Preincubation Method Research Articles

Antimutagenic potential of the Thai herb, Mucuna collettii Lace

Mucuna collettii Lace is a Thai herb with a long record of consumption among mature Thai males for the promotion of sexual potency. The mutagenic and antimutagenic potentials of Mucuna collettii extract were carried out by using the Ames test pre-incubation method in the presence and absence of S9 mixture. Salmonella typhimurium strains TA 98 and TA 100 were applied as the tester strains. Prior to mutagenic and antimutagenic tests, the survival of the tester strains was performed by treating with the plant extract. Results showed Mucuna collettii extract exhibited strong cytotoxic effects in a dose-dependent manner. Toxicity of the plant was confirmed in mice in which negative adverse effect was found in kidney, uterus, ovary, and testis. Mucuna collettii extract in the presence and absence of S9 mixture was negative for mutagenic Ames test. Mucuna collettii extract in the presence and absence of S9 mixture was positive for antimutagenic Ames test towards either one or both of the tested mutagens: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and benzo(a)pyrene. The antimutagenic activity of the plant extract was confirmed in rec-assays. Micronucleus test demonstrated that Mucuna collettii extract at high dose and a long incubation time could induce micronucleus formation in tested animals, but less than the response of the positive control. The overall mutagenic and antimutagenic assays are further evidences for the antimutagenic potential of Mucuna collettii.

The effects of wounding type, preculture, infection method and cocultivation temperature on the Agrobacterium‐mediated gene transfer in tomatoes

AbstractTo reduce the complexity of Agrobacterium‐mediated gene transfer in tomatoes, effects of various parameters, such as shoot regeneration medium (SRM), wounding type, infection method, preculture and cocultivation temperature, have been evaluated. Transformation frequency was analysed by the Agrobacterium strain LBA4404, harbouring a recombinant binary expression vector pIG121Hm‐GS, which contained the glutathione synthetase gene under the control of the CaMV 35S promoter. The transformation frequency was highly dependent on the wound type of the explants, the infection method and the cocultivation temperature. On the other hand, a commonly used, preincubation method of the explants, on the preculture medium, did not show any significant improvement regarding transformation frequency. Optimal transformation frequency was observed when fresh perforated cotyledonary explants were directly infected with a bacterial solution A, followed by cocultivation at 24°C in a coculture medium for 2 days and subsequent shoot regeneration on a selective SRM1. The presence of transgene, in putative transgenic plants, was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. By using the most effective treatment from each category, an average of 20.7% kanamycin‐resistant and PCR‐positive shoots were recovered from the three tomato cultivars examined. The optimisation of these parameters may offer a simple, consistent, efficient and much less laborious protocol for tomato transformation.

Mutagenicity of benzyl chloride in the Salmonella/microsome mutagenesis assay depends on exposure conditions

Benzyl chloride (BCl) is a clear yellowish, volatile liquid that is widely used as an intermediate for the production of benzyl alcohol and benzyl compounds used in perfumery, dyes and pharmaceuticals. In previous studies BCl has shown weak and inconsistent mutagenicity in the Salmonella/microsome mutagenesis assay (Ames test). The aim of the present study was to investigate the potential mutagenic activity of BCl using modifications of the standard Ames test in order to adapt the method to the volatile nature of the test compound. Tests were performed using (a) the standard plate-incorporation method, (b) incubation of the treated plates in closed containers, (c) a vaporization–diffusion method to expose Ames test plates to volatilised BCl and (d) the pre-incubation method. Using the standard plate-incorporation method, BCl showed no (in the absence of metabolic activation) or very weak (in the presence of metabolic activation) mutagenic activity in Salmonella typhimurium tester strain TA 100. The use of the pre-incubation method did not improve detection of mutagenic activity of BCl. The use of closed containers significantly increased the response, but the most marked response was obtained by testing BCl in volatilised form in the vaporization–diffusion method. Using the latter approach there appeared to be less toxicity of the BCl treatments to the tester bacteria. Our findings suggest that BCl may show greater mutagenic activity in the gaseous phase. This work underlines the importance of using appropriate methods for the evaluation of volatile compounds. The modifications described here are easy to realize in practice and should prove useful for the investigation of other volatile materials or atmospheric contaminants.

