Net Revertants Research Articles

Identification of a mutagenic chlorination by-product produced from (E)-1, 3-dichloropropene (a component of nematocide DD) by using high resolution LC/MS and multivariate analysis

The estimated domestic usage of DD (a typical nematocide) has been the greatest among all the agricultural chemicals in Japan. DD is involved in a document which is used to establish tap water quality standard in Japan. The document indicates that DD is potentially detectable in raw water for tap waters. DD in raw water will be treated with chlorine at waterworks, which raises concerns about formation of mutagenic chlorination by-products through the treatment. The objective of this work was to identify a mutagenic chlorination by-product by using high resolution mass spectrometers and multivariate analyses. Mutagenicity of the chlorination by-product was evaluated with the Ames Salmonella mutagenicity assay. (E)-1,3-Dichloropropene (DCP) was used as a model compound of DD. The mutagenicity of chlorine treated model aqueous solutions of DD increased with increasing chlorine dosages up to 3.00 mol-Cl2/mol-DCP. From the chlorine treated aqueous solutions of DD, 1,3-dichloroacetone (DCA) was identified as a major mutagen by a cochromatography with an authentic standard, precursor ion analyses and first-generation product ion analyses with the high resolution mass spectrometers. The mutagenicity of DCA against TA100 strain without microsomal activation was 160,000 net revertant colony/μmol (the toxicity equivalent factor was 0.14). Based on the mutagenicity of the chlorine treated DCP sample, the specific mutagenicity of DCA, and the DCA concentration in the sample, the mutagenic contribution of DCA to the chlorine treated DCP sample was calculated. High contribution (98%) clearly shows that DCA is a major mutagen in the chlorine treated DCP sample. Because the exploration of DCA was performed by using the unrealistic high concentration samples of DCP and chlorine, formation of DCA in the practical concentration samples (0-200 μg-DCP/L, 0.10 or 1.27 mg-Cl2/L) was examined. It was proven that DCA formation reaction takes place in the practical concentrations of DCA and chlorine.

Involvement of nitro-compounds in the mutagenicity of urban Pm2.5 and Pm10 in Turin

Fine particles can be active carriers of toxic compounds into the alveoli of the lungs. Among these compounds are numerous mutagens and carcinogens. The direct mutagenicity per unit mass of fine particulate matter (PM) is significantly higher than that of coarse particles, especially in urban areas. In this study, the mutagenic properties of urban PM2.5 and PM10 were evaluated, and the role of nitro-compounds was estimated. PM2.5 and PM10 samplings, and measurements of NOx and some PAHs were performed daily in 2007 in Turin, following a consolidated in vitro test – the Salmonella mutagenicity assay – conducted with organic extracts of PM2.5 and PM10. The mutagenic properties were assessed for each month of sampling with Salmonella typhimurium strain TA98 and TA98-derived strains: a nitroreductase-deficient mutant strain (TA98NR) and an additional nitroreductase-producing plasmid strain (YG1021). The annual measured mean levels of PM2.5 and PM10 were 34 ± 20 and 48 ± 18 μg/m 3. The PM2.5/PM10 ratio ranged from 0.36 to 0.89. The Salmonella assay showed higher mutagenicity in autumn/winter (20 ± 15 TA98NR; 54 ± 39 TA98; 173 ± 161 YG1021 net revertants/m 3) compared with spring/summer (2 ± 2 TA98NR; 7 ± 8 TA98; 24 ± 27 YG1021 net revertants/m 3) ( p < 0.01). There are also statistically significant seasonal differences in the gravimetric analysis data. The number of TA98 net revertants per μg of PM2.5 is 6.5 times greater than per μg PM10. Moreover, the bioassay results showed an amplified response in the YG1021 strain and a reduced response in the TA98NR strain. The net revertant ratio TA98NR/YG1021 is 11 ± 4 for organic extracts of PM2.5 and 13 ± 6 for extracts of PM10 ( p < 0.01). There is a significant correlation between the NOx and PAH concentrations. These findings illustrate the relevant role of nitro compounds, and they underline the priority in improving preventive measures to reduce air pollution by nitrated molecules.

