatives (benzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2,4-dihydroxybenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone and 2,2'-dihydroxy-4,4'-dimethoxybenzophenone) currently used in plastic products as additives to serve as ultraviolet absorbing agents The evaluation of the initiation activity used a light absorption umu-test, a luminescent umu-test and the Ames test The promotion activity was examined by a Bhas assay, a method that uses Bhas 42 cells for the formation of transformation foci The luminescent umu-test indicated positive initiation activity of 2-hydroxy-4-methoxybenzophenone, and pseudo-positive activity of 2,4-dihydroxybenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone In the Ames test, 2-hydroxy-4-octyloxybenzophenone showed pseudo-positive initiation activity Conversely, 2,4-dihydroxybenzophenone indicated weak promotion activity at 10 microg/ml concentration

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge.

Abstract: A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF = 1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF = 1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60 min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI = 2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.

Mutagenicity of UV-irradiated maltol in Salmonella typhimurium

Abstract: We investigated the photomutagenicity of maltol (3-hydroxy-2-methyl-4H-pyran-4-one) in bacterial cells. Maltol has a caramel-butterscotch odour and is used as a food additive to impart flavour to bread and cakes. Unirradiated maltol was not mutagenic up to 5 mg/plate in the Ames test. When maltol was irradiated with either UVA (a black light, 320-400 nm, 230 μW/cm 2 ) for 5-30 min or UVC (a germicidal lamp, 610 μW/cm 2 ) for 3 min in sodium phosphate buffer (pH 7.4) prior to the exposure of bacterial cells, it was mutagenic to Salmonella typhimurium TA100, TA104 and TA97. Mutagenic activation of maltol by UVA-irradiation was more evident in neutral and alkaline conditions (pH 7.0-9.0) than in acidic conditions. On the other hand, photomutagenicity was not observed when maltol was irradiated with UVA in 100 mM NaCl solution or water. The mutagenic photoproduct was stable for at least 60 min after UVA-irradiation. However, addition of thiol compounds (cysteine or glutathione) to the UVA-irradiated maltol diminished the mutagenicity. Mutational spectrum analysis revealed that the predominant base-substitutions induced were G:C→T:A transversions and G:C→A:T transitions. An increase of 8-hydroxydeoxyguanosine formation in salmon sperm DNA exposed to maltol and UVA in vitro was detected by HPLC-ECD, but it was too small to explain the photomutagenicity. We are considering the formation of DNA adducts as the photomutagenic mechanism.