MUTAGENICITY OF WATER-SOLUBLE FePt NANOPARTICLES IN AMES TEST

A mutagenicity test was conducted on water-soluble FePt nanoparticles capped with tetramethylammonium hydroxide in a bacterial reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli strain WP2uvrA/pKM101, with and without metabolic activation by S9 mix in the preincubation method. Mutagenicity was weakly positive in the TA100 strain without S9 mix (maximum specific activity was 61.6 revertants/mg), but negative in other cases.

Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase

We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the αβα-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL = 590 at 25 °C, pH = 7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the β-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.

Study on the mutagenicity and antimutagenicity of β-ionone in the Salmonella/microsome assay

β-Ionone (BIO) is a degraded (C 13) sesquiterpenoid compound found in a variety of edible and aromatic plants. BIO and other ionone derivatives have been used in fragrance products and as flavoring food additives. In this study we investigated the mutagenic and antimutagenic activities of BIO using the Salmonella/microsome assay. Mutagenicity was evaluated by two tests with Salmonella typhimurium strains TA100, TA98, TA97a and TA1535, without and with addition of S9 mixture. A first assay was performed by the plate incorporation procedure and a confirmation test by the pre-incubation method. In either test, no increase in the number of his + revertant colonies over the negative (solvent) control values was noted with any of the four tester strains thereby indicating that BIO was not genotoxic in the Salmonella assay. Antimutagenic activity was investigated by testing (by the plate incorporation method) different non-toxic doses of BIO against one or more non-toxic doses of direct-acting (sodium azide: SA, 4-nitroquinoline- N-oxide: 4-NQNO, 2-nitrofluorene: 2-NF and nitro- o-phenylenediamine: NPD) as well as indirect-acting (cyclophosphamide: CP, benzo[ a]pyrene: B[ a]P, aflatoxin B1: AFB1, 2-aminoanthracene: 2-AA, and 2-aminofluorene: 2-AF) mutagens. BIO did not alter the effects of any direct-acting mutagen or B[ a]P and 2-AF. Mutagenic effects of AFB1 and CP, however, were markedly and dose-dependently antagonized by BIO. It has been reported that, in the rat liver, activation of B[ a]P and 2-AF depend on CYP1A1 activity, and that CYP2B subfamily is involved in the metabolic activation of CP and AFB1. It has also been described that BIO is a potent inhibitor of CYP2B1/2 and a weaker inhibitor of CYP1A1. Therefore, antagonism of CP-and AFB1-induced mutagenic effects by BIO could have been mediated—at least in part—by the inhibition of CYP2B enzymes.

Modified bacterial mutation test procedures for evaluation of peptides and amino acid-containing material

Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.

Salmonella/human S9 mutagenicity test: a collaborative study with 58 compounds

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems. The bacterial test strains consisted of Salmonella typhimurium TA100, TA98 or YG7108. The data indicated that the mutagenicity of chemicals in the rat and human liver S9 fractions varied considerably, depending on the chemicals in question. In addition, a large inter-individual diversity in the mutagenic response to mutagens, depending on the chemical structures of the mutagens, was also demonstrated using two selected human S9 fractions. Most of the mutagens tested in this study (75%; 36 out of 48 compounds that were judged to be mutagenic in at least one S9 fraction) were less mutagenic in the presence of the two human S9 fractions than in the presence of the rat S9 fraction. On the other hand, the other compounds (25%), including some aromatic amines and nitrosamines, showed a more potent mutagenicity in the presence of either one of the two human S9 fractions than in the presence of the rat S9 fraction. These data strongly suggest that the use of human liver S9 fraction in mutagenicity testing systems may be useful for a better understanding of the mutagenic effects of chemicals on humans.

Antiamoebic and toxicity studies of a carbamic acid derivative and its therapeutic effect in a hamster model of hepatic amoebiasis.