Mutagenicity assessment of contaminated soil in the vicinity of industrial area

In the industrial area of Chinhat, Lucknow (India) wastewater coming from pesticide manufacturing and other industries is used to irrigate the agricultural crops. This practice has been polluting the soil and pollutants might reach the food chain. Gas chromatographic analysis revealed the presence of certain organochlorine pesticides in soil samples. Samples were extracted using different solvents, i.e., hexane, acetonitrile, methanol, chloroform, and acetone (all were HPLC-grade, SRL, India). Soil extracts were assayed for mutagenicity using Ames Salmonella/mammalian microsome test. Mutagenicity was observed in the test samples and TA98 was the most responsive strain for all the soil extracts (irrigated with wastewater) in terms of mutagenic index in the presence (+S9) and absence (-S9) of metabolic activation. In terms of slope (m) of linear dose-response curve for the most responsive strain TA98 exhibited highest sensitivity against the soil extracts in the presence and absence of S9 fraction. Hexane-extracted soil sample (wastewater) exhibited maximum mutagenicity in terms of net revertants per gram of soil in the presence and absence of S9 mix as compared to the other soil extracts. Groundwater-irrigated soil extracts displayed low level of mutagenicity as compared to wastewater-irrigated soil. The soil is accumulating a large number of pollutants due to wastewater irrigation and this practice of accumulation has an adverse impact on soil health.

A level change in mutagenicity of Japanese tap water over the past 12 yr

A relative comparison study of mutagenicity in Japanese tap water was conducted for 1993 and 2005 surveys. It intended to assess the effects of advanced water treatment installations to water works, improvement of raw water quality and improvement of residual HOCl concentration controlling. Sampling points (taps) were the same in both surveys. The results of 245 samples obtained by the Ames Salmonella mutagenicity test (Ames test) were analyzed. The Ames tests were conducted by using Salmonella typhimurium TA98 and TA100 strains with and without exogenous activation (S9). With the exception of TA100-S9, the other conditions needed no discussion as a factor in the mutagenicity level change. The average mutagenicity in 1993 and 2005 under the conditions of TA100-S9 were 2600 and 1100 net revertant L −1, respectively. This indicated that the mutagenicity level of Japanese tap water decreased during the 12-yr period. Particularly a remarkable decrease in mutagenicity was observed in the water works where the advanced water treatments were installed during the 12-yr period. The advanced water treatments were effective in decreasing the mutagenicity of tap water. Mutagenicity also decreased in the water works with conventional water treatments; the improvement of residual HOCl concentration controlling was also considered to be effective in decreasing the mutagenicity of tap water.

Genotoxicity Assessment of Wood-Preserving Waste–Contaminated Soil Undergoing Bioremediation

ABSTRACT Bioremediation represents one of the most cost-effective technologies for treatment of petroleum hydrocarbons in contaminated surface soils. A major concern for regulatory agencies when evaluating bioremediation is how to determine acceptable levels for residual organics in soil. Although guidelines have been developed for some organics in soil, limited information is available to define acceptable levels of the metabolites of biological degradation. The products of oxidative degradation are likely to be more water soluble and may also be more toxic. The purpose of the current study was to monitor changes in compound concentration and genotoxicity in soils undergoing bioremediation. The site selected for this study was a former wood-preserving site in the northwestern United States. Soil samples were collected over a 4-year period from two 6075-m2 land treatment units. Conditions for biodegradation were enhanced by the addition of water and nutrients, as well as by frequent tilling to add oxygen. Due to the location of the facility, the temperature was conducive to a more rapid rate of biodegradation for approximately 6 months per year. Soil samples were collected using a grid system and solvent extracted. Polycyclic aromatic hydrocarbons were quantified in soil extracts using gas chromatography–mass spectrometry (GC/MS), and genotoxicity measured using the Salmonella/microsome assay. After 2 years of treatment, concentrations of total and carcinogenic polycyclic aromatic hydrocarbons (PAHs) were reduced to approximately 10% the concentration in the untreated soil. However, the mean weighted activity of the untreated soil was 293 net revertants per g soil, whereas the extracts of soil collected after 2 years induced a mean weighted activity of 325 net revertants per g soil. Thus, although biodegradation clearly reduced the concentration of total and carcinogenic PAHs in the surface soils, the results from the genotoxicity bioassay indicate that there was a lag in the reduction of mutagenicity in treated soils.

