Showing papers on "Ames test published in 1994"

Evaluation of the Allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater

Abstract: The Allium anaphase-telophase test was evaluated to find out if it could be recommended in the screening of wastewater for genotoxicity. Five mutagenic or carcinogenic chemicals usually found in wastewater were tested in the Allium anaphase-telophase test. Sodium dichromate (25 μM), benzene (100 μM), dichloromethane (175 μM) and 1,1,1-trichloromethane (175 μM) increased the frequency of chromosome aberrations in the root cells, whereas formaldehyde (1 mM) was found to be non-mutagenic in this test system. Other studies where chemicals were tested in the Allium test were reviewed. For 15 chemicals the results were compared with results from the Ames test, the Microscreen assay, and carcinogenicity tests in rodents. The sensitivity of the Allium test was calculated to be 82%. In conclusion the Allium test is recommended for the screening of wastewater because it has a high sensitivity, is cheap, rapid, easy to handle, and because it can be used on wastewater without pretreatment of the sample.

5-Sulfooxymethylfurfural as a possible ultimate mutagenic and carcinogenic metabolite of the Maillard reaction product, 5-hydroxymethylfurfural

Abstract: Heat treatment of foods containing reducing sugars and amino acids during cooking or sterilization triggers a sequence of non-enzymatic reactions collectively known as the Maillard reaction. 5-Hydroxymethylfurfural (HMF), one of the major intermediate products in the Maillard reaction, has been found to possess cytotoxic, genotoxic and tumorigenic activities, but the mechanisms of its toxic actions remain unclear. Formation of an electrophilic allylic ester bearing a good leaving group such as sulfate has been proposed as a possible metabolic activation pathway for HMF. In order to further test this possibility, we compared the mutagenic and carcinogenic activities of HMF and its chemically synthesized sulfuric acid ester, 5-sulfooxymethylfurfural (SMF). SMF induced dose-dependent increases in the number of His+ revertants in Salmonella typhimurium TA100. This intrinsic mutagenicity of SMF was significantly inhibited by ascorbic acid added to the assay media. In the presence of chloride ions, the bacterial mutagenicity of the highly polar sulfuric acid ester of HMF may also be mediated by formation of a lipophilic chloromethyl derivative. When topically applied to mouse skin, both sulfooxymethyl and chloromethyl derivatives of HMF exhibited higher skin tumor initiating activity than the parent hydroxymethyl compound. 5-Chloromethylfurfural was found to be a strong hepatocarcinogen in infant male B6C3F1 mice.

Suppression of Hydroperoxide-induced Cytotoxicity by Polyphenols

Abstract: A variety of synthetic and dietary polyphenols protect mammalian and bacterial cells from cytotoxicity induced by hydroperoxides, especially hydrogen peroxide (H2O2). Cytotoxicity of H2O2 on Chinese hamster V79 cells was assessed with a colony formation assay. Cytotoxicity and mutagenicity of H2O2 on Salmonella TA104 were assessed with the Ames test. SOS response induced by H2O2 was investigated in the SOS chromotest with Escherichia coli PQ37. The polyphenol-bearing o-dihydroxy (catechol) structure, i.e., nordihydroguaiaretic acid, caffeic acid ester, gallic acid ester, quercetin, and catechin, were effective for suppression of H2O2-induced cytotoxicity in these assay systems. In contrast, neither ferulic acid ester-bearing o-methoxyphenol structure nor alpha-tocopherol were effective, indicating that o-dihydroxy or its equivalent structure in flavonoids is essential for the protection. There are many reports describing that polyphenols act as prooxidants in the presence of metal ions. Our results suggest, however, that they act as antioxidants in the cells, when no metal ions are added to the medium.

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: Studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen

Abstract: Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 μM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase (Ki ∼ 120 μM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.

The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity.

Abstract: Hydrogen peroxide (H2O2) has been implicated as a major contributor to coffee mutagenicity and genotoxicity in vitro. We have used three assays to show the gradual formation of H2O2 in freshly prepared roasted ground coffee and in instant coffees over time reaching levels of 400-450 microM after a 1-h incubation period. Formation of H2O2 occurs through an auto-oxidation process where polyphenolics, in the presence of transition metals, reduce atmospheric oxygen. However, because of these polyphenolics, coffee also possesses in vitro antioxidant activity as shown by its capacity to inhibit lipid peroxidation in Fenton-catalysed hydroxylation reactions. The pro- and antioxidative effects of coffee are also reflected in its mutagenic and antimutagenic activity in the Ames test. Coffee is directly mutagenic in strains TA100 and TA102 due to H2O2 formation. However, coffee is also an antioxidant and antimutagen. This beverage exerts a strong protective effect against the mutagenicity and cytotoxicity induced by the oxidant t-butylhydroperoxide (t-BOOH). Thus, coffee, like many antioxidants, exhibits dual effects in vitro which are highly dependent upon parameters such as dose, atmospheric oxygen, transition metals as well as the biological and chemical endpoints used for measurement. Consequently, the data obtained on the pro- and antioxidant properties of foods and beverages from in vitro bioassays must be interpreted with caution and the results are not easily extrapolated in vivo to assess the impact on human health.

