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The aim of this study was to evaluate mutagenicity and genotoxicity of soils from 12 different land use types such as electronic waste dismantling workshop, open burning site and car dismantling workshop (CDW), based on soil concentrations of polycyclic aromatic hydrocarbons (PAHs), and polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/Fs). Soils of CDW contained the sum of 10 PAHs level (94392 μg/kg) exceeded The New Dutch List (Dutch Intervention Value (40000 μg/kg)), dominated by high molecular PAHs (99.6%) and benzo(a)pyrene (82.6%), indicating its potential carcinogenic risks. In addition, Ames test and SOS Chromotest further manifested that soil of CDW had a significantly higher mutagenic potency (MP) of 13.8 and 7.43 on both strains of TA98 and TA100 with S9 mix, respectively, and a relatively higher genotoxicity with S9 mix (SOS inducing potency = 1.16), amongst the 12 different soil types. BaP TEQ PAHs in soils were significantly correlated with MP of TA98, with and without S9 mix (r = 0.858 and r = 0.976 at p

Stearic acid grafted chitosan oligosaccharide (CSO-SA) nanomicelles could be promptly internalized into cancer cells; therefore, it is regarded as a promising drug carrier for cancer therapy. However, the toxicity of CSO-SA is not clear. In the present study, the genotoxic effects of CSO-SA nanomicelles (with high substitution degree of SA, 42.6±3.8%) were evaluated with a battery of genotoxicity assays. Mutagenicity was not found in Ames Salmonella/microsome mutagenicity assay (Ames test), while mild but definite positive results were observed in mouse bone marrow micronucleus assay and single cell gel electrophoresis (SCGE or comet assay) in A549 cells. CSO-SA was also found to induce apoptosis and oxidative stress through the induction of reactive oxygen species (ROS) in a dose-dependent manner in A549 cells. Preincubation with the free radical scavenger N-acetyl-l-cysteine (NAC) decreased the intracellular ROS level and alleviated the DNA damage in A549 cells. Expression levels of cleaved poly ADP-ribose polymerase (PARP), caspase-9 and caspase-3, markers of apoptosis, were significantly higher in CSO-SA treated cells. In conclusion, these results suggested significant genotoxicity of high doses of CSO-SA nanomicelles in vivo and in vitro. Oxidative stress was, at least partially, the possible mechanism underlying the genotoxicity induced by CSO-SA.

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5'-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein-protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.

N -Acyloxy- N -alkoxyamides (see structure 1 , below) are direct-acting mutagens for which a QSAR has been established that predicts with accuracy their activity in the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium TA100. Steric bulk next to oxygen on the alkoxyl side-chain in structure 4 has no impact on activity, but branching at the position adjacent (alpha) to the ester-carbonyl of the leaving group in structure 5 strongly inhibits mutagenicity. Both results reflect the manner in which these molecules interact with DNA. The alkoxyl group has greater flexibility, which minimises steric effects within the major groove. Bulk adjacent to the carbonyl of the ester group must impose conformational constraints that impede reaction at the N7 position of guanine. A new, expanded QSAR shows a clear dependence of activity on log P , although with a smaller coefficient relative to indirect-acting mutagens.

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK(+/-) mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK(+/-) mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague-Dawley rats for 14 days at doses up to 2g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure.

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.

Tetrachlorinated butadienes (TetraCBDs), pentachlorinated butadienes (PentaCBDs) and hexachloro-1,3-butadiene (hexachlorobutadiene or HexaCBD) are environmental contaminants that can occur in groundwater and drinking water at specific sites. While some toxicological data exist for HexaCBD, only few or no toxicity data are available for TetraCBDs and PentaCBDs. In view of structural alerts for potential genotoxicity and carcinogenicity, the genotoxicity of these substances was examined in the Salmonella typhimurium mutagenicity assay (Ames test) and in the in vitro chromosome aberration test. All of the tested polychlorinated butadienes induced chromosome aberrations. Such an effect of HexaCBD is reported here for the first time. In addition, 1,1,3,4-TetraCBD and 1,2,3,4-TetraCBD were positive in the Ames test while the other polychlorinated butadienes including HexaCBD were negative. From these findings it is concluded that certain incompletely chlorinated butadienes have a different genotoxic profile than the completely halogenated HexaCBD, which is of relevance for the risk assessment of these compounds.