Mise en œuvre de la pluridisciplinarité dans les services de santé au travail

The estimated domestic usage of DD (a typical nematocide) has been the greatest among all the agricultural chemicals in Japan. DD is involved in a document which is used to establish tap water quality standard in Japan. The document indicates that DD is potentially detectable in raw water for tap waters. DD in raw water will be treated with chlorine at waterworks, which raises concerns about formation of mutagenic chlorination by-products through the treatment. The objective of this work was to identify a mutagenic chlorination by-product by using high resolution mass spectrometers and multivariate analyses. Mutagenicity of the chlorination by-product was evaluated with the Ames Salmonella mutagenicity assay. (E)-1,3-Dichloropropene (DCP) was used as a model compound of DD. The mutagenicity of chlorine treated model aqueous solutions of DD increased with increasing chlorine dosages up to 3.00 mol-Cl2/mol-DCP. From the chlorine treated aqueous solutions of DD, 1,3-dichloroacetone (DCA) was identified as a major mutagen by a cochromatography with an authentic standard, precursor ion analyses and first-generation product ion analyses with the high resolution mass spectrometers. The mutagenicity of DCA against TA100 strain without microsomal activation was 160,000 net revertant colony/μmol (the toxicity equivalent factor was 0.14). Based on the mutagenicity of the chlorine treated DCP sample, the specific mutagenicity of DCA, and the DCA concentration in the sample, the mutagenic contribution of DCA to the chlorine treated DCP sample was calculated. High contribution (98%) clearly shows that DCA is a major mutagen in the chlorine treated DCP sample. Because the exploration of DCA was performed by using the unrealistic high concentration samples of DCP and chlorine, formation of DCA in the practical concentration samples (0-200 μg-DCP/L, 0.10 or 1.27 mg-Cl2/L) was examined. It was proven that DCA formation reaction takes place in the practical concentrations of DCA and chlorine.

Drinking water mutagenicity and urinary tract cancers: a population-based case-control study in Finland.

The detection of mutagenic and carcinogenic chlorination by-products in chlorinated drinking water has raised concern in many countries over the potential health effects of long-term exposure to these products. The relation between estimated exposure to historical drinking water mutagenicity and cancer was studied in Finland by using a population-based case-control study comprising 732 bladder cancer cases, 703 kidney cancer cases, and 914 controls. The cases were obtained from the nationwide Finnish Cancer Registry for the years 1991-1992. The controls, frequency matched by age and sex, were randomly selected from the national population registry. Information on past drinking water sources and confounding factors was acquired through a questionnaire. Historical exposure to drinking water mutagenicity was estimated by using information on past residence, past water source, and historical data on water quality and treatment. Odds ratios were calculated for an increase of 3,000 net revertants per liter (net rev/liter) in average exposure from 1950 to 1987, adjusting for age, sex, socioeconomic status, and smoking in logistic regression models. A small, statistically significant, exposure-related excess risk was found for kidney cancer for men (odds ratio = 1.49, 95 percent confidence interval (CI) 1.05-2.13) for 3,000 net rev/liter exposure level. For women, the association remained nonsignificant, with a lower odds ratio of 1.08 (95% CI 0.69-1.68). For bladder cancer, the odds ratio for both men and women was 1.22 (95% CI 0.92-1.62) for 3,000 net rev/liter exposure. However, a higher odds ratio of 2.59 (95% CI 1.13-5.94) for 3,000 net rev/liter exposure was observed for male nonsmokers.

Mutagenic potential of binary and complex mixtures using different enzyme induction systems.

A series of experiments was conducted to investigate the mutagenic potential of binary and complex mixtures in the presence and absence of inducible liver enzyme systems prepared with several different chemical inducers. Liver homogenate (S9, or 9000 x g supernatant) fractions were obtained from Sprague-Dawley rats induced with either Aroclor 1254 (AR), phenobarbital (PB), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or corn oil (UI). the mutagenic potential of test samples was measured with Salmonella typhimurium strain TA98 using each of the various S9 fractions. Test samples included benzo[a]pyrene (BaP), pentachlorophenol (PCP), a binary mixture of BaP and PCP, two five-component mixtures, a methylene chloride extract of wood preserving waste-amended soil, and a methanol extract of coal gasification waste. At a dose of 25 micrograms/plate, BaP produced 55, 83, 217, and 161 net revertants per plate with UI-, PB-, AR-, and TCDD-induced S9, respectively. The complex mixture extracted from the wood preserving waste-amended soil induced approximately equal responses with all four S9 mixes. At a dose of 250 micrograms/plate, the methanol extract of a coal gasification waste produced 56 net revertants using the uninduced S9; however, when Ar- and TCDD-induced S9 was used, 129 and 67 net revertants were observed, respectively. These data demonstrate the relative importance of the various induced cytochrome P-450 isozymes for the metabolism of mutagenic chemicals and complex mixtures.

