Mutagenic Fractions Research Articles

Multicriteria approach to select polyaromatic river mutagen candidates.

The identification of unknown compounds remains one of the most challenging tasks to link observed toxic effects in complex environmental mixtures to responsible toxicants in effect-directed analysis (EDA). Here, a workflow is presented based on nontarget liquid chromatography-high resolution mass spectrometry (LC-HRMS) starting with molecular formulas determined in a previous study. A compound database search (ChemSpider) was performed to retrieve candidates for each formula. Subsequently, the number of candidates was reduced by applying MS-, physical-chemical, and chromatography-based selection criteria including HRMS/MS fragmentation and plausibility, ionization efficiency with different ion sources and detection modes, acid/base behavior, octanol/water partitioning, retention time prediction and finally toxic effects (mutagenicity caused by aromatic amines). The workflow strongly decreased the number of possible candidates and resulted in the tentative identification of possible mutagens and the positive identification of the nonmutagen benzyl(diphenyl) phosphine oxide in a mutagenic fraction. The positive identification of mutagens was hampered by a lack of commercially available standards. The workflow is an innovative and promising approach and forms an excellent basis for possible further advancements.

Assessment of the Hydrocarbon Degrading Abilities of Three Bioaugmentation Agents for the Bioremediation of Crude Oil Tank Bottom Sludge Contaminated Libyan Soil

Bioremediation is a widely used environmental friendly treatment method for petrogenic hydrocarbon contaminated soils but its application to the treatment of crude oil tank bottom sludge (COTBS) contaminated soil is limited especially in Mediterranean countries such as Libya. Therefore in this study, the hydrocarbon degrading abilities of three bioaugmentation agents Pseudomonas sp (4M12), Pseudomonas xanthomarina (4M14) and Arthrobacter nitroguajacolicus (1B16A) (isolated from COTBS polluted soils) applied as part of a biostimulation-bioaugmentation (BS/BA) strategy were assessed in COTBS contaminated Libyan soils. Biostimulated (BS) and natural attenuation (NA) microcosms were also set up for comparative purposes. Gas chromatograph mass spectrometer (GC- MS) analysis revealed a total soil petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbon (PAHs) content of 30,703 mg kg-1 and 13,816 mg kg-1 respectively. Two carcinogenic fractions (naphthalene and benzenamine, 4, 4`methylenbis [2-methyl-]) and 4 mutagenic fractions (pyrene, phenanthrene, fluorene and anthracene) were detected. Substantial PAH degradation occurred in 4M14 and 4M12 samples within 15 days in contrast to up to 23 days in 1B16A, NA. However, substantial reduction in TPH (> 97%) was only observed in 4M12 and 4M14 inoculated microcosms within 15 days compared to 25-30 days in 1B16A inoculated, BS and NA microcosms. 4M14 inoculated microcosms were most efficient at complete removal (D100) of all carcinogenic and mutagenic fractions; 4M14 (9-10 days), 4M12 (9-15 days), 1B16A (15-23 days), BS (18-21 days) and NA (18-22 days). Pseudomonas xanthomarina was therefore shown as the best candidate for use in a BS/BA approach for treating COTBS contaminated Libyan soils. This study shows the importance of pre-screening bioaugmentation agents for the removal of carcinogenic and mutagenic fractions prior to use; in order to carry out safe, efficient and sustainable COTBS bioremediation in Libya.

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.

Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments

Polar fractions of a sediment extract of the industrial area of Bitterfeld, Germany, have been subjected for effect-directed identification of mutagens using the Ames fluctuation assay with TA98. Mutagenicity could be well recovered in several secondary and tertiary fractions. Dinitropyrenes and 3-nitrobenzanthrone could be confirmed to contribute great shares of the observed mutagenicity. In addition, a multitude of polar polycyclic aromatic compounds has been tentatively identified in mutagenic fractions including nitro-PAHs, azaarenes, ketones, quinones, hydroxy-compounds, lactones and carboxylic acids although their contribution to mutagenicity could not be quantified due to a lack of standards. Diagnostic Salmonella strains YG1024 and YG1041 were applied to confirm the contribution of nitro-aromatic compounds. We suggest the inclusion of dinitropyrenes and 3-nitrobenzanthrone into sediment monitoring in order to minimize the mutagenic risk to aquatic organisms and to human health.