Amoebiasis is an important public health problem in developing countries. Entamoeba histolytica, the causative agent of amoebiasis, may develop resistance to nitroimidazoles, a group of drugs considered to be the most effective against this parasitic disease. Therefore, research on new drugs for the treatment of this common infection still constitutes an important therapeutic demand. In the present study we determined the effects of a carbamate derivative, ethyl 4-chlorophenylcarbamate (C4), on trophozoites of E. histolytica strain HM-1:IMSS. C4 was subject to various toxicity tests, including the determination of mutagenicity for bacterial DNA and changes in the enzymatic activities of eukaryotic cells. Genotoxicity studies were performed by the mutagenicity Ames test (plate incorporation and preincubation methods) with Salmonella enterica serovar Typhimurium, with or without metabolic activation produced by the S9 fraction of rat liver. C4 toxicity studies were performed by measuring enzymatic activity in eukaryotic cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan test with Fischer 344 rat hepatocytes. C4 did not induce either frame-shift mutations in S. enterica serovar Typhimurium TA97 or TA98 or base pair substitutions in strains TA100 and TA102. The compound was not toxic for cultured rat hepatic cells. Trophozoites treated with 100 microg of C4 per ml were inhibited 97.88% at 48 h of culture; moreover, damage to the amoebae was also confirmed by electron microscopy. The antiamoebic activity of C4 was evaluated by using an in vivo model of amoebic liver abscess in hamsters. Doses of 75 and 100 mg/100 g of body weight reduced the extent of the amoebic liver abscess by 84 and 94%, respectively. These results justify further studies to clearly validate whether C4 is a new suitable antiamoebic drug.

Final Report of the Amended Safety Assessment of Dioscorea Villosa (Wild Yam) Root Extract1

Dioscorea Villosa (Wild Yam) Root Extract is an extract of the rhizomes of the wild yam, D. villosa. A manufacturing process was described in which cut up and ground rhizomes are combined with an eluant (e.g., oleyl alcohol), the plant material precipitated with addition of a miscible solvent, washed, and redissolved in the original eluant. The extract contains glycoside and steroidal saponins (< or =0.4%), diosgenin (< or =3.5%), alkaloids, tannins, phytosterols, and starch. Levels of heavy metals, 1,4-dioxane, chloroform, methylene chloride, trichloroethylene, and benzene are reported to be below limits of detection. Although only one use was reported to the U.S. Food and Drug Administration (in a body and hand preparation), industry reported uses in body and hand creams, lotions, powders, and sprays at a concentration of 0.00001% (equivalent to 0.000002% plant solids), and in moisturizing creams, lotions, powders, and sprays at concentrations up to 15% (equivalent to 0.5% plant solids). Preparations fromD. villosaare used in herbal medicine for treatment of a variety of ailments and by the pharmaceutical industry in the preparation of steroids. Using Dioscorea Villosa (Wild Yam) Root Extract prepared via a specified process, it is possible to produce a stable extract with a narrow range of diosgenin content. The extract produced using this methodology was tested in acute and short-term toxicity tests, dermal irritation tests, a sensitization test, an ocular irritation test, a rat uterotropic assay, and genotoxicity tests. An acute oral toxicity test produced hypoactivity, piloerection, and dyspnea and a death in 1 of 10 rats at 2 g/kg using the specified extract, but no toxicity in rats given 0.5 g/kg. A dermal toxicity test using the specified extract demonstrated no acute toxicity in rats. Both a 7-day local tolerance test and a 28-day dermal toxicity test in rats produced no significant adverse effects at the maximum tested concentration of 10%. A single application of undiluted extract to the intact and abraded skin of rabbits produced sufficient irritation for the test material to be rated"irritant,"but a 10% dilution was not irritating. Undiluted extract was only mildly irritating to the conjuctiva of the rabbit eye; irritation in the iris and cornea was mild and transient. Undiluted extract was not irritating during the induction phase of a guinea pig sensitization study, nor did challenge with a 25% dilution elicit any sensitization. The specified extract at concentrations up to 500 mg/kg/day did not have any estrogenic activity in the juvenile rat uterotrophic assay. Genotoxicity assays in bacterial and mammalian systems were negative, except that Ames test strain TA 1537 was positive at one dose level using the plate incorporation method, but not using a preincubation method. Although the concentration at which the actual plant extract is used in cosmetic products is low, one of the primary safety concerns with this plant extract is the possible metabolic/endocrine activity, e.g., estrogen-like or progesterone-like activity as a result of the presence of small amounts of plant phytosterols such as diosgenin. Extracts prepared as described in this safety assessment, with an upper limit of 3.5% diosgenin, did not have any estrogenic activity, demonstrating that it is possible to produce material that does not present this specific safety concern. Although extracts from pesticide-free plants were not considered genotoxic and it was the view of the Cosmetic Ingredient Review (CIR) Expert Panel that there do not appear to be any components that could be carcinogenic, pesticide residues could raise this issue. It was urged that manufacturers limit pesticide residues to the limit previously used for lanolin of not more than 40 ppm (with not more than 10 ppm for any one residue). Based on these data, it was concluded that Dioscorea Villosa (Wild Yam) Root Extract is safe as used in cosmetic formulations. This conclusion regarding safety, however, is valid only for extracts prepared in a manner that produces a similar chemical profile as that described in this report, particularly as regards diosgenin. Extracts not prepared in a manner that produces a similar chemical profile would be considered safe if they have a similar safety test profile.