Excretion of mutagens, nicotine and its metabolites in urine of cigarette smokers.

Urine samples from 26 cigarette smokers on a restricted diet were collected in the late afternoon. Urine extracts on XAD-2 resin were tested for mutagenicity in the microsuspension assay using Salmonella typhimurium strain TA98 in the presence of metabolizing and deconjugating enzymes. Levels of urinary nicotine plus metabolites and cotinine were determined. Eighteen samples were clearly mutagenic, i.e. capable of doubling the number of spontaneous revertants at one of the assayed doses of urine. Urinary mutagenic activity ranged from 193 to 8462 net revertants/mmol of creatinine, while urinary nicotine plus metabolites and cotinine levels varied from 0.007 to 1.366 and from 0.011 to 0.297 mg/mmol creatinine. Urine samples with nicotine metabolite levels of < 0.33, 0.33- < 0.66 and > 0.66 mg/mmol creatinine had mean values +/- SD of mutagenic activity of 490 +/- 222 (n = 10), 964 +/- 560 (n = 9) and 2692 +/- 2807 (n = 7) revertants/mmol of creatinine, respectively, the statistical comparison between the groups being positive (Mann-Whitney U-test, P < 0.05). The mutagenic activity of urine samples from smokers correlated well with urinary nicotine plus metabolite levels (r = 0.658, P < 0.01). A less close correlation was found between urinary mutagenic activity and other indicators of tobacco smoke exposure, such as urinary cotinine (r = 0.504, P < 0.05), number of cigarettes smoked during the day of urine collection (r = 0.399, P < 0.05) and machine smoking-derived nicotine deliveries of the total number of cigarettes smoked (number of cigarettes multiplied by the nicotine content of each cigarette, as indicated by the manufacturer; r = 0.439, P < 0.05). These results suggest that the mutagenic activity of smokers' urine may be predicted by the urinary level of nicotine plus metabolites. The low degree of reliability of many presumptive indicators of exposure to tobacco smoke and the different urinary excretion kinetics of tobacco smoke mutagens with respect to cotinine (a frequently used biomarker for monitoring exposure to tobacco smoke) are both emphasized.

Mutagenic potential of environmental samples before and after remediation of a solvent‐contaminated site

AbstractSoil, sediment, and paint sludge samples were collected during a 4‐year period from an abandoned solvent recovery site prior to and after site remediation. Samples were sequentially extracted with methylene chloride and methanol and redissolved in dimethyl sulfoxide for biological analysis. The mutagenic potential of the extracts was evaluated using the Salmonella/microsome assay with and without metabolic activation. Noncontaminated background soil samples collected off‐site induced specific mutagenic activities that ranged from below detection to 28 net revertants per milligram of residue. Although several sludge samples induced specific activities that were relatively low, the maximum mutagenic response induced by a sludge sample was 1,258 net revertants per milligram of residue. Compared to background soil, elevated levels of mutagenic activity were also detected in the extracts of several samples collected from a drainage area downgradient from the waste storage pad and in the extract of sediment samples from a downgradient storm‐water retention pond. The weighted activity of the noncontaminated background soil samples ranged from below detection to 15 revertants per gram of soil. The combined weighted activity of the sludge sample extracts ranged from below detection to 43,567 revertants per gram. The combined weighted activity of the extracts of samples collected after site remediation ranged from below detection to 1,273 revertants per gram. A visual inspection of the site following remediation suggested that all obvious signs of chemical contamination had been removed. However, the results from biological analysis indicate that soil collected following site remediation induced a weighted activity more than 30 times the maximum weighted activity observed in the noncontaminated background soil. These data suggest that additional site remediation is required if the genotoxic potential of soil is to be reduced to levels approximating those of noncontaminated background soil.