Marked antimutagenic potential of aqueous green tea extracts : mechanism of action

Abstract: In the present study aqueous extracts of green tea, at the concentrations customarily consumed by humans, were evaluated for their antimutagenic activity against major classes of dietary and occupational carcinogens. Green tea extracts caused a very marked and concentration-dependent inhibition of the Aroclor 1254-hepatic S9-mediated mutagenicity of heterocyclic amines (IQ and Glu-P-1) and polycyclic aromatic hydrocarbons (benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene) and of the isoniazid-induced S9-mediated mutagenicity of nitrosamines (nitrosopiperidine and nitrosopyrrolidine). Similar inhibition was seen in the mutagenicity of the two aromatic amines, namely 2-amino-fluorene and 2-aminoanthracene, whether Aroclor 1254-S9, isolated microsomes or cytosol served as the activation system. Finally, the mutagenicity of the direct-acting mutagens 9-aminoacridine and MNNG was also suppressed by green tea extracts, but the effect was less pronounced when compared with the indirect-acting mutagens. Green tea extracts caused a marked and concentration-dependent decrease in the O-dealkylation of methoxyresorufin, ethoxyresorufin and pentoxyresorufin. A similar inhibition of the NADPH-dependent reduction of cytochrome c was also observed. Following the termination of the microsomal metabolism of the various promutagens, incorporation of green tea extracts into the activation system resulted in a comparatively modest inhibition of their mutagenic response. It is concluded that aqueous extracts of green tea possess marked antimutagenic potential against a variety of important dietary and environmental mutagens. Two mechanisms appear to be responsible. The first involves a direct interaction between the reactive genotoxic species of the various promutagens and nucleophilic tea component(s) present in the aqueous extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity of 1,3-dithiane and 1,4-dithiane in the CHO/SCE assay and the Salmonella/microsomal test

Abstract: 1,3-Dithiane and 1,4-dithiane are the sulfur-containing Maillard reaction products (MRPs) which have been found in boiled beef extracts. In this study the genotoxicity of these products was examined using the Salmonella/microsomal test and the CHO/SCE assay. 1,3-Dithiane showed a potent direct-acting mutagenicity toward S. typhimurium TA98 and TA100, but 1,4-dithiane had a lower mutagenicity toward both tester strains. Both compounds were shown to be non-mutagenic with hepatic metabolic activation with the exception of 1,3-dithiane toward strain TA100. To compare the mutagenic potential of 1,3-dithiane and 1,4-dithiane with other types of MRPs, 24 MRPs were examined for their mutagenicity to S. typhimurium TA98 and TA100 in the presence or absence of S9 mix. 2,6-Dimethylpyrazine, furan, 2-acetylpyrrole, and thiazole were shown to be mutagenic. However, these four MRPs exhibited a lower mutagenicity in TA98 than 1,3-dithiane and 1,4-dithiane. Furthermore, SCE frequencies in CHO cells were very significantly induced by 1,3-dithiane in the absence of S9 mix, but the SCE-inducing capability of 1,3-dithiane was reduced or even disappeared with metabolic activation. 1,4-Dithiane did not significantly induce SCE frequencies in the presence or absence of S9 mix. Thus, we concluded that 1,3-dithiane was a potent mutagenic MRP in the Salmonella/microsomal test, whereas it was a weak SCE inducer in the CHO/SCE assay.