大気浮遊粒子，河川水および土砂の変異原性モニタリング―７年間（１９９６〜２００３年）の結果―

Airborne particles (n=541) , river waters (n=541) and surface soils (n=544) were collected from five geographically different regions of Japan (Hokkaido, Kanto, Chubu, Kinki and Kyushu regions) between 1996 and 2003. Extracts from those samples were measured and compared by a pre-incubation method using Salmonella typhimurium strains and a standard reference. Airborne particles were collected by high volume air samplers. All organic extracts of airborne particles except for some of those from Kinki region were mutagenic toward both TA98 and TA100 strains with and without S9 mix. Higher mutagenicity of airborne particulate extracts was observed for the samples collected at sites in heavy traffic areas and nearby industrial steel works. Mutagenic activity of airborne particles showed a gradual decrease over the period of the 7 years. Most of the blue rayon-absorbed materials from river waters exerted strong mutagenicity toward YG1024 strain with S9 mix, and the potencies varied widely among sampling sites. Blue rayonabsorbed materials showed no strong mutagenicity in TA100 or YG1029 strains, with or without S9 mix. Non-agricultural surface soils were collected from roadsides, parks and other open areas from each region. All organic extracts of surface soils were mutagenic toward either TA98 or TA100 strains with or without S9 mix. Mutagenic activities of surface soil extracts showed a wide range, and the effect of additional S9 mix on the activities depended on samples.

Stereochemical effects in the metabolic activation of nitrosopiperidines: correlations with genotoxicity

The genotoxicity of nitrosopiperidine and six alkyl derivatives was studied by use of the Ames tester strains TA100 and TA1535 in the pre-incubation method. Among the compounds investigated, those exhibiting genotoxic activity under the experimental conditions employed were only genotoxic in the presence of S9 mix (10%). The results obtained were correlated with models of the metabolic activation of nitrosamines in an attempt to rationalize the genotoxicity of these compounds. The results show that the presence of substituents in the nitrosopiperidine molecule may be one of the modulating factors affecting the genotoxicity of these cyclic nitrosamines, and may help provide some chemical clues for the identification of risk compounds from among a large group of structurally related molecules.

Application of Microtiter Plate Assay to Evaluate Inhibitory Effects of Various Compounds on Nine Cytochrome P450 Isoforms and to Estimate their Inhibition Patterns