Bioassay-based risk assessment of complex mixtures

In order to compare a standard chemical-based risk assessment with in vitro genotoxicity assays, two complex environmental mixtures from a wood-preserving site were analyzed in the Salmonella/microsome and E. coli prophage induction assays. Using GC/MS, sample 003 was found to contain relatively low levels of polycyclic aromatic hydrocarbons (PAHs) and elevated levels of polychlorinated dibenzo- p-dioxins (PCDDs), while sample 005 had higher levels of PAHs and relatively low levels of PCDDs. The complex mixtures were sequentially extracted with methylene chloride and methanol for analysis in Salmonella, or extracted with a 1:1 hexane:acetone mixture for analysis in the E. coli prophage induction assay. At a dose of 1.0 mg/plate in Salmonella strain TA98 with metabolic activation, the methanol extract of sample 003 induced 197 net revertants, while sample 005 induced 436 net revertants. In the prophage induction assay, with activation, the hexane:acetone extract of sample 003 induced a genotoxic response that was slightly lower than that observed with sample 005. The estimated incremental carcinogenic risk for ingestion of PAHs was 1.5E – 3 for sample 003, while for sample 005 the estimated risk was 1.5E – 2. Thus, the sample which induced the maximum response in both bioassays also had the highest estimated cancer risk. However, the frequency of PAH-DNA adducts in both skin and liver tissues was appreciably higher with sample 003 than with sample 005. A combined testing protocol, using both biological and chemical analysis, therefore provides more accurate information from which to assess risk than the use of either method alone.

底質のＡｍｅｓ変異原性モニタリングのための試料調製方法とその応用

The Salmonella/microsome assay (Ames test) is a useful method for screening mutagens in complex environmental samples such as river water and sediment. However, no quantitative and generalized method to evaluate the mutagenicity of sediment has yet been established. In this study, the simple and effective method for monitoring the mutagenicity in sediment was investigated, and applied to river sediments and drainage sediments collected in Kanagawa prefecture.The following method was proved to be simple, sensitive and giving good results for the Ames test for various sediment samples : sediment samples were extracted twice using ultrasonic agitation with dichloromethane/ethanol (4 : 1) and then dichloromethane, and crude extracts were dissolved to n-hexane/ethylacetate (95 : 5) and separated to the insoluble fraction, the passing fraction which should be refined by Cu column from a silica-cartridge, and the eluted fraction from the silica-cartridge with ethylacetate/methanol (1 : 1).Eleven samples in the tested 13 samples showed the mutagenicities to TA98 or TA100 on addition of S9mix, and the ranges were 10-792 net revertants to TA98 and 13-1,990 net revertants to TA100 per g dry sediment. Fractionation procedure was effective not only for reducing interference caused by coexisting substances and lowering the detection limits of mutagenicity but also for getting information on characteristics of the mutagens contained in the sediment. The developed preparation method was confirmed to be adaptable to various sediment samples.

Chlorination byproducts of 4,4′-methylene dianiline(MDA) and their mutagenicity

A mutagenicity study was carried out on 4,4′-methylene dianiline(MDA) an epoxy resin hardener and chlorinated MDA using an Ames Salmonella/microsome assay. As reaction products of MDA with chlorine in the water six spots were observed by thin layer chromatograph(TLC) and high performance liquid chromatography(HPLC) analysis revealed eighteen peaks on the HPLC chromatogram. MDA exhibited mutagenic activity for strain TA100 in the presence of S9 mix. In the mutagenicity test of chlorinated MDA corresponding to 7.8∼250 μg/plate, at 250 μg/plate the specimen showed mutagenic activity for strain TA98 without S9 mix, but more diluted specimens had no mutagenicity. At 31 and 250 μg/plate, the specimen showed weak mutagenic activity for the strain TA98 in the presence of S9 mix. The strain TA100 without S9 mix was killed on the culture plate with 250 μg/plate. However strong mutagenicity such as net revertant colonies 1270 per plate was obtained with the diluted specimen of 7.8 μg/plate. At 31 μg/plate, the specimen showed mutagenic activity for the strain TA100 with S9 mix. The mutagenicity test of five fractionated specimens from the TLC analysis, spot No. 1 and No. 2 showed no mutagenicity for strain TA98 and TA100 with and without S9 mix. The specimen of Spot No. 3 exhibited weak mutagenic activity for strain TA98 without S9 mix, the specimen of spot No. 4 at 125 μg/plate showed mutagenicity for strain TA98 and TA100 with and without S9 mix, and diluted samples showed high mutagenic activity for strain TA100 with and without S9 mix. Several newly identified mutagenic substances were found to be produced by the chlorination of MDA. GC/MS analysis of chlorinated MDA revealed eleven compounds on the chromatogram. Benzaldehyde, benzoquinones and chloroanilines were identified. However, the structural formula of the highly mutagenic substances could not be determined by GC/MS.