Davilla nitida and Davilla elliptica (Dilleniaceae) are plants that occur predominantly in the cerrado region of South America. They are used in popular medicine to treat stomach diseases, diarrhea and swelling, particularly of the lymph nodes and testicles. Chemical investigation of these two plant species led to the identification of the compounds myricetin-3-O-α-l-rhamnoside (myricitrin), quercetin-3-O-α-l-rhamnoside (quercitrin), myricetin, quercetin and gallic acid derivatives in the leaves of D. nitida and D. elliptica. Therefore, it was concluded that the two species of Davilla possess qualitatively similar chemical profiles. In the present study, the mutagenic and genotoxic potential of these plants and of their isolated compounds was tested in the Salmonella typhimurium assay (Ames test) with strains TA100, TA98, TA102 and TA97a, in the micronucleus test with peripheral blood cells of mice treated in vivo, and in plasmid DNA to analyze DNA strand-breaks. In the assessment of mutagenic potential by the Ames test, extracts from both plant species and a D. nitida ethyl-acetate fraction induced positive responses. On the other hand, none of the extracts showed genotoxic activity in the mouse cells. In the presence of metal ion, D. nitida and D. elliptica aqueous and ethyl-acetate fractions, as well as their isolated compounds, induced single- and double-strand-breaks in plasmid DNA in a cell-free system.

Piplartine {5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propen-1-yl]-2(1H)-pyridinone} is an alkamide present in Piper species that exhibits promising anticancer properties. It was previously shown that piplartine is mutagenic in yeast and cultured mammalian cells. This study was performed to increase the knowledge on the mutagenic potential of piplartine using the Salmonella/microsome assay, V79 cell micronucleus and chromosome aberration assays, and mouse bone-marrow micronucleus tests. Piplartine was isolated from the roots of Piper tuberculatum. This extracted compound was unable to induce a mutagenic response in any Salmonella typhimurium strain either in the presence or absence of metabolic activation. Piplartine showed mutagenic effects in V79 cells, as there was an increased frequency of aberrant cells and micronuclei formation. In addition, piplartine administered at 50 mg/kg did not induce micronucleus formation in vivo, but a dose of 100 mg/kg induced an increase in the levels of micronucleus polychromatic erythrocytes (MNPCEs). Overall, these results provide further support that piplartine induces in vivo and in vitro mutagenicity in eukaryotic models.

The isoflavonoid fractions obtained from a methanolic extract of Belamcanda chinensis (L.) DC (syn. Iris domestica Goldblatt & Mabb.) rhizomes inhibited the chemically induced mutations in Salmonella typhimurium TA98 and TA100 in the Ames test. We have studied direct mutagenesis induced by N-nitroquinoline, and indirect mutation induction caused by metabolically activated 2-AF. The fractions enriched in isoflavonoids, obtained by sequential liquid-liquid extraction with diethyl ether and butanol, followed by ODS column separation, inhibited indirect mutagenesis in TA98 almost completely. In TA100 the maximum inhibition ranged between 80% and 100% depending on the test fraction. The inhibition of direct mutagenesis was lower, reaching about 50% in TA98 and in TA100, but it was dose-dependent only in the latter strain. Three in vitro anti-oxidant spectrophotometric assays-DPPH free-radical scavenging test, the phosphomolybdenum assay, and the linoleic acid peroxidation assay were also performed to support the process of bioactivity-guided fractionation and to provide more information about the potential mechanisms of action of the herb under study. The isoflavonoid fractions have the capability to scavenge free radicals, to reduce transition-metal ions and to protect polyunsaturated fatty acids from peroxidation. The analysis of the fractions obtained with high-performance liquid chromatography with photodiode-array and mass-spectrometric detection revealed several potentially bioactive isoflavones, either as glycosides or aglycones, depending on the polarity of the solvents used for fractionation. The main compounds were tectoridin and iridin in the glycoside fractions and the aglycones irigenin, tectorigenin, and 5,6,7,3'-tetrahydroxy-4'-methoxyisoflavone. The activities reported here can be regarded to be of additional value when using this plant as a phyto-estrogenic and chemopreventive agent.

Climbazole is an imidazole antifungal agent that can provide anti-dandruff benefits when incorporated into a shampoo matrix. A series of genotoxicity studies were performed to support the human safety of this azole antifungal drug. Climbazole was not mutagenic in the Salmonella typhimurium or Escherichia coli Ames assay and did not induce micronuclei in human lymphocytes. In the mouse lymphoma assay (MLA), climbazole was negative (non-mutagenic) with and without metabolic (S9) activation after a 4 h exposure; an increase in small colony mutants was observed without metabolic activation after a 24 h exposure at concentrations of 15 and 17.5 μg/mL. An in vivo mouse micronucleus test was negative up to a maximum tolerated dose (MTD) of 150 mg/kg climbazole administered orally. In the in vivo/in vitro unscheduled DNA synthesis assay, climbazole showed no evidence of DNA damage in the livers of rats at doses up to the MTD of 200 mg/kg orally. A toxicokinetic study was performed in mice with oral administration of [14C]-climbazole (150 mg/kg). Radioactivity (20.42 μg-equiv./g plasma) was detected 15 min after oral administration of [14C]-climbazole, and the peak concentration was 62.96 μg-equiv./g plasma at 8 h after dosing. The measured amounts of radioactivity in plasma, at all sample times from 15 min up to 24 h, exceeded the concentrations that induced increases in mutation frequency after 24 h exposure of mouse lymphoma cells in vitro (15 and 17.5 μg/mL). These observations lend support to the conclusion that climbazole does not present a genotoxic risk in vivo. Furthermore, these data are consistent with the published data for other azole antifungals that work by preventing the synthesis of ergosterol and, as a class, are generally non-genotoxic, except some isolated positive results of questionable significance. Collectively, these data are supportive of the view that climbazole does not present a genotoxic or carcinogenic risk to humans.