Mutagenic activity of peloids in the Salmonella Ames test

The mechanism of action of peloids (medicinal muds) applied in spa therapy has been mostly unknown, especially in aspect of chemical features. Recently, several organic compounds have been detected from spa waters using gas chromatographic methods, without exact knowledge on biological activity. They may have important roles in therapy, but may possess carcinogenic and other toxic properties, as well. This aim requires severe methodological developments in this field. In the present paper two peloid samples from Hévíz (West-Hungary) and Kolop (East-Hungary) were tested. We performed two types of experiments on peloids, at first without any pre-treatment ( in toto), and in the second set of experiment aqueous, hydrochloric acidic, methanol and toluene extracts of peloids were prepared for mutagenicity testing. The solvent extraction study was repeated twice. The objective of this study was to detect complex genotoxic effects of peloids, the risk assessment and protection of the exposed population. In the in toto experiment there was no statistically significant mutagenicity. The solvent extraction studies represented statistically significant mutagenicity in several fractions. Mutagenicity in samples was fluctuating. In the first study more mutagenic fractions were found. In the repeated experiment the mutagenic pattern was almost completely different, only minimal overlapping occurred.

Oxidative mutagenicity of polar fractions from polycyclic aromatic hydrocarbon–contaminated soils

Soils at hazardous waste sites contain complex mixtures of chemicals and often are difficult to characterize in terms of risk to human and ecological health. Over time, biogeochemical processes can decrease the apparent concentrations of pollutants but also can lead to accumulation of new products for which toxicity and behavior in the environment are largely unknown. A bioassay-directed fractionation technique was used to assess the contribution of redox-active bacterial metabolites to the toxicity of soil contaminated with polycyclic aromatic hydrocarbons (PAHs). A reverse mutation assay with Escherichia coli WP2 uvrA/pKM101 (IC188) and E. coli WP2 uvrA oxyR/pKM101 (IC203) was used to screen fractions for genotoxicity. Strain IC203 carries the delta oxyR30 mutation, which prevents the expression of antioxidant proteins in response to oxidative stress and increases its reversion by compounds that generate reactive oxygen species (ROS). Polar fractions of PAH-contaminated soil extracts were mutagenic to strain IC203 but not to strain IC188, suggesting the involvement of ROS in genotoxicity. Genotoxic potencies ranged from 300 to 1700 revertants per milligram of fraction. Catalase was able to decrease IC203 reversion, implicating the involvement of hydrogen peroxide as a key ROS. Oxidized PAH compounds, including quinones, were identified in the mutagenic fractions but were not by themselves mutagenic. Deasphalted whole extracts and recombined fractions were not mutagenic, indicating that interactions between compounds in different fractions can mitigate genotoxicity.

Identification of a new mutagen, 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl, in river water flowing through an industrial area in Wakayama, Japan

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.

Biodirected mutagenic chemical assay of PM 10 extractable organic matter in Southwest Mexico City

The concentration of breathable particles (PM 10) in urban areas has been associated with increases in morbidity and mortality of the exposed populations, therein the importance of this study. Organic compounds adsorbed to PM 10 are related to the increased risk to human health. Although some studies have shown the lack of correlation between specific mutagenic compounds in an organic complex mixture (OCM) and the mutagenic response in several bioassays, the same organic compounds selectively separated in less complex groups can show higher or lower mutagenic responses than in the OCM. In this study, we fractionated the OCM, from the PM 10 in four organic fractions of increasing polarity (F1–F4). The Salmonella bioassay with plate incorporation was applied for each one using TA98, with and without S9 (mammalian metabolic activation), and YG1021 (without S9) strains. The most polar fraction (F4) contained the greatest mass followed by F1 (non-polar), F2 and F3 (moderately polar). The concentrations of the OCM as well as the F4 were the only variables correlated with PM 10, atmospheric thermal inversions, fire-prone area, NO 2, SO 2, CO, rain and relative humidity. This indicated that polar organic compounds were originated in gas precursors formed during the atmospheric thermal inversions as well as the product of the incomplete combustion of vehicular exhausts and of burned vegetation. The percentages of the total PAH, and the individual PAH with molecular weight ≥ 228 g mol −1 (except retene) correlated with the percentages of indirect-acting mutagenicity in TA98 + S9. The percentages of the total nitro-PAH and most of the analyzed individual nitro-PAH correlated with percentages of the direct-acting mutagenicity in both TA98 − S9 and YG1021, the latter being more sensitive. In general, the highest mutagenic activity (indirect and direct) was found in F3 (moderately polar) and in F4 (polar). The non-polar fraction (F1) did not exhibit any kind of mutagenicity. In 77% of the cases, mutagenic activity was higher in the sum fractions with respect to their OCM. The combinations between F1, F2 and F4, with F3 under different or equal proportions suggested that mutagenicity reduction, in the combined matter of January (with TA98 + S9 and YG1021) and of May (with YG1021), was due to concentrations of mutagens and non-mutagens in each fraction, and not to an antimutagenic effect. The organic compounds present in the non-polar fractions showed no antagonism, inhibition or reduction in the most mutagenic fractions in both indirect- and direct-acting mutagenicity, and the less polar organic compounds in F3 reduced mutagenicity in F4, in both months.

