Genotoxicity of water extracts from the River Yamuna at Mathura,India

Water samples were collected from the River Yamuna at Mathura, India, and concentrated by using XAD resins (Amberlite XAD-4 and XAD-8) and liquid-liquid extraction procedures. The genotoxicities of the extracted water samples were evaluated by the Ames Salmonella/mammalian microsome test, DNA repair of defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain, both with and without metabolic activation. The XAD-concentrated water samples collected in the summer showed maximum mutagenic responses compared with those collected in other seasons, whereas the liquid-liquid extracted samples exhibited maximum mutagenic activity during the postmonsoon season. The damage brought about during DNA repair of defective mutants in the presence of XAD-concentrated water samples was found to be remarkably high compared with the liquid-liquid extracted water samples at a dose level of 20 microL/mL of culture. All the mutants invariably exhibited significant decline in their colony-forming units compared with their isogenic wild-type counterparts. Survival was decreased by 86.7% and 65.1% in the polA(-) strain after 6 h of treatment with XAD-concentrated and liquid-liquid extracted water samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with test samples. The damage was more pronounced in lexA mutants when the phage was treated with XAD-concentrated samples. The recA, lexA, and polA mutants of E. coli K-12 were found to be sensitive to the test samples, suggesting damage to the DNA of exposed cells as well as to the role of recA(+), lexA(+), and polA(+) genes in coping with the hazardous effect of the pollutants. The results demonstrated substantial genotoxicity and mutagenicity in the water samples tested.     

Evaluation of mutagenicity of wastewater in the vicinity of pesticide industry

Pesticide industrial wastewater samples were taken from the Chinhat industrial area nearby Lucknow city, India. GC–MS analysis revealed the presence of pesticides lindane, α-endosulfan, β-endosulfan, chlorpyriphos, monocrotophos, dimethoate and malathion. A pesticide mixture and wastewater extracts were studied to determine the mutagenicity by Ames Salmonella test, survival of DNA repair defective E. coli K-12 mutants and bacteriophage λ systems. Wastewater samples were concentrated with XAD-resins as an adsorbent and liquid–liquid extraction procedure. The XAD concentrated sample exhibited maximum mutagenic activity in comparison to liquid–liquid extracted sample. TA98 strain was the most responsive strain for both test samples with (+S9) and without (?S9) metabolic activation, while other strains exhibited weak response. A significant decline of DNA repair defective E. coli K-12 mutants, bacteriophage λ was observed with test samples in the survival. The intracellular damage was highest when treated with XAD concentrated sample as compared to liquid–liquid extract after 6 h treatment.     

Genotoxicity of some wastewaters in India

Heavy metal concentrations in wastewaters from the industrial estate of Aligarh city (U.P.) India were determined. The analysis of test samples revealed significantly higher levels of Fe, Zn, Cu, Cr, and Ni compared with the pure domestic samples. Raw wastewaters were screened for mutagenic potential using the Ames Salmonella/microsomal test. Mutagenic activity was observed with industrial as well as domestic waste samples. Salmonella typhimurium strains TA102 and TA104 were the most sensitive both in the absence and presence of S9 fraction. A significant decrease in the survival of DNA repair defective Escherichia coli mutants recA, lex A, and polA was observed as compared to their wild-type counterparts in the presence of wastewater. © by John Wiley & Sons, Inc.     

Genotoxicity of quinocetone,cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His⁺ mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His⁺ mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ⩾10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.     

Mutagenicity of industrial wastewaters collected from two different stations in northern India

Mutagenicity of wastewaters taken from two different cities was compared by means of Ames plate test and Ames fluctuation test. TA100 and TA98 strains of S. typhimurium exhibited the highest sensitivity against the Saharanpur sample (SWW) in terms of the slope (m) of the dose-response curve in the plate incorporation assay. However, the most sensitive strain against the test samples from Aligarh (AWW) was TA98. Interestingly, TA100 and TA98 strains also displayed the highest susceptibility towards the samples from Saharanpur in the fluctuation test. However, TA102 and TA100 responded maximally to AWW in this bioassay. Interestingly, S9 supplementation resulted in the decline in mutagenic potential of SWW contrary to significant increase with AWW by both the tests. Both samples were found to generate different types of ROS as predominant species. While SWW were shown to generate a high concentration of superoxide radicals and hydrogen peroxide, hydroxyl radicals were predominantly occurring in AWW. From our result, we conclude that both the test water samples were highly genotoxic. In view of the complementary nature of these two testing systems, we recommend both bioassays for the genotoxicity assessment of complex water samples.     