Inonotus obliquus is a mushroom commonly known as Chaga that is widely used in folk medicine in Siberia, North America, and North Europe. Here, we evaluated the antimutagenic and antioxidant capacities of subfractions of Inonotus obliquus extract. The ethyl acetate extract was separated by vacuum chromatography into three fractions, and the fraction bearing the highest antimutagenic activity was subsequently separated into four fractions by reversed phase (ODS-C18) column chromatography. The most antimutagenic fraction was then separated into two subfractions (subfractions 1 and 2) by normal phase silica gel column chromatography. Ames test analysis revealed that the subfractions were not mutagenic. At 50 μg/plate, subfractions 1 and 2 strongly inhibited the mutagenesis induced in Salmonella typhimurium strain TA100 by the directly acting mutagen MNNG (0.4 μg/plate) by 80.0% and 77.3%, respectively. They also inhibited 0.15 μg/plate 4NQO-induced mutagenesis in TA98 and TA100 by 52.6–62.0%. The mutagenesis in TA98 induced by the indirectly acting mutagens Trp-P-1 (0.15 μg/plate) and B(α)P (10 μg/plate) was reduced by 47.0–68.2% by the subfractions, while the mutagenesis in TA100 by Trp-P-1 and B(α)P was reduced by 70.5–87.2%. Subfraction 1 was more inhibitory than subfraction 2 with regard to the mutagenic effects of 4NQO, Trp-P-1, and B(α)P. Subfractions 1 and 2 also had a strong antioxidant activity against DPPH radicals and were identified by MS, 1H NMR and 13C NMR analyses as 3β-hydroxy-lanosta-8, 24-dien-21-al and inotodiol, respectively. Thus, we show that the 3β-hydroxy-lanosta-8, 24-dien-21-al and inotodiol components of Inonotus obliquus bear antimutagenic and antioxidative activities.

Zero-valent nickel compounds are organometallic chemicals that are used in synthetic applications and may also occur as intermediates in nickel-catalyzed hydrogenation reactions used in food processing. Few studies have been performed on their possible genotoxic actions. We have tested two commercially available examples of this class of compounds. Solubility and stability were examined. Mutagenicity testing did not confirm a previous report that bis(1,5-cyclooctadiene)nickel is positive in the Ames assay. No stimulation of lipid peroxidation was observed in studies of bovine erythrocytes exposed in vitro. Our results do not indicate that zero-valent nickel compounds have genotoxic effects.

3,3′-Dichlorobenzidine (DCB), which has been assigned as a possible carcinogen to humans (Group 2B) by IARC, is produced as a raw material in the manufacture of polymers and dye intermediates. In our previous paper, we identified DCB as an indirect-acting mutagenic constituent in the water concentrates from the Waka River, which flows through an industrial area in Wakayama, Japan. In this study, we have identified a novel mutagen in the water samples from the Waka River. Organic chemicals in the river water were adsorbed to blue rayon at the site where effluents from chemical plants and a sewage plant were discharged into the river. The adsorbate was highly mutagenic in Salmonella YG1024 in the presence and absence of S9 mix, inducing 440,000 and 170,000 revertants/g blue rayon equivalent, respectively. Two mutagenic fractions, which accounted for 18% and 12% of the total mutagenicity of the water concentrate in YG1024 with S9 mix, were separated by HPLC with a reversed-phase column following Sephadex LH20 column chromatography. Both fractions were further separated by HPLC using reversed-phase columns. On the basis of spectral analysis and co-chromatography using an authentic chemical standard, one mutagen in the former fraction was identified as DCB and one mutagen in the latter fraction was deduced to be a novel chemical, a 5-nitro derivative of DCB (5-nitro-DCB; 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl). 5-Nitro-DCB showed strong mutagenicity in YG1024 especially with S9 mix, inducing 24,200 revertants/μg. 5-Nitro-DCB was detected in water concentrates in the range from less than detection limit to 6.9 μg/g of blue rayon. DCB was also detected in the range from 13.2 to 104 μg/g of blue rayon. These results demonstrate that Waka River water might be continually contaminated with the indirect-acting mutagens DCB and 5-nitro-DCB as major mutagenic constituents of the river water.