Using a microtiter plate (MTP) assay consists of recombinant cytochromes P450 and fluorescent probes, we evaluated inhibitory effects of commercially available model-compounds, 18 typical substrates and 8 selective inhibitors, on nine cytochromes P450 (CYPs) activities. The IC(50) values obtained from the assay were used to estimate inhibition constant (Ki) values, assuming competitive inhibition. The Ki values calculated from IC(50) (the Ki(-cal)) with the MTP assay using recombinant CYPs were compared with the Ki values (the Ki(-rep)), reported for human liver microsomes (HLM). Regarding all the inhibitory effects of the 26 test compounds on each CYP activity, a good correlation (r(2)=0.7306) was found between Ki(-cal) and Ki(-rep). The inhibitory patterns of some compounds on the five major CYP isoforms were estimated, using the MTP assay with the preincubation method. Furafylline and erythromycin, both mechanism based inhibitors, strongly inhibited CYP1A2 and CYP3A4 activity, respectively and their inhibitory effects increased depending on the preincubation time. In contrast, the inhibitory effects of phenacetin, diclofenac, S-mephenytoin, dextromethorphan, bufuralol and terfenadine, typical substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively, on each recombinant CYP activity decreased after preincubation. Therefore, the MTP assay is a useful high throughput screening method to evaluate inhibitory effects of new drug candidates on 9 CYP isoforms in HLM. In addition, the MTP assay with the preincubation method might be beneficial to estimate inhibitory patterns on CYP isoforms of new drug candidates and to estimate main CYP isoforms responsible for metabolism of these compounds.

MutagenicityTesting of 1,3-Butadiene, 1,4-Pentadiene-3-ol, Isoprene, 2,4-Hexadiene, cis- and trans-Piperlylene.

A mutagenicity test was conducted for 1,4-pentadiene-3-ol, 2,4-hexadiene, isoprene, cis- and trans-piperylene in the bacterial mutation assay using Salmonella typhimurium (S. typhimurium) strain TA 100, TA98, TA1535 and Escherichia coli (E. coli) WP2uvrA/pKM101 with and without metabolic activation by S9 mix in the preincubation method. The mutagenicity of 1,3-butadiene was tested by the gas exposure method. Mutagenicity was weakly positive for 1,4-pentadiene-3-ol in TA 100 with S9 mix and was pseudopositive for WP2uvrA/pKM101 without S9 mix. Mutagenicity was very clear for 1,3-butadiene in 10 fold concentrated TA1535 with S9 mix. The 2,4-hexadiene, isoprene, cis-piperlylene and trans-piperlylene were not mutagenic on S. typhimurium TA98, TA100, TA1535 or E. coli WP2uvrA/pKM101 with or without metabolic activation. It can be concluded from these results, that chemicals, containing double bond carbon (C=C) chemical generally tend to show weak mutagenicity in the presence of the metabolic activation system.

Development of an In Vitro System Detecting Pro-embryotoxin

An in vitro system for detection of embryotoxins has been developed by using primary cultures of embryo fibroblasts. Various embryotoxins, including benzo[a]pyrene and thalidomide, have trivial cytotoxicity in embryo fibroblast systems, which is at least in part due to a lack of capacity for metabolic activation. Introduction of steps for microsomal pre-incubation and calcium-precipitation prior to chemical contact resulted in the clear appearance of embryotoxicity toward thalidomide and benzo[a]pyrene. This preincubation method will offer advantages for the detection of embryotoxins, which require maternal metabolic activation, and for understanding the mechanisms of their metabolic activations.

In vitro genotoxicity testing of (1-chloroethenyl)oxirane, a metabolite of β-chloroprene

