Fate and toxicity assessment of linear alkylbenzene sulfonates in drinking water using the ames test

The genotoxic activity of the methanolic water extracts of prechlorinated water from Barcelona (NE Spain) using the Ames test was studied. High performance liquid chromatography (HPLC) and mass spectrometry in the mass spectroscopy/fast atom bombardment mode (MS/FAB) was employed to tentatively identify organic compounds responsible of genotoxic activity. Methanolic extracts of prechlorinated water were highly mutagenic in the Ames test, mainly with the TA98 strain for concentration lesser than 1 L. On the other hand, the TA100 strain showed higher mutagenicity for tap water extracts and concentrations higher than 1 L. Also, a strong toxic effect was observed when methanolic extracts were analyzed by the Ames test. Toxicity showed a reduction of the genotoxic ratio by a characteristic negative slope for the concentration vs genotoxicity curve. Toxicity was usually observed using the TAlOO strain and at a higher concentration than mutagenicity does. Both mutagenicity and toxicity in the Ames test showed a characteristic pattern depending on their origin (tap or prechlorinated water). It was possible to separate mutagenic from toxic fractions by HPLC. These subfractions were analyzed by MS/FAB in order to identify the organic compounds responsible for these effects, but unsuccessful results were obtained for mutagenic subfractions. Alkylbenzenesulfonates (LA3) were the sole compounds identified in toxic subfractions. The correlation between toxicity of samples (TA100 strain) and the presence of LAS was proved by comparison of toxicity from a standard LAS and those observed from real samples. An EC ₅₀ of 9.8 mg/L for LAS has been established by the Ames test using the TAlOO strain. © 1993 John Wiley & Sans, Inc.     

Contribution of coffee aroma constituents to the mutagenicity of coffee

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.     

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by hte metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and β-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.     

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).     

Release of mutagens after chemical or microbial degradation of beech wood lignin

The microbial or chemical degradation of lignin from untreated samples of beech wood dusts (Fagus silvatica) resulted in the release of different mutagenic responses in the Salmonella/mammalian plate incorporation assay. In the first experiment using chemical degradation of lignin, dust samples were pre-extracted using acetone-water; the lignin portions were degraded into simpler compounds which were further fractionated on a Sephadex-LH20 column. The compounds isolated from the second phase of Sephadex, representing substances with a 3-4 ring structure and/or those of the same molecular weight, were highly mutagenic towards Salmonella typhimurium TA100 in the presence of metabolic activation. These substances were also active to some extent in strain TA1537 both in the presence and absence of Aroclor-induced rat liver homogenates. In contrast, no direct- or indirect-acting mutagenicity was found when testing with strains TA97 and TA98. Strain TA1535 responded positively only to direct-acting mutagens in the fraction tested. The mutagenic fraction was found to be toxic to the cells when tested in a histidine-rich medium. Repurification of this mutagenic fraction, using silica-gel column chromatography, revealed much higher mutagenic activity than the test material towards strain TA100. In the second pilot experiment, Phanerochaete chrysosporium and Chaetomium globosum, which are known for their ability to degrade lignin, were each incubated with wood dusts in a mixture of physiological saline and nutrient broth for either 3 or 30 days. Significant mutagenic activity was observed with the dust extract after incubation with Ph. chrysosporium but not with Ch. globosum which is a known degrader of beech lignin. These results are discussed regarding hypotheses on the carcinogenicity of beech wood dusts.     

Mutagenicity assay in Salmonella for thirteen 2-substituted-1 H-phenanthro (9,10-d) imidazoles

Some 2-substituted-1 H-phenanthro [9,10-d] imidazole compounds synthesized as a predrugs were tested in mutagenicity assays in Salmonella strains TA97, TA98, and TA100 using a plate incorporation assay both with and without S9 mix. The 10 substances were mutagenic in TA97 and five of them were mutagenic only with metabolic activation, whereas one of them did not require the addition of S9. The eight substances were mutagenic in TA98 only with S9. For TA100, seven substances showed positive results both with and without S9, however another four required S9, whereas only one of them did not required metabolic activation. In summary, all of 13 substances derived from phenanthro [9,10-d] imidazole were found to be mutagenic for at least one or two of the three strains and their mutagenicity are discussed.     