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.

The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX1 system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48 h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1–4.8 °C below normal; the temperature returned to normal within 8 h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.

An increase in the number of revertant colonies in the Ames test is generally taken as a strong indication of mutagenic activity of a test compound. However, irrelevant positive findings may constitute a major problem in regulatory drug testing. In this study, mixtures containing only amino acids such as glycine, lysine, arginine and isoleucine, routinely used as peptide preservatives in polypeptide pharmaceutical products, were investigated for mutagenesis in the Ames Salmonella typhimurium test. The results demonstrated that in the presence of metabolic activation, all the solutions containing arginine induced an increase in the number of revertant colonies in strains TA98, TA100 and TA1535 compared with the solvent control. More specifically, for strain TA98, all arginine doses tested, i.e. from 0.4 to 8 mg/plate induced a statistically significant increase in the number of revertants. This increase was biologically significant from 1.2 to 8 mg/plate. For strain TA100, the five highest test doses, i.e., from 1.2 to 8 mg/plate, induced statistically and biologically significant increases in the number of revertants. A statistically significant increase in colony number was also observed in strain TA1535, but only at the maximal test dose of 8 mg/plate arginine. These increases were observed with arginine from two different sources, suggesting that the observed effect would not be due to the presence of potential impurities in the type of arginine used. Our findings show that a functional metabolic activation system was required to induce an increase in the number of colonies. The presence of vitamin C inhibited the arginine-induced increase in the number of revertant colonies in S. typhimurium strain TA98, suggesting a potential involvement of oxidative stress.

The mutagenicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its N-nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were evaluated using the Salmonella tryphimurium reverse mutation assay (Ames assay) with strains TA97a, TA98, TA100, and TA102. Using a preincubation procedure and high S9 activation (9%), RDX was observed to induce weak mutagenesis to strain TA97a with a mutagenicity index (MI) of 1.5-2.0 at a dose range of 32.7-1090microg/plate. MNX induced moderate mutagenesis to strain TA97a with an MI of 1.6-2.8 at a dose range of 21.7-878microg/plate. TNX also induced moderate mutagenesis in strain TA97a with an MI of 2.0-3.5 to TA97a at a dose range of 22.7-1120microg/plate. TNX also caused weak mutagenesis to strain TA100 with S9 activation at the dose of 1200microg/plate. MNX and TNX induced weak to moderate mutagenesis to strain TA102. Strain TA97a was found to be the most sensitive strain among these four strains. No cytotoxicity of RDX, MNX, and TNX was observed at the concentrations used in this study. Doses were verified by HPLC.

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.

pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.

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.

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.

The combination of short-term bioassays and analytical chemical techniques has been successfully used in the identification of a variety of mutagenic compounds in complex mixtures. Much of the early work in the field of bioassay-directed fractionation resulted from the development of a short-term bacterial assay employing Salmonella typhimurium; this assay is commonly known as the Ames assay. Ideally, analytical methods for assessment of mutagenicity of any environmental matrix should exhibit characteristics including high capacity, good selectivity, good analytical resolution, non-destructiveness, and reproducibility. A variety of extraction solvents have been employed in investigations of mutagenicity of air particulate; sequential combination of dichloromethane followed by methanol is most popular. Soxhlet extraction has been the most common extraction method, followed by sonication. Attempts at initial fractionation using different extraction solvents have met with limited success and highlight the need for fractionation schemes applicable to moderately polar and polar mutagenic compounds. Fractionation methods reported in the literature are reviewed according to three general schemas: (i) acid/base/neutral partitioning followed by fractionation using open-column chromatography and/or HPLC; (ii) fractionation based on normal-phase (NP) HPLC using a cyanopropyl or chemically similar stationary phase; and (iii) fractionation by open-column chromatography followed by NP-HPLC. The HPLC methods may be preparative, semi-preparative, or analytical scale. Variations based on acid/base/neutral partitioning followed by a chromatographic separation have also been employed. Other lesser-used approaches involve fractionation based on ion-exchange and thin-layer chromatographies. Although some of the methodologies used in contemporary studies of mutagenicity of air particulate do not represent significant advances in technology over the past 30 years, their simplicity, low cost, effectiveness, and robustness combine to result in their continued application in modern laboratories.