Comparative assessment of the SOS Chromotest kit and the Mutatox test with the Salmonella plate incorporation (Ames test) and fluctuation tests for screening genotoxic agents

Abstract: Genuine needs for rapid, simple, and cost-efficient biotesting procedures to screen an ever-increasing number of chemicals and environmental samples are making the search for such assays a constant endeavor. With respect to genotoxicity screening, we compared, in this study, the performance of two novel assays (Vibrio fischeri M169 Mutatox™ assay and the Escherichia coli PQ37 SOS Chromotest kit assay) with two well-established Ames testing procedures (plate incorporation and fluctuation assays). Testing material included 14 chemicals (10 potentially directly acting and 4 indirectly acting compounds) reflecting different chemical classes (2 inorganics, 2 pesticides, 2 halogenated hydrocarbons, 2 alkylating agents, 2 aromatic amines, 1 chlorophenol, and 3 polycyclic aromatic hydrocarbons). Comparative assessment criteria included (1) interprocedural agreement in detecting presence or absence of genotoxicity, (2) accuracy in being able to recognize animal (non)carcinogens, and (3) sensitivity (detection of lowest actively genotoxic concentration). In terms of qualitative responses, both the SOS Chromotest (86% agreement) and Mutatox assays (93% agreement) were good predictors of the Ames testing mutagenicity. For their capability to correctly discriminate between (non)carcinogens, accuracy was 82% (9 of 11 chemicals) for Mutatox, 73% (8 of 11 chemicals) for Ames testing, and 64% (7 of 11 chemicals) for the SOS Chromotest. In general, the Salmonella-based assays proved more sensitive (6 times out of 9 chemicals) than the Mutatox (3 times out of 9 chemicals) and the SOS Chromotest (never more sensitive). Overall, this study demonstrates reliable performances by both the SOS Chromotest and Mutatox for chemical genotoxicity screening when results are referenced to the well-validated Ames assay. Although additional comparative data with other chemicals will be required, it appears likely that these more practical and cost-efficient procedures can be presently useful to screen genotoxic activity of various xenobiotics and environmental samples. © 1994 by John Wiley & Sons, Inc..

Potent clastogenicity of the human carcinogen etoposide to the mouse bone marrow and mouse lymphoma L5178Y cells: comparison to Salmonella responses.

Abstract: The suspect human carcinogen, etoposide, is known to be genotoxic, producing both gene and chromosomal mutations, probably by virtue of its ability to inhibit topoisomerase II activity. The present paper describes assays conducted using the Salmonella assay, the mouse lymphoma tk+/- assay (gene and chromosomal mutation analysis and molecular analysis of tk-/- mutants) and the mouse bone marrow micronucleus assay. Nonreproducible, weak, dose-related increases in mutation frequency in strain TA98 (but not TA1538 or TA1537) of Salmonella typhimurium were observed. Etoposide was highly mutagenic at the heterozygous thymidine kinase (tk+/-) locus of L5178Y mouse lymphoma cells at concentrations below 0.1 micrograms/ml. Mostly small colony mutants were induced, consistent with the potent clastogenicity also observed. Molecular analysis of mutants indicated that 83% and 92% of large and small colony mutants, respectively, had lost the entire target gene sequence. Chromosomally aberrant L5178Y cells were approximately 2 to 600-fold more prevalent than small tk-/- mutant colonies. This suggests that the viable target for etoposide-mediated clastogenesis in the selective assay is approximately one-fifth of chromosome 11b, itself being approximately one-fortieth of the mouse genome. An unusually potent response was observed for etoposide in the mouse bone marrow micronucleus assay (63.1 +/- 18 MPE/1,000 PE 24 hours after an oral dose of 1 mg/kg). The minimum detectable dose level in the assay was between 0.01 and 0.1 mg/kg. At dose levels between 1 and 15 mg/kg, an inverse dose response was observed. This reduction in assay response was not due to the small concommitant decrease in the incidence of polychromatic erythrocytes, a conclusion based on studies with N-methyl-N-nitrosourea. Animals sampled 48 hours after dosing with etoposide (10 mg/kg) had no polychromatic erythrocytes in the bone marrow. These observations for the micronucleus assay await explanation. The chemical structure of etoposide is displayed and discussed within the context of such strong mutagenic activity being associated with a nonelectrophilic agent.

In vitro and in vivo genotoxicity evaluation of hormonal drugs v. mestranol

Abstract: Genotoxicity of a widely used estrogen, Mestranol, was undertaken using in vitro, in vivo and host-mediated assay with bacteria as indicator organism. Analyses of chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes and chromosome aberrations, micronuclei and sister chromatid exchanges (SCEs) in bone-marrow cells of mice showed the drug to be capable of attacking the genetic material. However, both Ames Salmonella/S9 assay with and without S9 mix and host-mediated assay using same tester strains of Salmonella, did not show any significant increase/decrease in the His+ revertants.

Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains

Abstract: The molecular mechanisms involved in quinone cytotoxicity, especially mutagenicity, are still largely unknown. In order to better understand the molecular aspects of the mechanisms of quinone mutagenicity and cytotoxicity, we examined them by using a series of 13 simple structural naphthoquinone (NQ) derivatives for 9 Ames Salmonella mutagenicity tester strains in the presence of absence of liver homogenate S9 mix from rats induced with phenobarbital and 5,6-benzoflavone. Most NQs used in this study showed mutagenicity with and/or without S9 mix. The most potent mutagenic NQ was 2,3-dichloro-1,4-NQ, with mutagenicity of 18 induced revertants/nmol/plate for strain TA104 without S9 mix. Among the strains used, TA104, which is sensitive to oxidative mutagens, was the most sensitive to the NQs, and the second most sensitive strain was TA2637, which detects bulky DNA adducts. The relationship of mutagenic potency to the one-electron reduction potential with TA104 suggested that the higher redox potential NQs were more mutagenic than the lower redox potential NQs. Significant reduction of the mutagenicity of 1,4-naphthoquinone without S9 mix was observed in the presence of catalase. Enhancement of the mutagenic potential of the NQs by the pKM101 plasmid implicated in error-prone repair was also observed. The most cytotoxic NQ was 2,3-dichloro-5,8-dihydroxy-1,4-NQ, and the least cytotoxic NQ was beta-NQ-4-sulfonic acid potassium salt, a 700-fold range in potency. The cytotoxic effect of the NQs was largely dependent on the structures of their substituents.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes.

Abstract: Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.

Mutagenic potential of root canal sealers: Evaluation through Ames testing

Abstract: The mutagenic potential of 12 commercially available dental cements and of two ‘pure substances’ (zinc oxide and eugenol) used in root canal filling were examined. The cements were prepared according to the manufacturers' indications and set for defined times. Ames tests were performed in their extracts by using Salmonella typhimurium strains TA 98 and TA 100. The results showed that most cements present strong bactericidal activity that disappears or decreases remarkably in time. One of the tested cements showed mutagenicity with both Salmonella strains. Two cements yielded doubtful results. The remaining cements and the two ‘pure substances’ showed no mutagenic potential. The authors conclude that it is convenient to examine endodontic cements with the Ames test and to eliminate those that present mutagenicity in time. © 1994 John Wiley & Sons, Inc.

Comparative mutagenicity of plant-activated aromatic amines using Salmonella strains with different acetyltransferase activities.

Abstract: Plant systems can activate aromatic amines into mutagens. In the plant cell/microbe coincubation assay, we earlier demonstrated that 2-aminofluorene and m-phenylenediamine were activated by plant cells into mutagens with reversion at the hisD3052 allele in Salmonella typhimurium strain TA98 as the genetic endpoint. New derivatives have been developed which possess elevated levels of acetyl-CoA:N-hydroxyarylamine O-acetyltransferase which are very sensitive to N-hydroxylated amines [Watanabe et al., 1990: Mutat Res 234:337-348]. The objectives of this research were to examine Salmonella strains with different acetyltransferase activities in the plant cell/microbe coincubation assay with a series of structurally related aromatic amines. The hypothesis tested was whether and to what degree a plant-activated metabolite of these aromatic amines could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. Every aromatic amine examined was activated by plant cells with YG1024 (the strain with elevated O-acetyltransferase activity) as the genetic indicator organism. The rank order of the mutagenic responses of YG1024 to the plant-activated aromatic amines was 2-aminofluorene >benzidine > m-phenylenediamine > 4-aminobiphenyl > 2,4-diaminotoluene > 2-naphthylamine. This rank order was expressed by TA98 but to a much lower degree. There was a very slight mutagenic response observed in TA98/1,8-DNP6 (a strain lacking O-acetyltransferase activity) with the plant-activated metabolites of benzidine and 4-aminobiphenyl. We conclude that the plant-activated aromatic amines are substrates for bacterial O-acetyltransferases. © 1994 Wiley-Liss, Inc.

Reduction and Mutagenic Activation of Nitroaromatic Compounds by a Mycobacterium sp

Abstract: Mycobacterium sp. strain Pyr-1 cells, which were grown to the stationary phase in media with and without pyrene, were centrifuged and resuspended in a medium containing 1-nitropyrene. Cells that had been grown with pyrene oxidized up to 20% of the added 1-nitropyrene to 1-nitropyrene-cis-9,10- and 4,5-dihydrodiols. However, cells that had been grown without pyrene reduced up to 70% of the 1-nitropyrene to 1-aminopyrene but did not produce dihydrodiols. The nitroreductase activity was oxygen insensitive, intracellular, and inducible by nitro compounds. Nitroreductase activity was inhibited by p-chlorobenzoic acid, o-iodosobenzoic acid, menadione, dicumarol, and antimycin A. Extracts from cells that had been grown without pyrene activated 1-nitropyrene, 1-amino-7-nitrofluorene, 2,7-dinitro-9-fluorenone, 1,3-dinitropyrene, 1,6-dinitropyrene, and 6-nitrochrysene to DNA-damaging products, as shown in Salmonella typhimurium tester strains by the reversion assay and by induction of the umuC gene. Activation of nitro compounds, as shown by the umu test, was enhanced by NADPH. This study shows that Mycobacterium sp. strain Pyr-1 metabolizes nitroaromatic compounds by both oxidative and reductive pathways. During reduction, it generates products that are mutagenic.