Simplified protocol for evaluating the genotoxic risk of hospital wastewater

To evaluate the genotoxicity of hospital wastewater, we drew up a simplified protocol based on two well-known tests: the SOS chromotest and the Ames fluctuation test. Three concentrations of wastewater samples were directly tested without extraction or concentration. By fixing three significance levels in genotoxicity response for each test, we could classify the samples in five categories ranging from nongenotoxic to highly genotoxic. This simplified classification thus constitutes an invaluable help in making the nonscientist public aware of the genotoxic risk of hospital wastewater and can be applied in a screening approach to chemicals in the environment.     

Absence of mutagenic activity in Salmonella and of clastogenic activity in CHO cells of Caramel Colours I, II, III and IV.

A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed.     

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.     

Tofisopam--evaluation of mutagenic and genotoxic properties

The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.     

Use of four short-term tests to evaluate the mutagenicity of municipal water

Some ways in which four short-term tests may be used to evaluate the mutagenicity of drinking water were explored by testing raw and treated water from Lake Bloomington, which serves the town of Bloomington, Illinois (population, 44,000). The water was collected from February 1976 to October 1977 and was concentrated by evaporation or by use of XAD-2 resin. The water was tested for the ability to induce reverse mutation in a prokaryote, Salmonella typhimurium; forward mutation in a mold, Neurospora crassa; mitotic gene conversion in a yeast, Saccharomyces cerevisiae; and reverse mutation in maize, Zea mays. Because of the large number of water samples (54) and the limited amounts of the samples, it was not possible to test all samples in all four tests by all the protocols. Thus, the sensitivities of the four tests to potential mutagens in the water samples could not be rigorously compared. However, the results do show that lake and tap water samples collected during 1976 were toxic but not mutagenic in N. crassa and neither toxic nor genotoxic in S. cerevisiae; lake water collected during 1977 was mutagenic in one line of Z. mays and slightly mutagenic in S. typhimurium strain TA1536 in the presence of rat liver S9. The results suggest that tests that detect a variety of genetic end points should be used when testing complex mixtures such as drinking water. The advantages and disadvantages of the tests and protocols are discussed in terms of their applicability to the study of the mutagenicity of drinking water.     

Genotoxic effects of N-nitrosodicyclohexylamine in isolated human lymphocytes

Dicyclohexylamine x nitrite is classified as an "experimental equivocal tumorigenic agent" by the National Toxicology Program. Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine x nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine x nitrite. Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes. Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography. Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H- and 13C-NMR. More than 97% purity was achieved. Stability and availability in the solvent were checked by GC/MS. N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15-100 micrograms/ml (= 71.4-476.2 microM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments. For the Ames test, arochlor-1254-, beta-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104. No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 micrograms (= 1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5. In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes. With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.     

The assessment of genotoxic effects of wastewater from a fertilizer factory

In this study we investigated cytotoxic, mutagenic and genotoxic effects of different concentrations of wastewater from the phosphoric gypsum depot near the factory for fertilizing agents 'INA Petrokemija' (Kutina, Croatia). The Ames test was performed on Salmonella typhimurium TA98 and TA100 strains, in the presence of S9 mix, glutathione and buffer, respectively. Cytotoxicity was studied on human laryngeal carcinoma cells (HEp2) and human cervical cells (HeLa). The level of lipid peroxidation in these two cell lines was evaluated in parallel. To establish the levels of primary DNA damage, the alkaline comet assay was performed on treated human peripheral blood leukocytes. No mutagenic effects of phosphoric gypsum on Salmonella typhimurium strains in the presence of S9 mix, GSH or PBS were observed. However, strong cytotoxic effect was observed on both human cell lines when they were treated with different concentrations of wastewater. Lipid peroxidation was induced and increased by prolonged time of incubation, highlighting that the damage was not repaired, but increased with the time of incubation. The results of the alkaline comet assay indicate significant DNA damaging potential of wastewater for human leukocytes. Since phosphoric gypsum transport water in its present composition and acidity is highly toxic and acts as prooxidant, causing free radicals formation and DNA damage, urgent neutralization/purification of the wastewater to a level acceptable for disposal into the environment is mandatory.     