The genotoxic potential of E-5842, a sigma ligand compound being developed as an antipsychotic drug, was evaluated by means of an extensive battery of in vitro and in vivo assays. Negative results were obtained in an Ames test (up to 5000 μg/plate), a mouse lymphoma assay (up to 535.1 μg/ml (-S9) and 891.8 μg/ml (+S9)), an in vivo rat hepatocyte micronucleus assay (up to 100 mg/kg/day on 2 days), and a two-dose mouse micronucleus assay (up to 40 mg/kg/day on 2 days). In a single-dose mouse bone-marrow micronucleus assay (up to 400 mg/kg; 24, 48 and 72 h sampling) a slight and non-statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was observed 48 h after administration of a 200 mg/kg dose, in the absence of bone-marrow toxicity. This minor increase in MNPCE frequency was considered of questionable biological relevance, because it was observed under conditions of marked animal toxicity including mortality. In addition, it occurred in association with a strong hypothermic effect produced by administration of E-5842. A clear increase in the frequency of structural chromosomal aberrations was observed in human lymphocytes at concentrations ≥350.6 and 1685.4 μg/ml in the presence and absence of S9, respectively. Mitotic accumulation was observed at those concentrations at which clastogenic effects were observed, a condition that may have masked toxicity. Concentrations lacking clastogenic effects in this chromosome aberration assay (300.7 and 173.2 μg/ml in the presence and absence of S9, respectively) were well in excess of maximum human plasma concentrations attained in clinical studies at the maximum tolerated dose (19.1 ng/ml). A weight-of-evidence analysis, taking into consideration the results obtained in the different in vitro and in vivo assays and the conditions of clinical use, suggest that E-5842 would not pose a genotoxic risk under clinical conditions.

Several in vivo and in vitro studies have shown that grape extracts could prevent certain steps in carcinogenesis and a few mechanisms have been proposed for this activity. In this study, the potential antimutagenic activity of methanolic and aqueous extracts from two Greek grape varieties of Vitis vinifera against DNA damage induced by reactive oxygen species (ROS) was assessed as a potential novel chemopreventive mechanism, using Salmonella typhimurium strain TA102. The two grape varieties were Assyrtiko (white grapes) and Mandilaria (red grapes), while the oxidant mutagens used were bleomycin (BLM) and hydrogen peroxide (H 2 O 2 ). Since it has been considered that polyphenols present in grapes are their most potent biologically active compounds, we also tested the effects of polyphenol-rich fractions as well as some of the more common grape polyphenols on the activity of the two test mutagens. These polyphenols were quercetin, (+)-catechin, (−)-epicatechin, trans -resveratrol, gallic acid and protocatechuic acid. Almost all extracts showed inhibitory activity against both mutagens. On the other hand, polyphenol-rich fractions as well as individual polyphenols at concentrations found in the extracts either did not diminish or did enhance the activity of the mutagens. These results suggest that the protection of DNA from mutations induced by ROS may be one of the mechanisms accounting for the chemopreventive activity of grape extracts. However, it seems that this protective activity may not be attributed to polyphenols but rather to a synergism of many compounds in the grapes.

Electrophilic N -acyloxy- N -alkoxyamides are mutagenic in Salmonella typhimurium TA100 without the need for S9 metabolic activation and they react with DNA at guanine—N7 at physiological pH. Since these are direct-acting mutagens, structural factors influence binding and reactivity with DNA. Mutagenicity in TA100 can be predicted by a QSAR incorporating hydrophobicity (log P ), stability to substitution reactions at nitrogen (p K a of the leaving acid) and steric effects of para -aryl substituents ( E s ). A number of mutagens exhibit activities that deviate markedly from the predicted values and they fall into two classes: di- tert -butylated N -benzoyloxy- N -benzyloxybenzamides, which – because of their size – are most probably excluded from the major groove or are unable to achieve a transition state for reaction with DNA, and N -benzoyloxy- N -butoxyalkylamides with branching α-to the amide carbonyl, which are resistant to S N 2 reactions at the amide nitrogen.

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflamatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l -phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.

The genotoxicity of industrial wastewater samples from Aligarh and Ghaziabad cities was compared using the Ames plate incorporation test, the Ames fluctuation test and the Allium cepa test. While TA102 and TA104 strains exhibited the highest sensitivity against the Aligarh sample (AWW) in terms of the slope (m) of the dose-response curve in the plate incorporation assay, TA98 and TA97a were the most sensitive strains based on the induction factor, Mi(p). TA98 once again, was the most sensitive strain against the test sample from Ghaziabad (GWW) in terms of 'Mi(p)' while TA102 was the most sensitive strain on the basis of the slope (m). TA100 displayed the highest susceptibility towards the samples from Aligarh in the fluctuation test. However, TA102 and TA98 responded maximally to GWW in this bioassay. The mutagenicity of the test samples seemed to be partly mediated by reactive oxygen species (ROS) as evidenced by the use of free radical scavengers. Mannitol brought about the maximum decline in the number of revertants of TA102 by the Aligarh sample, whereas such a reduction in case of Ghaziabad sample was exhibited with superoxide dismutase. Both the test water samples induced various anaphase aberrations in the root cells of Allium cepa. Fragmentation of the chromosome was the predominant effect of the Aligarh water sample while the Ghaziabad sample induced chromosome stickiness. The crucial roles of heavy metals and pesticides in the genotoxicity of AWW and GWW, respectively, have been suggested. In view of the problem associated with the interpretations of data, we recommend that all the test bioassays should be carried out in the presence of ROS scavengers for the fool proof evaluation of the genotoxicity of water samples.