Mutagenic potential of allyl and allylic compounds: Structure-activity relationship as determined by alkylating and direct in vitro mutagenic properties

A series of compounds, each containing an allylic moiety, has been tested using Salmonella typhimurium TA 100 in a modified Ames mutagenicity assay system. In the absence of activating enzymes (S-9) mix, those allylic compounds possessing chemically good leaving groups show direct mutagenic activity. Their activity decreases in the following order: allyl methanesulfonate > -iodide > -bromide > -chloride. This is in good agreement with the alkylating properties measured in the nitrobenzyl-pyridine (NBP) test. For all allyl and allylic compounds found to be directly mutagenic, a decrease in, and sometimes total loss, of mutagenicity is registered after addition of S-9 supernatants. Compared to the direct mutagenic activity of allyl chloride, epichlorohydrin shows a much higher mutagenicity, whereas propyl chloride has proven to be nonmutagenic. The direct mutagenic effect of this type of compound is theoretically explained by sn-1, Sn-2 and SN-2' mechanisms.     

Mutagenic activity in roadside soils

Mutagenicity of soils sampled at median strips, roadsides and a park neighboring arterial roads in Kurume City was determined by Ames test. Organic extracts of soils were mutagenic in strains TA98, TA100, YG1041 and YG1042 with and without S9mix. No sample showed mutagenic responses in strains YG3003 or YG7108. Extracts from soils of median strips and beside intersections showed higher mutagenicity and concentrations of PAHs and heavy metals than others, and mutagenic activity of soils correlated significantly with concentrations of PAHs and heavy metals. However, PAHs accounted for less than 12% of total mutagenicity in strains TA98 and TA100 of soil extracts. These extracts showed much higher mutagenicity in YG strains than in TA strains. The results indicate that these soils may be polluted with nitroarenes and aromatic amines.     

Strong mutagenic effects of diesel engine emissions using vegetable oil as fuel

Diesel engine emissions (DEE) are classified as probably carcinogenic to humans. In recent years every effort was made to reduce DEE and their content of carcinogenic and mutagenic polycyclic aromatic compounds. Since 1995 we observed an appreciable reduction of mutagenicity of DEE driven by reformulated or newly designed fuels in several studies. Recently, the use of rapeseed oil as fuel for diesel engines is rapidly growing among German transportation businesses and agriculture due to economic reasons. We compared the mutagenic effects of DEE from two different batches of rapeseed oil (RSO) with rapeseed methyl ester (RME, biodiesel), natural gas derived synthetic fuel (gas-to-liquid, GTL), and a reference diesel fuel (DF). The test engine was a heavy-duty truck diesel running the European Stationary Cycle. Particulate matter from the exhaust was sampled onto PTFE-coated glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The gas phase constituents were sampled as condensates. The mutagenicity of the particle extracts and the condensates was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Compared to DF the two RSO qualities significantly increased the mutagenic effects of the particle extracts by factors of 9.7 up to 59 in tester strain TA98 and of 5.4 up to 22.3 in tester strain TA100, respectively. The condensates of the RSO fuels caused an up to factor 13.5 stronger mutagenicity than the reference fuel. RME extracts had a moderate but significant higher mutagenic response in assays of TA98 with metabolic activation and TA100 without metabolic activation. GTL samples did not differ significantly from DF. In conclusion, the strong increase of mutagenicity using RSO as diesel fuel compared to the reference DF and other fuels causes deep concern on future usage of this biologic resource as a replacement of established diesel fuels.     

Enzyme-mediated mutagenicity in Salmonella typhimurium of contaminants of synthetic indigo products

The mutagenic potential of two natural and seven synthetic, commercial indigo dye products was investigated. The natural products showed no mutagenicity in Salmonella typhimurium stains TA98 and TA100. In the presence of rat-liver homogenate from Aroclor 1254 pretreated rats all of the synthetic products were mutagenic towards strain TA98 but not towards strain TA100. The mutagenic effect produced was highly dependent on the amount of rat-liver homogenate added. Because of its high mutagenic potential, one product was further investigated. In the presence of rat-liver homogenate this product was weakly mutagenic towards strain TA1537 and strongly mutagenic towards strain TA1538. No mutagenicity was observed in strain TA1535. Experiments with purified synthetic indigo and natural indigo revealed that the mutagenic activity of the synthetic commercial products can be ascribed to one or more contaminants.     

Implication of nitro group reduction in the mutagenic and chromosome damaging activities of 22 new 5-nitroisoquinolines by the Salmonella mutagenicity test and the cytokinesis-blocked micronucleus assay.

