In-Silico Screening of High Production Volume Chemicals for Mutagenicity using the mcase QSAR Expert System

Computational screening is suggested as a way to set priorities for further testing of high production volume (HPV) chemicals for mutagenicity and other toxic endpoints. Results are presented for batch screening of 2484 HPV chemicals to predict their mutagenicity in Salmonella typhimurium (Ames test). The chemicals were tested against 15 databases for Salmonella strains TA100, TA1535, TA1537, TA97 and TA98, both with metabolic activation (using rat liver and hamster liver S9 mix test) and without metabolic activation. Of the 2484 chemicals, 1868 are predicted to be completely nonmutagenic in all of the 15 data modules and 39 chemicals were found to contain structural fragments outside the knowledge of the expert system and therefore suggested for further evaluation. The remaining 616 chemicals were found to contain different biophores (structural alerts) believed to be linked to mutagenicity. The chemicals were ranked in descending order according to their predicted mutagenic potential and the first 100 chemicals with highest mutagenicity scores are presented. The screening result offers hope that rapid and inexpensive computational methods can aid in prioritizing the testing of HPV chemicals, save time and animals and help to avoid needless expense.     

Metabolic activation of chemicals: in-silico simulation

The role of metabolism in prioritising chemicals according to their potential adverse health effects is extremely important given the fact that innocuous parents can be transformed into toxic metabolites. Our recent efforts in simulating metabolic activation of chemicals are reviewed in this work. The application of metabolic simulators to predict biodegradation (microbial degradation pathways), bioaccumulation (fish liver metabolism), skin sensitisation (skin metabolism), mutagenicity (rat liver S-9 metabolism) are discussed. The ability of OASIS approach to predict metabolism (toxicokinetics) and toxicity (toxicodynamics) of chemicals resulting from their metabolic activation in a single modelling platform is an important advantage of the method. It allows prioritisation of chemicals due to predicted toxicity of their metabolites.     

Metabolic activation of chemicals: in-silico simulation†

The role of metabolism in prioritising chemicals according to their potential adverse health effects is extremely important given the fact that innocuous parents can be transformed into toxic metabolites. Our recent efforts in simulating metabolic activation of chemicals are reviewed in this work. The application of metabolic simulators to predict biodegradation (microbial degradation pathways), bioaccumulation (fish liver metabolism), skin sensitisation (skin metabolism), mutagenicity (rat liver S-9 metabolism) are discussed. The ability of OASIS approach to predict metabolism (toxicokinetics) and toxicity (toxicodynamics) of chemicals resulting from their metabolic activation in a single modelling platform is an important advantage of the method. It allows prioritisation of chemicals due to predicted toxicity of their metabolites.

     

Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test

The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone), the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.     

Antioxidant and antimutagenic polyisoprenylated benzophenones and xanthones from Rheedia acuminata

Dichloromethane extract of the stem bark of Rheedia acuminata yielded three benzophenones with antioxidant activity, the new one named acuminophenone A (1), guttiferone K (2) and isoxanthochymol (3), along with the known xanthones formoxanthone C (4) and macluraxanthone (5). The structures were established through interpretation of their spectroscopic data, the stereochemistry of compounds (1) and (2) were resolved by experimental and computational experiments and their antioxidant activities were measured using the DPPH, ABTS and TEAC assays. The antioxidant results showed that metabolites 1, 4 and 5 had a better antioxidant activity than the reference compound quercetin. In addition, we evaluate the mutagenicity and antimutagenicity of the CH2Cl2 extract as well as of the free radical scavenger compounds 1, 4 and 5 by the AMES Salmonella/microsomal test. No mutagenicity was found in the CH2Cl2 extract using Salmonella typhimurium strains TA98, TA100, TA102, TA1537 and TA1538, with or without S9 metabolic activation. The pure compounds neither showed mutagenicity in TA 102 strain and the most important result was the strong reduction of mutagenic effect induced by hydrogen peroxide in S. typhimurium TA102, with or without S9, showed by the compounds 1 (more than 93%) and 4 (more than 88%) at 0.02 microg/plate.     