p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage induced by gamma-ray and ultraviolet irradiation, and also by genotoxic chemicals such as adriamycin. For the purpose of constructing an easy-operating genotoxicity test system using human cell lines, we developed a p53R2-dependent luciferase reporter gene assay, and demonstrated dose-dependent luminescence caused by adriamycin in two human cell lines that express wild-type p53, MCF-7 and HepG2. The performance of this assay system was evaluated with 80 chemicals including those known in the Ames test as genotoxic or non-genotoxic. When the luciferase activity of cells treated with the test sample was over 200% to that of control cells in a dose-dependent increasing manner, the sample was judged positive as a genotoxic chemical. Forty of 43 Ames-positive chemicals induced luciferase activity in this assay system. Eight Ames-negative chemicals also induced luciferase activity. These eight chemicals are genotoxic in other in vitro test systems using mammalian cells. It is suggested that this assay system can be applied to rapid screening of chemicals for potential human genotoxicity.

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.

The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied.

Benzene-ethanol extracts from particulates produced by coal burning were separated into four fractions by silica-gel column chromatography using n-hexane (240 ml), n-hexane-dichloromethane (3:1, v/v) (200 ml), dichloromethane (200 ml) and methanol (450 ml), as the corresponding eluents. The mutagenicity of each fraction was assayed by the Ames test using the Salmonellatyphimurium YG1024 strain. The nitropolycyclic aromatic hydrocarbons (NPAHs) of each fraction were assayed by high-performance liquid chromatography with chemiluminescence detection. The highest activity was observed in the n-hexane-dichloromethane fraction (Fr. 2). The mutagenic contribution of this fraction was 69.9% of the total of the four fractions. Ten of 11 NPAHs detected were in Fr. 2 and one (1-nitropyrene) was most concentrated in Fr. 3. Among the NPAHs examined, 3-nitrobenzanthrone made the largest mutagenic contribution. This is the first report of detection of 3-nitrobenzanthrone in coal burning-derived particulates.

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.

Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung. In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied. Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay. To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored. Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo[a]pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029. This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3). The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner. In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479+/-33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S. typhimurium YG1024 or YG1029. At a higher total asphalt fume exposure level (1150+/-63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control. However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test. These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content.

Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin. However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles. Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances. These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays. In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. Limited evidence for very weak activity was also obtained in the absence of S9 mix. trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen. Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes. Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear. Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations.

Anti-mutagenic and anti-carcinogenic effects of beer on heterocyclic amine (HCA)-induced carcinogenesis were studied in vitro and in vivo. Four commercial beers (two pilsner-type, black, and stout) showed inhibitory effects against five HCAs, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), in the Ames assay using Salmonella typhimurium TA98 in the presence of rat S9 mix. The inhibitory effects of dark-colored beers (stout and black beer) were greater than those of pilsner-type beers. Dark-colored beers suppressed CYP1A2 activity in a dose-dependent manner, suggesting that inhibition of HCA activation is partly responsible for their strong anti-mutagenic effects. Anti-mutagenic effects were also observed when the pooled human S9 mix or activated IQ was used in the assay. The micronucleus test using Chinese hamster lung CHL/IU cells showed that the addition of freeze-dried samples of pilsner-type and stout beer to the culture medium significantly reduced the number of cells with micronuclei induced with PhIP or Trp-P-2. Single-cell gel electrophoresis assay (comet assay) revealed that oral ingestion of pilsner-type and stout beers for 1 week significantly inhibited DNA damage in the liver cells of male ICR mice exposed to MeIQx (13 mg/kg, i.p.). A decrease in the formation of DNA adducts was also observed using a 32P-postlabeling method. Male Fischer 344 rats orally received PhIP (75 mg/kg, five times a week for 2 weeks) and aberrant crypt foci (ACF) formation in the colon was analyzed after 5 weeks. The number of ACF was significantly reduced in rats fed a diet containing freeze-dried beer. These results suggest that beer inhibits the genotoxic effects of HCAs and may reduce the risk of carcinogenesis caused by food borne carcinogens.