Genotoxicity and carcinogenicity in rats and mice of 2-amino-3,6-dihydro-3-methyl-7H-imidazolo[4,5-f]quinolin-7-one : an intestinal bacterial metabolite of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline

Abstract: Background Compounds formed on the surface of fried or grilled meat and fish may be associated with increased risk of colon cancer. Normal intestinal bacteria can convert one of these compounds, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), to the 7-hydroxy metabolite, 2-amino-3,6-dihydro-3-methyl-7H-imidazolo[4,5-f]quinolin-7-o ne (7-OHIQ), a direct-acting mutagen. Purpose We studied the genotoxicity and carcinogenicity of 7-OHIQ to determine if it is responsible for the colon-specific activity of IQ. Methods The effects of pure, synthetic 7-OHIQ on DNA were evaluated in the Ames Salmonella typhimurium TA98 test, with and without an induced rat liver S9 fraction, and in the Williams DNA repair test using freshly explanted rat hepatocytes. 7-OHIQ was also subjected to an in vivo bioassay for 21 months by long-term intrarectal infusion in male F344 rats, using IQ and N-nitrosomethylurea (NMU) given intrarectally as positive tumor-producing controls. The standard NIH-07 rodent diet was supplemented with 15% corn oil to maximize any effect of the infused materials on the colon. A parallel bioassay involved intraperitoneal injection of 7-OHIQ in newborn mice, followed by dietary administration from week 11 to week 67. Again, IQ and NMU were used as positive controls. Results We confirmed that 7-OHIQ is a direct-acting mutagen in the Ames test, with added S9 liver fraction giving higher mutagenicity. 7-OHIQ was negative in the Williams test, whereas IQ was positive. 7-OHIQ did not induce colon cancer in rats, and in the newborn mouse test it produced only a low incidence of liver neoplasms. Conclusions 7-OHIQ is not genotoxic, for to be so classified it must be definitely positive in both the Ames and Williams tests; moreover, it is not carcinogenic, in marked contrast to IQ and NMU.

Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities.

Abstract: Benomyl (methyl [1-[(butylamino)carbonyl]-1H-benzimidazol-2- yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate (samples tested up to 500 and 1200 micrograms/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (> or = 5000 micrograms/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20,000 micrograms/plate) or without (up to 16,000 micrograms/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 micrograms/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 microgram/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10 ppm, respectively, would be positive in the Salmonella/Ames test with activation when tested at 5000 micrograms/plate. Purified carbendazim is not mutagenic.

Inhibitory activity of cigarette-smoke condensate on the mutagenicity of heterocyclic amines.

Abstract: Cigarette-smoke condensate (CSC) is a complex mixture containing over 3800 identified chemicals including nicotine, water, mutagens, antimutagens, cytotoxins and inert chemicals. Although CSC is mutagenic in the Ames test, its effect on the activity of other mutagens has not been characterized. Using the Ames Salmonella bacterial mutagenesis assay, we found CSC exerts a significant inhibitory effect on mutagens requiring bioactivation. Those studied included heterocyclic amines (Glu-P-1, Glu-P-2, IQ, MeIQ, Trp-P-1 and Trp-P-2), benzo[a]pyrene (B[a]P) and aflatoxin B1. However, CSC had no effect on the activity of direct-acting mutagens (2-nitrofluorene, sodium azide, 4-nitro-1,2-phenylenediamine, 4-nitroquinoline N-oxide and methyl methanesulfonate). With indirect-acting mutagens, the reduced number of revertants observed in the presence of CSC was not attributable to cytotoxicity. CSC exhibited a potent inhibitory effect on the cytochrome P-450 dependent monooxygenases, ethoxyresorufin O-deethylase and B[a]P hydroxylase. This suggests inhibition of the cytochrome P-450 isozymes as one possible mechanism for the antimutagenicity of CSC. Fractionation studies of CSC revealed that the neutral, weakly acidic (phenolic) and basic fractions are all effective as antimutagens against Glu-P-1, a representative heterocyclic amine. This indicates that several classes of chemicals contribute to the inhibitory effect of CSC on the mutagenicity of the heterocyclic amines.