Comparison of CoMFA Models for Salmonella Typhimurium TA98, TA100+TA98+S9 and TA100+S9 Mutagenicity of Nitroaromatics

Abstract

Comparative Molecular Field Analysis (CoMFA) was applied to a comprehensive data set of heterogeneous nitroaromatics tested in Salmonella typhimurium TA98 and TA100 with and without S9 microsomal activation. The four CoMFA models developed agree with postulated mechanisms of mutagenicity, and explain over 70% of the corresponding mutagenic variance. The standard deviation coefficient contours common in the four models included high electronic density regions equivalent to C4-C5 in the pyrene ring, and an electron deficient site equivalent to C6. These areas are associated with high mutagenicity. Electron deficient areas may be related with the nitroreductive bioactivation of nitroaromatics. Electron rich sites may be involved with oxidative mechanisms analogous to the bioactivation pathway of polycyclic aromatic hydrocarbons. The contribution of steric factors to mutagenicity follows the order TA98 + S9 > TA98 > TA100 + S9 > TA100. The models indicated that increasing bulk perpendicular to the aromatic plane would decrease mutagenicity, but increasing the aromatic ring system along a region corresponding to C6-C7 in 1-nitropyrene would increase mutagenicity.     

A strategy for ranking environmentally occurring chemicals. Part VI. QSARs for the mutagenic effects of halogenated aliphatics.

A strategy for the systematic analysis and priority ranking of environmental chemicals has been applied to a class of 58 halogenated aliphatic hydrocarbons. A training set of ten compounds representing this class, was selected by statistical design. The training set compounds were then subjected to biological testing in the Salmonella typhimurium reverse mutation assay (Ames test). The measured biological data, recorded as dose-response curves, were analyzed to determine the mutagenic potency (slope of the initial portion) and the mutagen dose (MD 50) required to increase the number of revertants above the background by 50%. For each compound, four mutagenic potency estimates and four MD 50 values were determined, all originating from the tester strains TA 100 and TA 1535 with and without metabolic activation. The obtained responses were analyzed with multivariate techniques to give QSAR models relating the mutagenic potency data to the physico-chemical properties of the compounds. Finally, the derived QSARs were used to predict the mutagenic potencies and the MD 50S for the non-tested compounds in the class.     

Formation of a direct mutagen, diazo-N-nitrosoetilefrin, by interaction of etilefrin with nitrite

Reaction of an antihypotensive drug, etilefrin [alpha-[(ethylamino)methyl]-m-hydroxybenzyl alcohol], with nitrite under mildly acidic conditions produced N-nitrosoetilefrin [alpha-[(N-nitrosoethylamino)methyl]-m-hydroxybenzyl alcohol] (a mixture of syn and anti forms) (Iab) and diazo-N-nitrosoetilefrin [1-(4-diazo-3-oxo-1,5-cyclohexadienyl-2-(N-nitrosoethylamino )ethanol] (a mixture of syn and anti forms) (IIab). Treatment of etilefrin with an equivalent amount of nitrite at pH 3 and 37 degrees C for 4 h gave Iab (yield, 30%) and IIab (yield, 5%). Treatment of etilefrin with 4 eq of nitrite under the same conditions gave Iab (23%) and IIab (53%). Compounds Iab and IIab were each composed of two isomers due to the configuration of the N-nitroso group. While compound Iab was not mutagenic, compound IIab showed mutagenicity to Salmonella typhimurium TA98 and TA100 strains without metabolic activation. Specific mutagenic activity of IIab was 300 his+ revertant colonies for both TA98 and TA100 strains with a dose of 0.1 mumol. Addition of a microsomal activation system little affected the activity. It is noteworthy that this orally administered drug can produce a direct-acting mutagen by reaction with nitrite, which is present in the digestive tract.     

Mutagenicity of silver nanoparticles synthesized with curcumin (Cur-AgNPs)