We have conducted an evaluation of three of the most widely used commercial toxicity prediction programs, Toxicity Prediction by Komputer Assisted Technology (TOPKAT), Deductive Estimation of Risk from Existing Knowledge (DEREK) for Windows (DfW) and CASETOX. The three programs were evaluated for their ability to predict Ames test mutagenicity using 520 proprietary drug candidate (Test set 1) and 94 commercial (Test set 2) compounds. The study demonstrates that these three commercially available programs are useful, with limitations in their ability to predict mutagenicity over a wide range of chemical space, i.e. global predictivity. Individually, each of the programs performed at an acceptable level for overall accuracy, i.e. the ability to predict the correct outcome. However, analysis of the predictions indicates that the overall accuracy figure is heavily weighted by the ability of the programs to correctly predict non-mutagens, whereas none of the programs individually performed well in the prediction of novel mutagenic structures, i.e. Ames positive compounds. The performance of these programs' in predicting Ames positive mutagens appeared to be independent of the chemical utility of the compound, i.e. industrial, agricultural or pharmaceutical. The combination of program predictions provided some improvement in overall accuracy, sensitivity and specificity.

2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna (Lawsonia inermis). Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation. HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster. However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24 h after intraperitoneal injection. In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ. HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays. It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism. Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests. However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h. There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests. As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism. Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer.

The effect of lignin on cytotoxicity, mutagenicity and SOS response induced by 4-nitroquinoline- N -oxide (4NQO), 3-(5-nitro-2-furyl)acrylic acid (5NFAA), 2-nitrofluorene (2NF) as well as hydrogen peroxide was investigated in bacterial assay systems, i.e. the Ames test with Salmonella typhimurium TA98, TA100, TA102 and the SOS chromotest with Escherichia coli PQ37. Lignin preparations obtained from beech wood significantly decreased the mutagenicity induced by 4NQO, 2NF and H 2 O 2 . In the case of mutagenicity induced by 5NFAA the effect was lower. Antimutagenic properties of lignin samples tested were shown also by SOS chromotest where lignin inhibited the ability of both 4NQO and H 2 O 2 to induce the SOS response. Derivatives of lignin including those from soft and hard wood, as well as from annual plants differ in their efficiency to inhibit the induction of the SOS response. The modified lignins isolated from beech and spruce wood exhibit a high level of protection. Lignins from annual plants—corn cobs and straw—only marginally evoked an antimutagenic response, but their effect was increased by hydrothermic treatment of both annual plants. The results obtained indicate the prospective utilization of lignin preparations as additive in chemo-prevention. The antimutagenic effect of lignin samples varies with the method of isolation and modification, as well as with the genetic origin of the lignin.

Nine structurally related pyridone derivatives were assayed for photogenotoxicity and phototoxicity in the Ames test, the chromosomal aberration test in V79 cells and the neutral red uptake (NRU) test in 3T3 cells. All nine compounds absorb light to a comparable degree at wavelengths between 380 and 430 nm. Seven of the nine compounds were found to produce high quantities of singlet oxygen (1O(2)) upon irradiation in the presence of oxygen. These seven compounds were highly phototoxic in the NRU test, three were clearly and two were marginally photomutagenic in the Ames test, five were assessed as clearly and two as equivocally photoclastogenic in the chromosomal aberration test. Two compounds showed substantially lower 1O(2) yields. The pyridone ring of these two compounds is attached to a non-aromatic ring, while for the seven other compounds the chromophore system including the pyridone ring consists of two or three aromatic rings. One of the two compounds with low 1O(2) yields was distinctly less phototoxic and did not induce photogenotoxic effects. The other, structurally an indolo derivative and not the common thieno derivative, was, however, similarly phototoxic as the seven compounds with high 1O(2) quantum yield and was also clearly photogenotoxic indicating that different action pathways, not involving singlet oxygen, have to be considered at least for this compound.

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.

A new assay for detection of mutagenic pollution of marine environments, based on the use of a series of genetically modified Vibrio harveyi strains, was developed recently. Here, we compared this assay with a commonly used Ames test, employing mutants of Salmonella enterica serovar Typhimurium. We found that survival of S. enterica serovar Typhimurium in artificial marine water and in different samples of marine water is dramatically reduced relative to that of V. harveyi strains. This indicates that V. harveyi strains are significantly more useful in testing samples of marine water. Moreover, sensitivity of both assays was compared. We found that using the V. harveyi assay it is possible to detect significantly (from a few to several times) lower concentrations of typical chemical mutagens than employing the Ames test. Although the higher sensitivity of the V. harveyi test relative to the Ames assay may be of minor importance when testing mutagenicity of certain chemicals in their pure form under laboratory conditions, this feature is very important in testing samples of marine water. Therefore, we assume that the V. harveyi assay may be an adequate test for detection of mutagenic pollution of marine environments.