The mutagenic (MUT) and chromosome-damaging (CHR) activities of 22 potential antimalarial drugs (5-nitroisoquinoline derivatives) were evaluated by the Salmonella test and the cytokinesis-blocked micronucleus assay (CBMN). The Salmonella mutagenicity test was performed with and without metabolic activation (S9 mix) in S. typhimurium strains TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain). The CBMN was carried out on human lymphocytes without metabolic activation. Four concentrations were tested: 1, 10, 100 and 1000 ng/ml. MUT was expressed as minimal mutagenic concentrations (MMC, microM) and CHR was expressed as minimal chromosome-damaging concentrations (MCDC, nM) to compare both activities. All the 5-nitroisoquinoline compounds were mutagenic in TA100. MMC ranged from 0.1 to 52.9 microM in TA100. A statistically significant decrease in MMC was observed in YG1042 (8 x 10(-3) to 3.5 microM), implicating reduction of the nitro group. Modulation of MUT by S9 mix was not significant in TA100 and YG1042. CHR was detected in 13 products for at least one concentration. Among the chromosome-damaging compounds, the MCDC ranged from 2.9 x 10(-3) to 3.6 nM. No relationship was found between MUT and CHR, suggesting two distinct pathways of DNA damage.     

The mutagenicities of seven coumarin derivatives and a furan derivative (nimbolide) isolated from three medicinal plants

Seven coumarin derivatives (imperatorin, heraclenin, xanthotoxin, marmesin, chelepin, oxypeucedanin, esculin) and a furan derivative (nimbolide) were screened on 6 Ames tester strains (TA92, TA94, TA97, TA98, TA100, TA102). The eight compounds are chemicals isolated from three Nigerian medicinal plants: Afraegle paniculata, Clausena anisata, and Azadirachta indica. Different preparations of the former are taken by Nigerians for gut disturbances, and a concoction of the latter called "Agbo" is taken as an antimalarial. Marmesin and imperatorin were mutagenic in all tester strains except TA94 and TA102. The mutagenicity potencies of marmesin and imperatorin were 20 and 0.2 respectively. Mutagenicity was highest in TA98 and TA100 in both compounds. Marmesin was optimally mutagenic at a dose of 1.04 micrograms, and imperatorin at 30.0 micrograms. Microsomal activation was not required for mutagenicity in both compounds.     

Mutagenicity of 3-nitrodibenzofuran and 3-aminodibenzofuran

Mutagenicities of 3-nitrodibenzofuran and 3-aminodibenzofuran were examined using Salmonella typhimurium TA98 and TA100. Strong mutagenicity was found in both compounds. The mutagenic potency of 3-nitrodibenzofuran was approximately 3.5-fold stronger in TA98 and twice stronger in TA100 than that of benzo[a]pyrene. Mutagenicity of 3-aminodibenzofuran was observed under metabolic activation and was 10 times stronger in TA98 and about 5 times stronger in TA100 than that of benzo[a]pyrene.     

Mutagenicity and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt.

Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution. In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay. Effective lethality was observed in the rec-assay. No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix). In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix. The mutagenic activity became stronger increasing with the reaction period. Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity. PS, ascorbic acid and Fe-salts were inactive when they were used separately. Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive. These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity.     

Mutagenic activity in waste from an aluminum products factory in Salmonella/microsome assay

The mutagenic activity of waste material originating from an aluminum products factory was determined by the Salmonella/microsome assay, using the bacterial strains TA100, TA98 and YG1024. The material was obtained by sweeping the factory floor at the end of the work shift. Organic compounds were extracted by ultrasound for 30 min in dichloromethane or 70% ethanol. After evaporation of solvent, these extracts were dissolved in dimethylsulfoxide, and tested for the mutagenic activity at varying concentrations. All the extracts from the factory had mutagenic activity, especially in the YG1024 strain, suggesting the presence of aromatic amines, later confirmed by chemical analysis. The TA98 strain also showed mutagenic activity, though it did not exhibit the highest mutagenicity index observed with the YG1024 strain. In TA100, mutagenic activity was not observed. This study should serve as an alert to management and those who are occupationally exposed, and as a warning that this type of waste should not be discarded in the environment without any control.     

Mutagenicity Assay in Salmonella for Thirteen 2-Substituted-1H-phenanthro (9,10-d) Imidazoles

Abstract

Some 2-substituted-1H-phenanthro [9,10-d] imidazole compounds synthesized as a predrugs were tested in mutagenicity assays in Salmonella strains TA97, TA98, and TA100 using a plate incorporation assay both with and without S9 mix. The 10 substances were mutagenic in TA97 and five of them were mutagenic only with metabolic activation, whereas one of them did not require the addition of S9. The eight substances were mutagenic in TA98 only with S9. For TA100, seven substances showed positive results both with and without S9, however another four required S9, whereas only one of them did not required metabolic activation. In summary, all of 13 substances derived from phenanthro [9,10-d] imidazole were found to be mutagenic for at least one or two of the three strains and their mutagenicity are discussed.     