IntroductionSilver nanoparticles (AgNPs) are of particular interest for their antibacterial properties and are produced by the action of reducing agents on silver ions. Curcumin from Curcuma longa (Zingiberaceae) has been used as a precursor for obtaining biogenic AgNPs, to act as a potential drug.ObjectivesThis study aimed to evaluate the toxicity of AgNPs synthesized with curcumin (Cur-AgNPs 0.081 mg/mL, ~130 nm) through the Salmonella/microsome (Ames test), one of the first required assays for evaluating toxicity.MethodsThe study design was experimental and in vitro. After defining the preliminary toxicity, the mutagenicity was assessed in a concentration range of 0.0010–0.0081 mg/plate Cur-AgNPs using histidine negative (His−) Salmonella Typhimurium strains TA97a, TA98, TA100, and TA102, with (+S9) and without metabolic activation (−S9), in triplicate. Assays were monitored by positive and negative controls. The results were statistically analyzed by Salanal software with p < 0.05 values considered significant.ResultsThe data obtained in the absence of metabolic activation showed that Cur-AgNPs is not mutagenic, but when exposed to the presence of S9, Cur-AgNPs became mutagenic to TA98 and TA100 strains, showing the significance of metabolizer enzymes to activate Cur-AgNPs on these bacteria, which recovered their abilities in synthesizing histidine (His+).ConclusionCur-AgNPs is mutagenic in the presence (+S9), but not in the absence (−S9) of metabolic activation, being able to act as indirect mutagens potentially to organisms that share the same genotype vulnerabilities found in TA98 and TA100 strains to cause a frameshift and base-pair substitution mutations, respectively.     

Synthesis and properties of novel bifunctional nitrosamines with omega-chloroalkyl groups

Novel N-nitroso-N-(acetoxymethyl)-omega-chloroalkylamines were synthesized and their chemical and biological properties were evaluated. The nitrosamines were expected to decompose through omega-chloroalkyldiazohydroxides in aqueous solution, and then to alkylate various cellular macromolecules. N-Nitroso-N-(acetoxymethyl)-2-chloroethylamine rapidly decomposed in aqueous solution, and the reaction rate was apparently independent of the pH of the solution. On the other hand, the rate of decomposition of chloropropyl and chlorobutyl homologs was pH-dependent, and increased in alkaline solution. When mutagenicity was assayed in Salmonella typhimurium TA1535 and TA92 for preliminary evaluation, all three compounds were directly mutagenic. The mutagenicity in Salmonella typhimurium TA1535, which can detect base-pair change mutation, clearly showed that these compounds induced DNA alkylation in vivo. The increase of alkyl chain length in chloroalkyl compounds increased the mutagenic activity, and the activities were stronger than those of the corresponding simple alpha-acetoxy nitrosamines lacking a chloro group, N-nitroso-N-(acetoxymethyl)alkylamines. Furthermore, the positive result in TA92 suggested that chlorinated nitrosamines cross-linked DNA like antitumor chloroethylnitrosoureas and that they are expected to be new lead compounds for antitumor agents.     

The mutagenic constituents of Rubia tinctorum.

Twenty compounds were isolated from the roots of Rubia tinctorum which are used as a commercial source of madder color. Among these compounds, mollugin (1), 1-hydroxy-2-methylanthraquinone (2), 2-ethoxymethylanthraquinone(11), rubiadin (13), 1,3-dihydroxyanthraqunone (14), 7-hydroxy-2-methylanthraquinone (16), lucidin (17), 1-methoxymethylanthraquinone (18) and lucidin-3-O-primeveroside (19) showed mutagenicity with Salmonella typhimurium TA 100 and/or TA 98. Since the mutagenic compounds isolated are anthraquinone derivatives with the exception of compound 1, structure-mutagenicity relationships of the anthraquinones were also studied. The results suggested that the greatest activity is exhibited by 1,3-dihydroxyanthraquinones possessing methyl or hydroxylmethyl group on carbon 2.     

Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677

Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been used as a non-caloric sugar substitute in Japan. Pezzuto et al. demonstrated that steviol shows a dose-dependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the presence of metabolic activation system (Aroclor induced rat liver S9 fraction). Our studies were carried out to identify the genuine mutagenic active substance from among the eight steviol derivatives. Steviol indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al. 15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence of S9 mixture. Interestingly, specific mutagenicity of the lactone derivative under the presence of S9 mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture.     