Mutagenicity and CYP1A induction by azobenzenes correlates with their carcinogenicity

Abstract: The genotoxicity of six azobenzenes was evaluated in the Ames test, in the presence of an activation system derived from Aroclor 1254-treated rats. Moreover, the ability of these azobenzenes to induce rat hepatic CYP1A activities and apoprotein levels, and stimulate their own bioactivation to mutagens, was also determined. In the presence of the Aroclor 1254-activation system, o-aminoazotoluene and 3-methoxy-4-aminoazobenzene were potent mutagens, whereas 4-amino-azobenzene and 4-diethylaminoazobenzene failed to elicit a positive mutagenic response. A very weak mutagenic response was induced by 2-methyl-4-dimethylaminoazobenzene and by azobenzene. o-Aminoazotoluene and 3-methoxy-4-aminoazobenzene were potent inducers of CYP1A activities and apoprotein levels, whereas the remaining four compounds displayed either very weak or no induction capability. None of the azobenzenes studied could induce its own activation to mutagens in the Ames test. All six azobenzenes displaced [3H]tetrachlorodibenzo-p-dioxin from the cytosolic Ah receptor, with o-aminoazotoluene and 3-methoxy-4-aminoazobenzene being the most effective. A correlation appears to exist between carcinogenic activity of azobenzenes in the rat on one hand, and of their mutagenic potential and hepatic CYP1 induction on the other. Possible mechanisms accounting for this relationship are discussed.

Evaluation of the genetic toxicity of middle distillate fuels

Abstract: Petroleum middle distillate (PMD) fuels are mixtures of hydrocarbons that distill between approximately 170-370 degrees C. Commercial products that fall into this category include kerosine, diesel fuel, jet fuel, and home heating oil. These products contain both saturated (paraffins and cycloparaffins) and aromatic species, but because of the boiling range normally contain very small amounts of the 3-6 ring polycyclic aromatic hydrocarbon (PAH) constituents, which are considered to be carcinogenic. Nevertheless, there is evidence of weak tumorigenic activity when these materials are repeatedly applied to mouse skin. In the current studies representative products were tested in two commonly used, short-term assays for genetic toxicity, the Salmonella/mammalian microsome mutagenicity assay and the mouse bone marrow micronucleus test. All samples were inactive in the micronucleus assay, and three were clearly inactive in the Salmonella test. Of the remaining two, one was marginally active in the Salmonella assay, and one was equivocal. The marginally active sample contained detectable levels of PAH due to the use of catalytically cracked materials as blending stocks. The results indicated that PMDs that do not contain cracked material were not mutagenic. Thus they may produce tumors via nongenotoxic processes. Those products that do contain cracked stocks may have sufficient PAH to be mutagenic in the Salmonella assay, and in those cases the PAH might also contribute to tumor formation.

Study of the genotoxic activity of five chlorinated propanones using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test

Abstract: Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of five chlorinated propanones identified in several chlorinated waters (monochloropropanone, 1,1-dichloropropanone, 1,3-dichloropropanone, 1,1,1-trichloropropanone and 1,1,3-trichloropropanone). In the SOS chromotest, all the compounds except monochloropropanone were found to induce primary DNA damage in Escherichia coli. With the fluctuation test, all five chloropropanones showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae only for 1,3-dichloropropanone and 1,1,3-trichloropropanone. Moreover, two structure-activity relationships are noticeable: (1) chloropropanones with chlorine substituents on both carbon positions (1,3-DCP and 1,1,3-TCP) are by far more genotoxic than chloropropanones substituted only on one carbon position (1,1-DCP and 1,1,1-TCP); (2) the increase of the number of chlorine substituents decreases the mutagenic activity (fluctuation test) of the chlorinated propanones studied.

Evaluation of pyrazole and ethanol induced S9 fraction in bacterial mutagenicity testing.

Abstract: A major constitutive enzyme in the liver of the uninduced rat is cytochrome P450-2E1. This isozyme has been shown to metabolize a number of carcinogens, including low molecular weight nitrosamines and a number of compounds normally regarded as non-mutagenic in the Ames test, e.g. aniline, urethane and benzene. Using the standard induction procedures [Aroclor 1254 or a combination of phenobarbitone (PB) and beta-naphthoflavone (beta-NF)] the level of CYP2E1 in rat liver is actually suppressed and it has been suggested that this may account for the negative findings with these compounds in the Ames test. S9 fractions were prepared from rats pre-treated with pyrazole or ethanol (inducers of CYP2E1) and then used in the Ames test (or pre-incubation modification) with urethane, acetaminophen, aniline, benzene, procarbazine and N-nitrosopyrrolidine. Both pyrazole and ethanol induced S9 were superior to PB/beta-NF-S9 and uninduced-S9 for the activation of N-nitrosopyrrolidine, a known CYP2E1 substrate. However, there was no evidence of mutagenic activity with urethane, aniline, benzene, procarbazine or acetaminophen. As these compounds have demonstrated genotoxicity in vivo, additional important metabolic pathways must be required which are not present in rat liver S9 fraction.