Comparative mutagenicity studies of azo dyes and their reduction products in Salmonella typhimurium

The arabinose-resistant and Ames assay systems of Salmonella typhimurium were used to evaluate the mutagenic potential of azo dyes and their aromatic amine reduction products. Azo dyes, namely direct black 38, direct blue 15, and direct red 2, were mutagenic in the arabinose-resistant and Ames assays with both hamster and rat liver S9 activation. Both assays gave relatively higher mutagenic responses with hamster S9. Reduction products of these dyes, namely benzidine, o-dianisidine, and o-tolidine, were mutagenic in the Ames assay. Benzidine was weakly mutagenic and o-dianisidine and o-tolidine were nonmutagenic in the arabinose-resistant assay. These results indicate that both arabinose-resistant tester SV50 and Ames tester TA98 were sensitive in detecting mutagenicity of azo dyes. The use of the standard plate protocol with Ames tester TA98 is more efficient than the modified azo dye protocol in detecting mutagenicity of aromatic amine reduction products. Additional modifications in either the standard plate or modified azo dye protocols may improve detection of mutagenicity of these compounds in the arabinose-resistant assay system.     

Mutagenicity of cysteine and penicillamine and its enantiomeric selectivity

We previously observed that postmitochondrial supernatant (S9) from rat liver and kidney homogenates transforms L-cysteine into a mutagen that reverts bacteria of the strain Salmonella typhimurium TA100 to histidine independence. In the present study the enantiomers of cysteine and penicillamine (beta, beta-dimethylcysteine) have been investigated for mutagenicity. The Salmonella typhimurium strain TA92 was found to be more sensitive than TA100 to the mutagenic action of L-cysteine and was therefore also included. This strain allowed the unambiguous realization of a (weak) mutagenic effect of L-cysteine even in the absence of mammalian enzyme preparations. D-cysteine did not show mutagenicity under any experimental conditions. However, it was strongly bacteriotoxic. On the other hand, both enantiomers of penicillamine exerted clear mutagenic effects. Qualitatively, their mutagenicity was similar to that of L-cysteine in the following respects: the penicillamines were directly mutagenic, their mutagenicity was enhanced by S9, kidney S9 enhanced the mutagenicity more than did liver S9, TA92 was more sensitive than TA100. Thereby it is noteworthy that the ratios of the specific mutagenicities in the two strains were virtually identical in the direct, kidney-S9-mediated and liver-S9-mediated tests suggesting that the ultimate mutagens under these different metabolic conditions were identical. On the other hand, substantial quantitative differences in the mutagenicity between the beta-thiol amino acids were observed. L-penicillamine was about eight times more mutagenic than the clinically used enantiomer, D-penicillamine. In the direct tests, the mutagenic potency of L-cysteine was equal to that of D-penicillamine. In the S9-mediated experiments, the mutagenic potency of L-cysteine was intermediate between those of L- and D-penicillamine.     

1,2-Dibromo compounds. Their mutagenicity in Salmonella strains differing in glutathione content and their alkylating potential

The mutagenic activities of several structurally related dibromo compounds were compared in Salmonella strains sensitive to base substitution mutagenesis (TA1535 and/or TA100) and in the glutathione (GSH)-deficient derivative TA100/NG-57, using a preincubation procedure. The compounds tested were 1,2-dibromoethane (DBE), 1,2-dibromopropane (DBP), 1,2-dibromo-1-phenylethane (DBPE) and model compounds for the half-mustards resulting from their conjugation with GSH, i.e. the N-acetyl-S-2-bromoalkyl-L-cysteine methyl esters SBE, SBP, and SBPE, respectively. The alkylating potential of all compounds was assayed with the 4-(p-nitrobenzyl)pyridine (NBP) alkylation test. Five of the compounds showed a good correlation between relative mutagenic activity in TA100 and electrophilic reactivity in the NBP-test, the order of decreasing potency being SBE greater than SBP greater than DBPE greater than DBP. SBPE displayed the highest reactivity in the NBP-test, but was devoid of mutagenic activity. The mutagenic activity of DBE was substantially decreased in the GSH-deficient strain TA100/NG-57 and could be restored by pretreating the cells with GSH. None of the other chemicals showed different mutagenic activities in TA100 and TA100/NG-57. From the results it can be concluded that 2-bromothioethers possess higher alkylating activities than the 1,2-dibromo compounds. Methyl substitution has a deactivating effect on the mutagenic activity. The results with the phenyl-substituted analogue, DBPE, show that a higher alkylating activity does not always lead to a higher mutagenic activity.     

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.