Minor contribution of alternariol, alternariol monomethyl ether and tenuazonic acid to the genotoxic properties of extracts from Alternaria alternata infested rice

Alternaria spp. are known to form a spectrum of secondary metabolites with alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TA) as the major mycotoxins with respect to quantity. In the present study we investigated the contribution of these compounds for the DNA damaging properties of complex extracts of Alternaria spp. infested rice. Five different Alternaria strains were cultured on rice and analyzed for their production of AOH, AME, ALT and TA. The extracts of two strains with distinctly different toxin profiles were selected for further toxicological analysis. An extract from A. alternata DSM 1102 infested rice, found to contain predominantly TA, exhibited substantial DNA strand breaking properties in cultured human colon carcinoma cells in the comet assay, whereas TA as a single compound did not affect DNA integrity up to 200μM. An extract of A. alternata DSM 12633 infested rice, containing in comparable proportions AOH, AME and TA, exceeded by far the DNA damaging properties of the single compounds. In contrast to AOH, AME and TA, both selected extracts induced an increase of DNA modifications sensitive to the bacterial repair enzyme formamidopyrimidine DNA glycosylase (FPG) in the comet assay, indicative for oxidative DNA damage. Toxicity-guided fractionation of the DSM 12633 extract confirmed that these effects were not caused by AOH, AME or TA. Taken together, the mycotoxins AOH, AME and TA, representing the major mycotoxins with respect to quantity in A. alternata infested food, play only a subordinate role for the genotoxic properties of complex extracts and appear not to be involved in the induction of FPG sensitive sites.     

Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage

Because of the widespread use of pesticides for domestic and industrial applications the evaluation of their genotoxic effects is of major concern to public health. Although various experimental data have provided evidence that pesticides can possess genotoxic properties in animals and in in vitro test systems after acute and chronic exposure, the information on the genotoxic effects of some of pesticides is limited and inconsistent. In the present study, the genotoxic potential of commonly used pesticides (i.e., dimethoate and methyl parathion from the organophosphate class, propoxur and pirimicarb from carbamates, and cypermethrin and permethrin from pyrethroids) have been evaluated. The genotoxic effects of these substances were examined using the single cell gel electrophoresis (comet) assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 10, 50, 100 and 200 µg/ml concentrations of the test substances for 0.5 h at 37°C and DNA damage was compared with that obtained in lymphocytes from the same donor not treated with substances. Hydrogen peroxide, 100 µM, was used as a positive control. Within the concentration ranges studied, no significant cytotoxic effects were observed. Dimethoate and methyl parathion at 100 and 200 µg/ml; propoxur at 50, 100 and 200 µg/ml, and pirimicarb, cypermethrin and permethrin at 200 µg/ml significantly increased DNA damage in human lymphocytes.     

Mutagenicity and clastogenicity of native airborne particulate matter samples collected under industrial,urban or rural influence

Airborne particulate matter has recently been classified by the IARC as carcinogenic to humans (group 1). However, the link between PM chemical composition and its carcinogenicity is still unclear. The aim of the present study was to evaluate and to compare genotoxic potencies of 6 native PM samples collected in spring–summer or autumn–winter, either in industrial, urban or rural area. We evaluated their mutagenicity through Ames test on YG1041, TA98, and TA102 tester strains, and their clastogenicity on human bronchial epithelial BEAS-2B cells using comet assay, γ-H2AX quantification, and micronucleus assay. Ames test results showed a strong positive response, presumably associated with nitro-aromatics content. In addition, at least 2 positive responses were observed out of the 3 genotoxicity assays for each of the 6 samples, demonstrating their clastogenicity. Our data suggest that PM samples collected in autumn–winter season are more genotoxic than those collected in spring–summer, potentially because of higher concentrations of adsorbed organic compounds. Taken together, our results showed the mutagenicity and clastogenicity of native PM_2.5 samples from different origins, and bring additional elements to explain the newly recognized carcinogenicity of outdoor air pollution.     

Fish hepatic micronuclei as an indication of exposure to genotoxic environmental contaminants

An In vivo assay protocol for detecting hepatic micronuclei in fish was performed to evaluate whether this genotoxic response could serve as a bioindicator of environmental exposure to genotoxic substances. The incidence of hepatic micronuclei was compared in brown bullheads (Ameiurus nebulosus) with external lesions collected from a contaminated site and fish showing no externally visible pathologies collected from reference sites. Laboratory experiments were performed by exposing hatchery-raised rainbow trout (Onchorhynchus mykiss) to a pulp mill effluent extract (XAD-4-NaOH) with known genotoxic activity. Both bullhead and trout were injected with allyl formate to induce hepatic necrosis and regenerative proliferation of hepatocytes prior to examining the liver tissue for micronuclei. An elevated incidence of hepatic micronuclei was observed in Hamilton Harbour bullheads showing visible lesions relative to the micronucleus incidence in bullheads from reference sites with no external pathologies. Similarly, rainbow trout exposed to pulp mill effluent extracts exhibited an elevated incidence of hepatic micronuclei compared to controls. These results suggest that the hepatic micronucleus assay could serve as a valuable biomonitoring tool for assessing the impacts of genotoxic environmental contaminants on fish. ©1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12 : 217–222, 1997     