Effect‐directed analysis of mutagens and ethoxyresorufin‐O‐deethylase inducers in aquatic sediments

Sediment extracts from a creek in the Neckar river basin (Germany), which received the discharge of treated hospital wastewater, were found to exhibit strong aromatic hydrocarbon (Ah) receptor-mediated effects in a rainbow trout liver cell line (RTL-WI) as well as high mutagenicity in the Salmonella/microsome assay after fractionation. The crude extract did not exhibit a clear mutagenic response. Apparently, cleanup or fractionation before mutagenicity testing is necessary to minimize the risk of false-negative results. Effect-directed fractionation and analysis were applied to characterize and identify the toxicants that cause these effects. Major ethoxyresorufin-O-deethylase induction potency and mutagenicity were detected in different polyaromatic fractions, indicating different sets of toxicants that induce metabolic activation and mutagenicity. Dioxin-like halogenated aromatic hydrocarbons, including polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, and priority polycyclic aromatic hydrocarbons, contributed to Ah receptor-mediated activity only to a minor extent. Benzo[a]pyrene, benzo[a]fluoranthene, and perylene could be confirmed as important contributors to mutagenicity. The nonpriority pollutants 11H-indeno[2,1,7-cde]pyrene, a methylbenzo[e]pyrene, and a methylperylene were tentatively identified as major components, representing 82% of the peak area of a highly mutagenic fraction of the sediment extract. This suggests that hazard and risk assessment of complex environmental mixtures should make increasing attempts to identify and consider hazardous key pollutants rather than focusing on a priori-selected key pollutants alone.

Analytical methods in environmental effects-directed investigations of effluents

Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components. Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for non-mutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon, and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are characterized by gas chromatography/mass spectrometry (GC-MS) and/or other spectrometric techniques for identification. LC-MS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the potential for LC-MS to identify unknown polar compounds has yet to be fully realized. Salmonella-based assays (some miniaturized) have been coupled with fractionation methods for most studies aimed at identifying mutagenic fractions and chemical classes in mixtures. Effects-directed investigations of mutagens have focused mostly on drinking water and sewage, whereas extensive investigations of non-mutagenic effects have also included runoff, pesticides, and pulp mill effluents. The success of effects-directed investigations should be based on a realistic initial objective of each project. Identification of chemical classes associated with the measured biological endpoint is frequently achievable; however, confirmation of individual compounds is much more difficult and not always a necessary goal of effects-directed chemical analysis.

Mutagenicity of Surface Soils in Urban Areas of Aichi Prefecture, Japan, and Bangkok, Thailand

To clarify the contamination levels of surface soil with mutagens in urban areas of Aichi prefecture, Japan, and Bangkok, Thailand, 60 and 67 surface soil samples were collected in Aichi and Bangkok, respectively, and mutagenicities of the organic extracts from these soil samples were examined in the Ames assay using Salmonella typhimurium (S. typhimurium) TA98 and TA100 with and without a mammalian metabolic system (S9 mix). Most of the soil samples collected in both areas showed mutagenicity in TA98 with and without S9 mix and in TA100 with S9 mix. Thirteen (22%) soil samples collected in Aichi showed high mutagenicity (more than 1000 revertants/g of soil) in TA98 with and/or without S9 mix, and six soil samples induced more than 4000 revertants/g of soil in TA98. Mutagenic potencies of most of the soil samples from Bangkok were moderate (100‐500 revertants/g of soil) or low (less than 100 revertants/g of soil) in both strains with and without S9 mix, and only one sample induced more than 1000 revertants/g of soil in both strains. In the characterization of mutagens, organic extracts of two and one soil samples from Aichi and Bangkok, respectively, were separated on an octadecylsilyl (ODS) column for HPLC, and mutagenicities of the resulting fractions were examined using S. typhimurium YG1024 without S9 mix. For the two soil samples from Aichi, retention times of the distinct mutagenic fractions were similar at 40‐74 min, and especially high mutagenicities were detected in three fractions corresponding to dinitropyrene isomers. In contrast, distinct mutagenicities were detected at retention times of 6‐29 min for the sample from Bangkok, and marked mutagenicity was found in the fraction corresponding to another nitroarene. These results indicate that surface soils in urban areas in Aichi and Bangkok are commonly contaminated with mutagens, but major mutagenic constituents in the surface soils are different.