Screening for agents inhibiting the mutagenicity of extracts and constituents of tobacco products

Abstract: The aim of this study was to screen for potential agents affecting the mutagenicity of tobacco products. The influence of a number of compounds which have been suggested to be antimutagenic some of which are present in tobacco products, was investigated on the mutagenicity of a cigarette smoke condensate (CSC) and, in some cases, an extract of oral Swedish moist snuff (SNUS), using a screening procedure of the Ames Salmonella/microsome assay (STY). For some of the compounds the V79/hprt mutagenicity assay with benzo[a]pyrene metabolites as mutagens was used to obtain complementary and confirmatory information on mammalian cells. The antimutagens used included two selenium compounds, sodium selenite and ebselen; the flavonoids and polyphenols, ellagic acid, (+)-catechin hydrate, scopoletin, chlorogenic acid and rutin trihydrate; the porphyrin derivatives, bovine hemin, biliverdine dihydrochloride, chlorophyllin and a plant extract containing chlorophyll; the terpenoids, beta-carotene, retinol and a mixture of the two epimers (4R) and (4S) of (1S,2E,6R,7E,11E)-cembra-2,7,11-triene-4,6-diols (CBD); and cyclohexanol and ubiquinone. Screening of antimutagenic activities using the STY involves problems with toxicity. In several cases in this study mutagenicity was decreased below the control level without signs of toxicity in the background growth of bacteria. Since the survival of mutants and slight bacteriostatic effects on the background growth cannot be determined accurately in the STY, a reduction in mutagenicity may simply be due to toxicity. Only in cases where a dose-response curve declines to a level at or above the background and then levels off, can toxicity be excluded. An antimutagenic effect determined using this test system is therefore often not sufficient for classifying a compound as antimutagenic until these findings are confirmed in other test systems and, preferably, the mechanism behind this effect is clarified. The results obtained with the selenium compounds were considered to be inconclusive since the reduction in the mutation rate declined below the background level and might only reflect the toxic effects of these compounds. For ellagic acid an almost complete inhibition of the mutagenicity of CSC and SNUS in STY was indicated. This indication of antimutagenicity was confirmed in V79 cells using two metabolites of the CSC constituent benzo[a]pyrene, i.e., trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE). Chlorogenic acid and (+)--catechin reduced the mutagenicity of CSC and chlorogenic acid also strongly inhibited SNUS mutagenicity. Scopoletin and rutin trihydrate inhibited the mutagenicity of CSC, but showed confounding effects with SNUS.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of tryptophan photoproducts in the Ames Salmonella assay.

Abstract: During the photolysis of tryptophan a large number of products is formed. In this study, aqueous solutions of tryptophan were irradiated by ultraviolet light during 5, 20 or 40 h. Each of the irradiated batches was divided into two aliquots, which were freeze-dried or extracted with chloroform. For each batch the latter extract was subsequently divided into a purified chloroform extract and a methanol extract. Aliquots of the purified chloroform extracts were fractionated and pooled, peakwise, into seven fractions. A recombined sample was also constructed. All extracts and samples were tested for mutagenicity using the standard Ames Salmonella assay. The results indicate an exposure time dependent increase in mutagenicity of the extracts, as seen with tester strain TA100 both with and without metabolic activation. The mutagenicity of the freeze-dried extracts well approximated the mutagenicity of the chloroform extracts, indicating that most mutagenicity can be extracted with chloroform. With the fractions the highest mutagenic responses were seen in the late, i.e., less lipophilic fractions. This response pattern seen in TA98 and TA100, mainly with S9 activation, was in contrast to the response of TA102 without S9, which was highest to the more lipophilic fractions. On a fraction level, no general exposure dependent increase of mutagenicity was observed. The results also show that photooxidation of tryptophan gives rise to a different spectrum of products compared to pyrolysis. Both processes result in compounds with strong biological effects. Photooxidation results in compounds with low genotoxicity and high Ah receptor affinity while pyrolysis generates compounds with high genotoxicity and low or no Ah receptor affinity.