Chemical changes of organic compounds in chlorinated water. XI. Thin-layer chromatographic fractionation of Ames mutagenic compounds in chlorine-treated 4-methylphenol solution

The diethyl ether extract from an aqueous solution of 4-methylphenol after treatment with hypochlorite was mutagenic to the Ames Salmonella test strain TA100 in the absence of liver homogenate. Gas chromatography-mass spectrometry (GC-MS) showed the occurrence of, at least, twenty compounds in the extract: chloro-4-methylphenols, chlorohydroxy-4-methylquinones and chlorinated 4-methylphenol dimers. The diethyl ether extract was fractionated into several fractions by silica gel and polyamide thin-layer chromatography (TLC). The fractionated components were then examined for mutagenicity by means of Ames assays, and were identified by GC-MS. TLC fractionation of the extract revealed that the major components present in the extract are not mutagenic, but minor components (less than 4% of the total extract) are mutagenic. GC-MS analysis indicated the presence of chlorinated 4-methylphenol dimers in the fraction which exhibited the highest mutagenicity.     

In silico exploratory study using structure–activity relationship models and metabolic information for prediction of mutagenicity based on the Ames test and rodent micronucleus assay

The mutagenic potential of chemicals is a cause of growing concern, due to the possible impact on human health. In this paper we have developed a knowledge-based approach, combining information from structure–activity relationship (SAR) and metabolic triggers generated from the metabolic fate of chemicals in biological systems for prediction of mutagenicity in vitro based on the Ames test and in vivo based on the rodent micronucleus assay. In the first part of the work, a model was developed, which comprises newly generated SAR rules and a set of metabolic triggers. These SAR rules and metabolic triggers were further externally validated to predict mutagenicity in vitro, with metabolic triggers being used only to predict mutagenicity of chemicals, which were predicted unknown, by SARpy. Hence, this model has a higher accuracy than the SAR model, with an accuracy of 89% for the training set and 75% for the external validation set. Subsequently, the results of the second part of this work enlist a set of metabolic triggers for prediction of mutagenicity in vivo, based on the rodent micronucleus assay. Finally, the results of the third part enlist a list of metabolic triggers to find similarities and differences in the mutagenic response of chemicals in vitro and in vivo.     

Integration of structure-activity relationship and artificial intelligence systems to improve in silico prediction of ames test mutagenicity

The Ames mutagenicity test in Salmonella typhimurium is a bacterial short-term in vitro assay aimed at detecting the mutagenicity caused by chemicals. Mutagenicity is considered as an early alert for carcinogenicity. After a number of decades, several (Q)SAR studies on this endpoint yielded enough evidence to make feasible the construction of reliable computational models for prediction of mutagenicity from the molecular structure of chemicals. In this study, we propose a combination of a fragment-based SAR model and an inductive database. The hybrid system was developed using a collection of 4337 chemicals (2401 mutagens and 1936 nonmutagens) and tested using 753 independent compounds (437 mutagens and 316 nonmutagens). The overall error of this system on the external test set compounds is 15% (sensitivity = 15%, specificity = 15%), which is quantitatively similar to the experimental error of Ames test data (average interlaboratory reproducibility determined by the National Toxicology Program). Moreover, each single prediction is provided with a specific confidence level. The results obtained give confidence that this system can be applied to support early and rapid evaluation of the level of mutagenicity concern.     

Decomposition of linear dodecylbenzenesulfonate by simultaneous treatment with ozone and ultraviolet irradiation: rapid disappearance of formed mutagens.

The decomposition products and mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA104 in the presence and absence of S9 mix of linear dodecylbenzenesulfonate (DBS) in aqueous solution after ozone treatment alone or simultaneous treatment with ozone and ultraviolet (UV) irradiation (ozone/UV treatment) were investigated. The decomposed DBS solutions after these treatments for 4 h were mutagenic for strains TA98, TA100 and TA104 both with and without S9 mix, but this mutagenicity disappeared rapidly during further ozone/UV treatment. Mutagenicity of the decomposed solution of DBS, however, was not substantially decreased by treatment with ozone alone. Formaldehyde and glyoxal were identified as the decomposition products of DBS in water by high-performance liquid chromatography after treatment with 2,4-dinitrophenylhydrazine. Although these two compounds were mutagenic for strain TA104 both with and without S9 mix, they disappeared after further ozone/UV treatment but not after ozone treatment alone. These results indicate that ozone/UV treatment is an effective procedure for purifying drinking water.     