Effects of different cooking methods on the formation of food mutagens in meat

SummaryHamburgers and chicken fillets were cooked in convection ovens, deep‐fried or contact fried and analysed for mutagenic activity using the Ames test. For the three different convection ovens, the cooking parameters studied included the presence of steam, air velocity, air temperature and holding time. For deep‐frying and contact frying, the cooking parameters included cooking temperature and cooking time. In cooked hamburgers, mutagenic activity was only detected in those that had been deep‐fried. In chicken fillets, mutagenic activity was detected in samples prepared with all cooking methods, being highest in the deep‐fried samples. Factorial analysis indicated that heat transfer was the most important factor affecting mutagenic activity. High temperature and high air velocity in the convection ovens enhanced mutagenic activity. The presence of steam reduced the mutagenic activity, except when high temperature was used in combination with high air velocity. In chicken fillets, high mutagenic activity correlated to high weight loss during cooking. Pan‐fried chicken fillets were analysed using high performance liquid chromatography (HPLC) and the mutagenic/carcinogenic heterocyclic amines 2‐amino‐3,8‐dimethylimidazo[4,5‐f ]‐quinoxaline (MeIQx), 2‐amino‐1‐methyl‐6‐phenylimidazo‐[4,5‐b]‐pyridine (PhIP) and the co‐mutagenic heterocyclic amine Norharman (9H‐pyrido[3,4‐b]‐indole) were identified. The HPLC fractions were tested for mutagenic activity and, apart from the mutagenic fractions corresponding to MeIQx and PhIP, several mutagenic fractions were detected that did not correspond to known heterocyclic amines.

Mutagenic nitrated benzo[ a]pyrene derivatives in the reaction product of benzo[ a]pyrene in NO 2–air in the presence of O 3 or under photoirradiation

In order to clarify the contribution of nitrated products to the direct-mutagenic activity of products of the reactions of benzo[ a]pyrene in NO 2–air under various conditions, heterogeneous reactions of BaP deposited on filter in the air containing 10 ppm of NO 2 have been conducted in dark or under photoirradiation. The reaction products have been analyzed by gas chromatography and mutagenicity of the products fractionated by preparative HPLC was assayed for Salmonella typhimurium strains TA98 and YG1024 in the absence of S9 mix. 3,6-dinitrobenzo[ a]pyrene and 1,3-dinitrobenzo[ a]pyrene, which are strong direct-acting mutagens, largely contributed to the total direct-acting mutagenicity of the dark reaction products in NO 2–air. On the other hand, both the dark reaction in the presence of O 3 and the photoreaction in NO 2–air resulted in the formation of much smaller amounts of nitrobenzo[ a]pyrenes than that observed in the dark reaction in the absence of O 3. These results show that the contribution of other direct-acting mutagens to the total direct-acting mutagenicity of the products in these reactions should be considered. Benzo[ a]pyrene lactones were identified in a highly mutagenic fraction of the products of the dark reaction in the presence of O 3 and photoreaction and a nitrobenzo[ a]pyrene lactone was also identified in a highly mutagenic fraction of the dark reaction products in the presence of O 3. Nitrated oxygenated benzo[ a]pyrene derivatives such as nitrobenzo[ a]pyrene lactone were considered to largely contribute to direct-acting mutagenicity of the products of the dark reaction in the presence of O 3 and photoreaction.