乙双吗啉的致突变作用

目的:研究乙双吗啉的遗传毒性.方法:乙双吗啉5,10和15 mg·kg~(-1),腹腔注射观察诱发的小鼠骨髓染色体/染色单体畸变;应用Ames试验观察对测试菌株TA97,TA98,TA100,TA102的诱变作用.结果:乙双吗啉显著诱发小鼠骨髓染色体/染色单体畸变,其诱发的畸变细胞率(ACF)显著增加(P<0.01);在不加S9条件下,乙双吗啉对TA98,TA102有一定的诱发回复突变的作用.结论:乙双吗啉是一种遗传毒物质.     

Mutagenic,genotoxic and cytotoxic studies of invasive corals Tubastraea coccinea and Tubastraea tagusensis

The high diversity of species in the marine environment gives rise to compounds with unique structural patterns not found as natural products in other systems and with great potential for pharmacological, cosmetic and nutritional use. The genus Tubastraea (Class Anthozoa, Order Scleractinia, Family Dendrophylliidae) is characterized as a hard coral without the presence of zooxanthellae. In species of this genus alkaloids derived from the compound aplysinopsin with pharmacological activity are known. In Brazil T. coccinea and T. tagusensis are characterized as non-indigenous and invasive and are currently found along the Brazilian coast, from Santa Catarina to Bahia states. This study aims to analyze the mutagenic, cytotoxic and genotoxic potential of methanolic and ethanolic extracts from T. coccinea and T. tagusensis collected in Ilha Grande Bay, Rio de Janeiro state, Brazil. Bacterial reverse mutation assay on the standard strains TA97, TA98, TA100, TA102 and TA104, in vitro micronucleus formation test and colorimetric assays for cytotoxic signals on the cell lines HepG2 and RAW264.7 were used. We also synthesized an oxoaplysinopsin derivate alkaloid (APL01) for comparative purposes. No mutagenic (250; 312.5; 375; 437.5 and 500 μg/plate) or genotoxic (0.05; 0.5; 5.0; 50 and 500 μg/mL) effects were observed in any sample tested for all measured concentrations. Cytotoxic responses were observed for eukaryotic cells in all tested samples at 500 and 5000 μg/mL concentrations. Cytotoxicity found in the WST-1 assay was independent of the metabolism of substances present in samples compositions. The cytotoxicity observed in the LDH release assay depended on metabolism.     

Mutagenicity analysis of water samples from Seydisuyu (Kirka, Turkey) stream under the influence of boron production complex.

Kirka region in Turkey is naturally high in boron content. There is also a boron production complex in our study area. Although boron was found to be neither genotoxic nor carcinogenic, mutagenicity of boron can still be discussed. In this connection, the aim of the present study was to evaluate the potential mutagenicity of Seydisuyu stream water in Kirka due to high boron content. Salmonella/microsome test was performed with and without metabolic activation. Total boron contents were determined by inductively coupled plasma optical emission spectrometer (ICP-OES). IRA 743 extracts of water samples from four stations of Seydisuyu stream were tested for mutagenicity in Salmonella typhimurium strains TA 98 and TA 100, and environmental boron toxicity was discussed.     

Genotoxicity of dicrotophos, an organophosphorous pesticide, assessed with different assays in vitro

Dicrotophos is a systemic insecticide with a wide range of applications. We investigated the genotoxicity of dicrotophos using the Ames test, the chromosome aberration test in CHO-K1 cells, and the comet assay in the Hep G2 cells, while this chemicals' toxicity to both the cell lines was evaluated with the MTT assay. Results showed that dicrotophos did not show any cytotoxicity to CHO-K1 cells, whereas it was cytotoxic to HepG2 cells incubated for 24 h but not for 2 h. For genotoxicity of dicrotophos, a significant change in the numbers of bacterial reveratnts using Salmomella typhimurium TA97a, TA98, TA100, TA102, and TA1535 as the tester strains, an increase in the frequencies of chromosome aberration in CHO-K1 cells, and an induced DNA damage in HepG2 cells were observed, indicating that dicrotophos was genotoxic in these three performed assays. From this study, we provide further evidence towards of genotoxic effects of dicrotophos.