The Effect Of Preozonation, Ozone/Hydrogen Peroxide Treatment, And Nanofiltration On The Removal Of Organic Matter From Drinking Water

Abstract Pilot scale experiments were performed to evaluate the ability of ozonation, ozone/hydrogen peroxide treatment and nanofiltration to reduce levels of organic matter, mutagenicity, total adsorbable halogens, color and turbidity from purified and bank-filtered surface water rich with humic material. Ozonation and ozone/hydrogen peroxide decreased the amount of organic material from drinking water by about 20 percent measured as TOC and CODMn. Color and turbidity level reductions were 49 and 11 percent, respectively. Ozonation reduced the AOX concentrations formed during postchlorination from 150 μgL−1 to 75 μgL−1. The addition of hydrogen peroxide further improved the removal to 37 and 26 μgL−1 depending on the ratio of H2O2/O3. The mutagenicity reduction followed the same pattern: without ozonation the chlorination-derived mutagenicity was 1,450 net revertant L−1 after the ozonation 700 and after the H2O2/O3 treatment from <100 to 400 net revertant L−1 depending on the H2O2/O3 ratio. Nanofiltratio...

水道水のＡｍｅｓ変異原性に関する研究 第２報 高性能吸着剤を用いた変異原性物質の濃縮・回収方法

Sample preparation method using a cartridge (Sep-Pak Plus Long) in which 2ml of a superior adsorbent CSP800 was packed was optimized for convenient and quantitative Ames mutagenicity assay of tap water.Mutagenicity activities were decreased considerably by free chlorine reduction by sodium sulfite. Recovery of mutagens from tap water augmented much in lower pH. There was no influence of adsorbing flowrate in the range from 8 to 80ml·min-1 on the recovery of mutagens. Therefore, it is optimal that tap water adjusted at pH2 without free chlorine reduction is fed to the cartridge at a flowrate of c.a. 50ml·min-1. By this method, the mutagens less than 8,000 net revertants.·l-1 could be recovered completely from 2l of water sample, and those more than that could be recovered from 1l. The adsorbed mutagens could be desorbed by 2ml-dimethyl sulfoxide (DMSO) at 0.15 ml·min-1 in upper flow.This newly developed method requires only 1 or 2l of water sample and less than an hour to concentrate mutagens with over 90% recovery. Furthermore, the desorbing DMSO solution can be used directly to Ames assay without solvent exchange, because concentration factor of mutagens by this method attains 500 or 1,000 times.

Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation

Naturally occurring dipeptides, cholecystokinine (CKK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10–30 min with 40 m m-nitrite (pH 3.5, 37°C), and the resultant products examined for mutagenicity in Salmonella typhimurlum TA100. Specific mutagenicities (net revertants per μmol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/μmol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/μmol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/μmol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/μmol, respectively), The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/μmol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

変異原性試験による廃棄物埋立処分場浸出水中の微量汚染物質サーベイランス

Mutagenic activity was examined on dichloromethane extracts of leachate samples collected from a municipal solid waste landfill by means of the Ames Salmonella assay. Mutagenicity was found among 51% of 101 leachate samples and most of the positive samples showed mutagenicity for both strains TA100 and TA98 irrespective of metabolic activation by S9, indicating that the active ingredients consisted mainly of direct mutagens. A four-year survey revealed that mutagenic activity in landfill leachate was high (200-1,500 net revertants·l-1) during the operation of the landfill, whereas it decreased gradually after the landfill was closed. Detailed study on clesed landfill sites showed that younger landfill sites discharged leachate with higher mutagenic activity. No correlation was observed between mutagenic activity and conventional water pollution indicators such as BOD, COD, TOC, DO, and water temperature except for T-N which showed a weak positive correlation. These results demonstrated the significance of the mutagenicity assay for evaluating the possible presence of micropollutants in landfill leachate.