Effects of deuterium labeling on azido amino acid mutagenicity in Salmonella typhimurium

Abstract: The mutagenic effects of azide (N 3 − ) anion in bacterial test systems require the formation of the novel mutagenic metabolite, 3-azido- L -alanine (AZAL) Although the mechanism of AZAL-induced mutagenicity is unknown, subsequent bioactivation of this metabolite appears likely Earlier studies have shown that other azide-containing amino acids are mutagenic as well In fact, the mutagenic potency of the synthetic AZAl homologue, l -2-amino-4-azidobutanoic acidobutanoic acid (HomoAZAL), was several timesthat of AZAL To gain insight into the biochemical processing and mutagenicity of azido amino acids in Salmonella typhimurium , several specifically deuterium-labeled azido amino acids have been prepared and tested for mutagenic potency In addition, the effect of (aminooxy)acetic acid (AOA) (a potent inhibitor of pyridoxal-dependent processes) on AZAL-induced mutagenesis was examined The results showed that 2-deuterium substitution of AZAL resulted in a slight increase in mutagenic potency, while AOA treatment resulted in no change in AZAL potency Taken together these findings did not support the involvement of pyridoxal-dependent processes in AZAL bioactivation In contrast, deuterium substitution adjacent to the azide group in HomoAZAL and 5-azido- l -norvaline (N 3 -Norval) resulted in a large decrease in mutagenic potency when compared to the non-deuterium labeled compounds These observations are consistent with a bioactivation mechanism involving rate-limiting CH bond cleavage in the formation of the ultimate mutagen Moreover, this effect of deuterium labeling points to processing of the azide-containing side chain as a key feature in the mutagenic activation mechanism

Bacterial mutagenicity of 2-chloro-1,3-butadiene (chloroprene) caused by decomposition products.

Abstract: Since the literature on genotoxicity of 2-chloro-1,3-butadiene (chloroprene) is controversial, the mutagenicity of this compound was reinvestigated with respect to its chemical stability. Because of the volatility of chloroprene, Ames tests withS. typhimurium TA 100 were carried out with gas-tight preincubation. Propylene oxide, a volatile direct mutagen, served as a positive control. Benzo[a]pyrene was used as a control for an indirect mutagen. Using this experimental regimen, freshly distilled chloroprene was not mutagenic. However, a mutagenic effect occurred linearly with increasing age of the chloroprene distillates. Aged chloroprene gave the same positive results whether preincubation was gas-tight or not. Analysis by gas chromatography (GC) revealed several decomposition products in aged chloroprene distillates. The direct mutagenicity towards TA 100 correlated with the integrated amounts of four of these substances; these substances always occurred in the same relative ratio. When chloroprene was kept under anaerobic conditions, products occurred with time which were partly different from those obtained under aerobic conditions. The direct mutagenicity of anaerobically aged chloroprene was only weak, but the mutagenic effect was enhanced about two- to threefold by addition of S9 mix. Partial identification of chloroprene decomposition products was done by gas chromatography-mass spectrometry (GC-MS): major byproducts of chloroprene, probably responsible for mutagenic properties of aged chloroprene samples, were cyclic chloroprene dimers.

Genetic toxicology testing of di(2-ethylhexyl) terephthalate.

Abstract: Di(2-ethylhexyl) terephthalate (DEHT) is a commercially produced chemical (Kodaflex DOTP) that is used as a general purpose, low-volatility plasticizer for polyvinyl chloride and other polymeric materials. Less than 30 million kilograms of DEHT are produced annually. DEHT is isomeric with di(2-ethylhexyl) phthalate (DEHP), a nongenotoxic rodent carcinogen whose mode of action has been suggested to derive from its ability to produce hepatocellular proliferation and/or hepatic peroxisome proliferation. Thus it is important to know the behavior of DEHT in genotoxicity assays in order to compare it with that of DEHP and other phthalate ester plasticizers. It is known from previously published studies that rats fed DEHT in the diet at 2,000 mg/kg produce urine that is negative in the Ames Salmonella bacterial mutagenicity assay in the presence and absence of induced rat liver S-9 and in the presence and absence of beta-glucuronidase/aryl sulfatase. Reported here are the results of direct testing of DEHT in the Ames plate incorporation assay, the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) in vitro mammalian cell mutagenicity assay, and an in vitro chromosome aberrations assay using CHO cells. The results for mono(ethylhexyl) terephthalate (MEHT), a metabolite of DEHT, in the Ames Salmonella bacterial mutagenicity assay are also presented. All test results for both DEHT and MEHT were found to be negative, and it is therefore concluded that DEHT, like its isomeric relative DEHP, is not genotoxic.