Mutagenic activity of 4-nitroquinoline- N-oxide in upper aerodigestive tissue in lacZ mice (Muta™Mouse) and the effects of 1,4-phenylenebis(methylene)selenocyanate

4-Nitroquinoline- N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 μg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117±26, 73±15, and 48±15 mutants/10 5 plaque-forming units (pfu) (ca. 15–40×background). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance ( p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies.

Genotoxicity of fractionated organic material in airborne particles from São Paulo, Brazil

Particulate matter less than 10 μm aerodynamic diameter (PM10) is associated with adverse health effects including increased respiratory problems and mortality. PM10 is also associated with increases in cancer in some urban areas. Identification of toxic compounds in PM10 is a step toward estimating exposure to these compounds and evaluating their public health risk. However, the toxic compounds on PM10 are part of a highly complex mixture of compounds that makes chemical characterization difficult. Before this study, there has been little investigation of genotoxic compounds in particulate matter from Latin American cities. Here, both bioassay (mutagenicity) and chemical analyses were conducted with organic solvent extracts of PM10 collected from São Paulo, a major Brazilian city. Sequential extraction in dichloromethane (DCM) followed by acetone (ACE) yielded 20.3% and 10.2% of the total mass, respectively. Non-polar and moderately polar organic material solubilized in DCM. ACE extracted more polar organic species and some inorganic ions. Both extracts were fractionated separately using cyanopropyl-bonded silica chromatography with organic solvents of increasing polarity. The mass distribution among the fractions was measured. The mutagenic activity of the fractions was assayed using the microsuspension procedure with the Salmonella typhimurium tester strain TA98, with and without addition of metabolic enzymes (S9). The DCM extract had about four times higher mutagenic activity than the ACE extract. In general, addition of S9 resulted in an increase in mutagenicity of DCM fractions, but a decrease for the ACE extract. Most of the activity was concentrated in fractions in the mid-range of polarity within both the DCM and ACE extracts. The fractions were analyzed by gas chromatography with mass selective detection (GC/MS) without derivatization. The most mutagenic fractions in the DCM extract contained ketones, aldehydes, and quinolines. The most mutagenic ACE fraction had ketones, carboxylic acids, and aldehydes.

Bioassay-directed fractionation and chemical identification of mutagens in bioremediated soils.

Soil from a Superfund site (Reilly Tar Site, St. Louis Park, Minnesota) contaminated with polycyclic aromatic hydrocarbons (PAHs) from creosote was treated with several bioremediation technologies including bioslurry (BS), biopile (BP), compost (CMP), and land treatment (LT). These treatment technologies are being evaluated in pilot scale laboratory systems by the U.S. Environmental Protection Agency's National Risk Management Research Laboratory in Cincinnati, Ohio. To evaluate the genotoxicity and identify the mutagens in the soil before and after the various treatments, fractionated extracts of five soils were bioassayed for mutagenic activity with a microsuspension modification of the Salmonella histidine reversion assay. Soils were extracted by sonication using dichloromethane (DCM). The five extracts were fractionated in triplicate (two for bioassay and one for chemical analysis) by reverse-phase high-performance liquid chromatography (HPLC) using hexane/DCM/methanol, and the fraction for bioassay were solvent-exchanged into dimethyl sulfoxide by nitrogen evaporation. Forty HPLC fractions for each sample were bioassayed in strain YG1041 with and without exogenous liver metabolic activation. As shown in a companion paper, the mutagenicity of two treatments (BS and BP) was significantly greater than the mutagenicity of the untreated soil. Mutagenic fractions (> 500 revertants) were analyzed by gas chromatography/mass spectrometry (GC/MS). PAH analysis of the soils indicated that all treatments were effective in reducing the total PAH concentration (48-74%). Qualitative GC/MS analysis of the mutagenic fractions from the BS and BP treatments indicated that they contained azaarenes, which are mutagens. The CMP and LT processes were the most effective and least toxic bioremediation procedures based on mutagenic potency and chemical analysis. This research demonstrated that the combination of bioassays and chemical analysis provided a more accurate determination of toxicity in these complex environmental mixtures.

Mint prevents shamma-induced carcinogenesis in hamster cheek pouch.