Bacterial mutagenicity and acute toxicity of solvent and aqueous extracts of soil samples from an abandoned chemical manufacturing site

AbstractUsing a grid pattern, a total of 50 soil borings (DCT 1 through DCT 50) were collected from an abandoned chemical manufacturing facility. Split samples were obtained from six cores collected from a centralized location near a former wastewater lagoon. An additional sample from the 2.1‐ to 2.4‐m depth of core DCT 20 was obtained as representative of the waste present on site. Three subsamples from each of the six cores or a total of 18 samples were sequentially extracted with methylene chloride and methanol for testing in mutagenicity and acute toxicity assays. A separate batch extraction with water was conducted and evaluated, using both chemical and biological test methods. The maximum mutagenic response was 3,237 net revertants induced with metabolic activation by 1 mg of the methanol extract of the 0‐ to 0.6‐m depth at sampling location DCT 32. The water extract of the 0‐ to 0.6‐m depth at sampling location DCT 32 induced 737 net revertants per mg. At sampling location DCT 24, the water extract induced 0.74, 1.32, and 2.5 toxic units in the Microtox® assay and 935, 69, and 39 net revertants per mg in the mutagenicity assay at the 0‐ to 0.3‐, 1.2‐ to 1.4‐, and 2.1‐ to 2.4‐m depths, respectively. The chemical analysis of the samples from location DCT 24 indicated that the concentration of 2,3,6‐trichlorobenzene acetic acid ranged from 16 ppm in the 0‐ to 0.3‐m depth and to 0.95 ppm and below the detection limits at the 1.2‐ to 1.4‐ and 2.1‐ to 2.4‐m depths. The methanol extract of the soil from the 0‐ to 0.3‐m depth induced 19.2 toxic units in the acute toxicity assay and 1,467 net revertants in the mutagenicity assay. These results suggest that the most accurate site assessment is obtained when a water extract is evaluated to measure leaching potential and a solvent extract is evaluated to determine the relative total hazard of the sample. In addition, the combined use of chemical analysis, mutagenicity, and acute toxicity bioassays provides more accurate information from which to make a risk assessment.

Bacterial mutagenicity of leachate water from municipal sewage sludge‐amended soils

AbstractThe objective of this study was to monitor the mutagenic potential of leachate from soils amended with municipal sewage sludge. The Salmonella/microsome assay was employed to monitor the mutagenicity of the organic extract of leachate collected from sludge‐amended lysimeters containing undisturbed monoliths of Weswood silty clay (Fluventic Ustochrept) and Padina loamy sand (Grossarenic Paleustalf) soils. The sludge was applied to 18 barrel‐sized lysimeters, while 6 unamended lysimeters served as controls. The leachate was concentrated on a combined XAD‐2/XAD‐7 resin, and the adsorbed organics were eluted with acetone. The leachate extracts were tested in the standard plate incorporation assay using strain TA98 with and without metabolic activation. Nine of 26 leachate extracts induced a positive response in the bioassay. The maximum specific activity was 207 net revertants per 10 mg induced without metabolic activation in the Weswood soil at the highest application rate (150 Mg/ha). The results suggest that land application of municipal sewage should be carefully managed to prevent leaching of mutagens into ground water.

Metabolism and bacterial mutagenicity of binary mixtures of benzo(a)pyrene and polychlorinated aromatic hydrocarbons

The results of both the Salmonella/microsome mutagenicity assay and high-performance liquid chromatography (HPLC) analysis were used to evaluate the interactions of binary mixtures of benzo(a)pyrene (BAP) and several different polychlorinated aromatic hydrocarbons. Binary mixtures of either 2-nitro-3,7,8-trichlorodibenzo-p-dioxin (2NTCDD) or pentachlorophenol (PCP) with BAP produced synergism, whereas strictly additive effects were observed with mixtures of octa- or hepta-chlorodibenzo-p-dioxin and BAP. At a dose of 50 micrograms per plate, BAP induced 120 total revertants, whereas the binary mixture of BAP and PCP induced 303 total revertants. The binary mixture of BAP at 1 microgram per plate and 2NTCDD at 0.5 microgram per plate induced 261 net revertants, whereas BAP alone induced 42 net revertants. HPLC analysis of the mixtures indicated that preincubation of BAP with 2NTCDD increased the quantity of benzo(a)pyrene-7,8-dihydrodiol, and 9,10-dihydrodiol metabolites detected. The data suggest that nonmutagenic components of a complex mixture may alter the metabolism of promixate mutagens. Thus, in the present study, 2NTCDD appears to have inhibited the detoxication of BAP metabolites.