The mutagenic activity of sulfoscanate (SSC) (4-isothiocyanate-4'-nitrodiphenyl sulphide) has been compared with that of the following reported drugs: (a) nitroscanate (NSC) (4-isothiocyanate-4'-nitrodiphenyl ether) which is a veterinary anthelmintic drug and (b) amoscanate (ASC) (4-isothiocyanate-4'-nitrodiphenyl amine) which is effective against schistosomes. SSC has been found to be a very potent mutagen towards TA98 and TA100 inducing 26.0 and 475.5revertants/nmole, respectively. NSC was found to induce mutations at a rate of 11.1 and 21.5revertants/nmole in TA98 and TA100, respectively. ASC was found to be non-mutagenic as such, but the urine of animals given the drug displayed mutagenicity. When SSC was tested in TA98/1,8-DNP(6), deficient in O-acetyltransferase, the activity decreased to 10.0revertants/nmole. However, in case of NSC the mutagenic activity was reduced to 0.24revertants/nmole, indicating the importance of O-acetyltransferase in generating N-acetoxyarylamine. In TA98NR, deficient in nitroreductase, the mutagenicity of SSC and NSC was totally absent. The positional isomers of SSC, 4-isothiocyanate-3'-nitro- and 4-isothiocyanate-2'-nitrodiphenyl sulphide, were found to be non-mutagenic in both TA98 and TA100. Our comparison of the mutagenic activity of SSC, NSC and ASC indicates that the pattern of activity is SSC>NSC>ASC.

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%). Anilines and phenols tended to induce only structural CA. p-tert-Butylphenol had a peculiar feature in inducing not only structural CA but also polyploidy at considerably high frequency (93.2%) after continuous treatment for 48 h, posing an aneugenic potential. Not all, but six of 11 carboxylic acids or esters also showed the simultaneous induction of structural CA and polyploidy. The majority of organic phosphates, alcohols or ethers, alkyl benzenes and non-cyclic alkanes had no CA induction activity. For chemicals which were negative in the bacterial reverse mutation assay (Ames test), the proportion of the chemicals that induced CA at a severely cytotoxic dose (doses manifesting more than 50% cytotoxicity) was similar to that of the CA-negative chemicals manifesting severe cytotoxicity, suggesting that severely cytotoxic chemicals do not always induce CA.

The genotoxic activity of environmental xenobiotics is manifested either in their direct interaction with cellular genetic material or in provoking secondary events, among which reactive oxygen species (ROS) production is a common phenomenon. Both pathways can be mediated by the activity of the cytochrome P450 monooxygenase system. We studied induction of the CYP 1A or CYP 2B monooxygenases in rat liver by the fungicides: thiram, captan, captafol, dodine and the drugs: nitrofurazone, furazolidone and the plant flavonoid: quercetin. A cytochrome P450 induction assay (CYPIA test) was used. S9 prepared from livers of rats treated with the test compounds were used to activate ethidium bromide (EtBr) (CYP 1A isoenzyme) or cyclophosphamide (CPA) (CYP 2B isoenzyme) in the Ames test. It was found that among the tested compounds, the most potent inducer of CYP 1A was furazolidone (3× 80 mg/kg). Less potent was thiram (1× 100 mg/kg), as well as quercetin (3× 80 mg/kg), and captafol (1× 30 mg/kg). On the other hand, thiram (1× 100 mg/kg), captafol (1× 30 mg/kg), and quercetin (3× 80 mg/kg) were most potent in the CYP 2B isoenzyme induction, while furazolidone (3× 80 mg/kg), and nitrofurazone (3× 80 mg/kg) appeared to be less potent in this respect. Captan and dodine (3× 80 mg/kg) did not affect the activity of any of the cytochrome P450 isoenzymes.

The basis for the observed mutagenicity of cyclohexanone oxime in the presence of hamster liver S9 in Salmonella typhimurium strain TA1535, but not in TA100, was explored. While the chemical had no effect on the appearance of the background lawn in either strain, it did cause a reduction in mutant colony counts in strain TA100, raising the possibility of selective toxicity to this strain. Viability of the two strains was determined directly by titering the cells in background lawns over a 3 day period. In order to do this, cells embedded in top agar overlays were released by extruding agar plugs through small holes in the bottoms of centrifuge tubes, followed by vigorous vortexing. Viable cell counts in background lawns of strain TA100, but not strain TA1535, were greatly reduced in the presence of cyclohexanone oxime. Most of the loss of viable TA100 cells occurred on days 2 and 3 following plating, after the cells had exhausted the histidine in the medium and stopped growing. Therefore, the observed loss of background lawn viable cells is unlikely to be the cause of the non-mutagenicity of cyclohexanone in strain TA100. Analysis of reversion spectra showed that cyclohexanone oxime-induced C→T transitions in the second position of the CCC triplet at the his mutation site in strain TA1535, but had no significant effect on any transition or transversion in strain TA100.