Shamma, a complex mixture of powdered tobacco, slaked lime, ash, oils, spices and other additives, has been linked to oral cancer in Saudi Arabia. Shamma varies in colour and odour due to the nature of the additives which characterize different brands. Using the Ames Salmonella assay, a chloroform extract of a brand named 'white shamma' (WSH) was found to be mutagenic, while that of a brand called 'brown shamma' (BSH), which is known to contain mint as a flavouring agent, was found to be non-mutagenic. Using HPLC, a mutagenic and a non-mutagenic fraction were isolated from the extract of BSH. The non-mutagenic fraction of BSH was found to neutralize the genotoxic effect of the mutagenic fraction when the two were recombined. A chloroform extract of mint showing no mutagenic activity in the Ames assay effectively inhibited the mutagenicity of carcinogens/mutagens like benzo[a]-pyrene, aflatoxin B1, methylmethane sulfonate and extract of WSH. A carcinogenicity assay designed to test the effects of WSH and BSH in the hamster cheek pouch model showed that the former was tumorigenic, while the latter was not. However, when crushed leaves of mint were mixed with powdered WSH (in 1:1 proportion), the tumorigenic effect of the latter was abolished. These data strongly suggested that mint has a chemopreventive effect against shamma-induced carcinogenesis, which could be due to its antimutagenic properties.

Human Lymphoblast Mutagens in Urban Airborne Particles

While it is known that urban airborne particles typically contain trace levels of bacterial mutagens and rodent carcinogens, little work has been done to identify chemicals in such particles that can genetically alter human cells. In this paper, we describe the analysis of an organic extract of a Washington, DC, airborne particle sample (SRM 1649) for human cell mutagens. Due to the chemical complexity of the extract, a bioassay-directed fractionation method was used to separate mutagenic constituents into chemically simplified fractions. Mutagenicity testing was done using the h1A1v2 cell line, a line of human B-lymphoblastoid cells that have been engineered to overexpress the human cytochrome P4501A1. Chemical analysis of mutagenic fractions was accomplished using GC−MS and HPLC−UV techniques. Our results indicate that ∼20% of the total mutagenicity the extract was accounted for in two fourth-order fractions that contained ∼3% of the total extract mass. These fractions were composed largely of polycycli...

Characterization of the carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in cooking aerosols under domestic conditions.

Lung cancer in women is the leading cause of cancer death in Taiwan. Most Chinese women are non-smokers and 60% of female lung cancer patients have adenocarcinomas. Epidemiological data indicate that the incidence of lung cancer among Chinese women may be correlated with cooking fumes. However, the carcinogenic compound(s) in cooking fume aerosols is not defined. In the present study, the cooking aerosols from Chinese stir-frying of fish were prepared under domestic conditions. To determine the mutagenic compounds in the cooking aerosol, mutagens were purified by two steps of high-performance liquid chromatography (HPLC), and their mutagenicity was monitored with Salmonella typhimurium TA98. The mutagen was eluted as a single peak. The chemical structure of the mutagenic fraction of cooking aerosol from frying of fish was characterized by UV spectra and electrospray mass spectrometry. The bacterial indirect-acting mutagenic compound in the cooking aerosol extract was determined to be 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). An amount of 0.25 ng MeIQx/g of meat per min was estimated based on the mutagenic response. These data indicated that significant amounts of MeIQx (268.1 ng/Chinese dish of frying fish) were present in cooking aerosol in a short time. Chinese women spend approximately 1 h preparing meals everyday, thus, they may be exposed to significant amounts of MeIQx from cooking aerosols in the kitchen.

Purity of recycled fibre‐Based materials

In order to study the purity of recycled fibre‐based materials, products containing recycled fibre as well as recycled pulp were examined with regard to their chemical impurities, toxicity and microbiological quality. The study was carried out to clarify both qualitatively and quantitatively the variations in microbiological quality. The levels of several classes of chemical substances were analysed and semi‐volatile and volatile substances identified. The toxicity and mutagenicity of virgin fibre and recycled fibre materials were screened using the Photobacter toxicity test and the Ames Salmonella mutagenicity test. Preliminary chemical characterization of the mutagens was carried out. Identification of the compounds found in the mutagenic fractions was performed by gas chromatography/mass spectrometry (GC/MS). The concentration of various substances analysed was found to be low, although the variety of substances present appeared to be very broad. Preliminary chemical characterization revealed that some samples contained compounds known to have mutagenic or other toxic activity. Also, the recycled fibre pulps contained large amounts of various microbes, the microbial load consisting mainly of aerobic spore‐forming bacteria. The paper‐making process was found to clearly have reduced the total microbial counts.