Searching for an enhanced predictive tool for mutagenicity

The Multiple Computer Automated Structure Evaluation (MCASE) program was used to evaluate the mutagenic potential of organic compounds. The experimental Ames test mutagenic activities for 2513 chemicals were collected from various literature sources. All chemicals have experimental results in one or more Salmonella tester strains. A general mutagenicity data set and fifteen individual Salmonella test strain data sets were compiled. Analysis of the learning sets by the MCASE program resulted in the derivation of good correlations between chemical structure and mutagenic activity. Significant improvement was obtained as more data was added to the learning databases when compared with the results of our previous reports. Several biophores were identified as being responsible for the mutagenic activity of the majority of active chemicals in each individual mutagenicity module. It was shown that the multiple-database mutagenicity model showed a clear advantage over normally used single-database models. The expertise produced by this analysis can be used to predict the mutagenic potential of new compounds.     

Antimutagenic,Cytoprotective and Antioxidant Properties of Ficus deltoidea Aqueous Extract In Vitro

Ficus deltoidea var. deltoidea is used as traditional medicine for diabetes, inflammation, and nociception. However, the antimutagenic potential and cytoprotective effects of this plant remain unknown. In this study, the mutagenic and antimutagenic activities of F. deltoidea aqueous extract (FDD) on both Salmonella typhimurium TA 98 and TA 100 strains were assessed using Salmonella mutagenicity assay (Ames test). Then, the cytoprotective potential of FDD on menadione-induced oxidative stress was determined in a V79 mouse lung fibroblast cell line. The ferric-reducing antioxidant power (FRAP) assay was conducted to evaluate FDD antioxidant capacity. Results showed that FDD (up to 50 mg/mL) did not exhibit a mutagenic effect on either TA 98 or TA 100 strains. Notably, FDD decreased the revertant colony count induced by 2-aminoanthracene in both strains in the presence of metabolic activation (p < 0.05). Additionally, pretreatment of FDD (50 and 100 µg/mL) demonstrated remarkable protection against menadione-induced oxidative stress in V79 cells significantly by decreasing superoxide anion level (p < 0.05). FDD at all concentrations tested (12.5–100 µg/mL) exhibited antioxidant power, suggesting the cytoprotective effect of FDD could be partly attributed to its antioxidant properties. This report highlights that F. deltoidea may provide a chemopreventive effect on mutagenic and oxidative stress inducers.     

Integrated biological–chemical approach for the isolation and selection of polyaromatic mutagens in surface waters

Many environmental mutagens, including polyaromatic compounds are present in surface waters, often in complex mixtures and at low concentrations. The present study provides and applies a novel, integrated approach to isolate polyaromatic mutagens in river water using a sample from the River Elbe. The sample was taken downstream of industrial discharges using blue rayon (BR) as a passive sampler that selectively adsorbs polyaromatic compounds and was subjected to effect-directed fractionation in order to characterise the compounds causing the detected effect(s). The procedure relies on three complementary fractionation steps, the Ames fluctuation assay with strains TA98, YG1024 and YG1041 with and without S9 activation and analytical screening. Several mutagenic fractions were isolated by combining mutagenicity testing with fractionation. The enhanced mutagenicity in the nitroreductase and/or O-acetyltransferase overexpressing strains YG1024 and YG1041 strains suggested amino- and/or nitro-compounds causing mutagenicity in several fractions. Analytical screening of mutagenic fractions with LC-HRMS/MS provided a list of molecular formulas typically containing one to ten nitrogen and at least two oxygen atoms supporting the presence of amino and nitro-compounds in the mutagenic fractions.

Further studies on polycyclic arene sulfides. Preparation and mutagenic activity of 9, 10, 10a, 11a‐tetrahydrotriphenyleno[1,2‐b]‐thiirene, 1a,2,3,10b‐tetrahydro‐5H‐thiereno[3,4]benzo[1,2‐b]fluorene and 1a,2,3,11b‐tetrahydroacenaphtho[1′,2′:6,7]naphth[1,2‐b]thiirene

The title compounds 6 , 8 and 10 , which are dihydroarene sulfides of the environmental pollutants triphenylene, benzo[b]fluorene and benzo[k]fluoranthene, have been synthesized from the corresponding epoxides and N,N‐dimethylthioformamide. The mutagenicity of the episulfides has been investigated using Salmonella typhimurium strains TA98 and TA100. While compounds 6 and 10 were mutagenic, the tetrahydrobenzo[b]fluorene episulfide 8 was inactive.