(1-Chloroethenyl)oxirane (CEO) is a metabolite of β-chloroprene (2-chloro-1,3-butadiene, CD). The purpose of this study was to evaluate the in vitro mutagenic and clastogenic (chromosome breaking) potential of CEO. For comparative purposes, the study also included an evaluation of the racemic compounds, 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Mutagenicity was evaluated in a bacterial reverse mutation test (Ames), using the pre-incubation method in the presence and absence of an exogenous metabolism system (Aroclor ®-induced rat liver S9). Four Salmonella typhimurium tester strains, TA97a, TA98, TA100 and TA1535 were used. The exposure concentrations in the sealed incubation vials ranged from 0 to 69 mM for CEO, 0 to 102 mM for EB, and 0 to 83 mM for DEB. All three compounds showed signs of toxicity, with DEB being substantially more toxic than either CEO or EB. Mutagenic activity was observed with all three chemicals in primarily the base pair substitution strains ( S. typhimurium TA100 and TA1535), but some activity was also seen in the frameshift elimination strains ( S. typhimurium TA97a and TA98). The observed mutagenic responses after exposure with CEO or EB were greater than the observed response for DEB, most likely because of the higher toxicity of DEB. Generally, the mutagenic responses were unchanged in the frameshift strains and base pair substitution strains in the presence of S9 metabolism. In vitro clastogenicity was evaluated using the cytochalasin-B blocked micronucleus test in cultured Chinese hamster V79 cells. The test was conducted without S9 metabolism because of the absence of substantial changes in the Ames test. Exposure concentrations ranged from 0 to 0.943 mM for CEO, 0 to 3.0 mM for EB, and 0 to 0.035 mM for DEB, with the upper exposure concentrations dictated by cytotoxicity. Cytotoxicity, measured as a reduction in the proportion of binucleated cells and altered cell morphology, was observed for CEO at concentrations ≥0.175 mM. Exposure to EB led to a reduced proportion of binucleated cells at concentrations ≥2.0 mM, and cell death was observed after DEB exposure at concentrations ≥0.025 mM. No clastogenicity was observed in the V79 cells when tested up to cytotoxic concentrations of CEO, whereas an elevated frequency of micronuclei was observed after exposure to either EB (≥1.0 mM) or DEB (≥0.0125 mM). These results suggest that CEO-induced mutagenicity, but not clastogenicity, may contribute to the observed β-chloroprene-induced carcinogenicity in the rodent bioassay studies.

Method for measuring mutagen formation potential (MFP) on chlorination as a new water quality index

A novel water quality index, the mutagen formation potential (MFP) is proposed for use in evaluation of the quality of drinking water which may contain pollutants capable of forming mutagens when chlorinated under the conditions used in water purification processes. A method for measuring MFP was established as follows. The water sample to be tested is diluted until the TOC reaches 3–4 mg l −1, the pH is adjusted to 7.0±0.2, sodium hypochlorite is added to obtain conditions where Cl/TOC=3–4 mg Cl (mg C) −1, and the water sample is left standing for 24±2 h at room temperature. Thereafter, 2 l of the chlorinated water sample at pH 2.0±0.1 is passed through a Sep-Pak Plus CSP-800 cartridge to adsorb any mutagens formed, and DMSO is applied to the cartridge to desorb the mutagens. Then, a 2 ml sample of the eluate is collected after the DMSO had begun to flow out of the cartridge and evaluated by the Ames Salmonella mutagenicity assay (preincubation method).

Antimutagens in gaiyou (Artemisia argyi levl. et vant.).

Antimutagens from gaiyou (Artemisia argyi Levl. et Vant., Compositae) were examined. The methanol extract prepared from aerial parts of this plant strongly reduced the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), when Salmonella typhimurium TA98 was used in the presence of the rat liver microsomal fraction. The antimutagens were purified chromatographically while monitoring the antimutagenic activity against Trp-P-2 with a modified Ames test employing a plate method. This purification resulted in the isolation of four strong antimutagens, 5,7-dihydroxy-6,3',4'-trimethoxyflavone (eupatilin), 5, 7,4'-trihydroxy-6,3'-dimethoxyflavone (jaceosidin), 5,7, 4'-trihydroxyflavone (apigenin) and 5,7, 4'-trihydroxy-3'-methoxyflavone (chrysoeriol) from the methanol extract. These antimutagenic flavones exhibited strong antimutagenic activity against not only Trp-P-2 but also against other heterocyclic amines, such as 3-amino-1,4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA(alpha)C) in S. typhimurium TA98. In contrast, they did not exhibit antimutagenic activity against benzo[a]pyrene (B[a]P), 4-nitroquinoline-1-oxide (4-NQO), 2-aminofluorene (2-AF), 2-nitrofluorene (2-NF) or furylfuramide (AF-2) in S. typhimurium TA98, or B[a]P, 4-NQO, 2-NF, AF-2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium azide (SA) in Salmonella typhimurium TA100, whereas they decreased the mutagenicity caused by aflatoxin B(1) (AFB(1)) and 2-aminoanthracene (2-AA) in both of these tester strains. Regarding the structure-activity relationship, the tested flavones had distinct differences in the intensities of their antimutagenic activities according to the differences of their substitution patterns. Namely, the intensity of antimutagenic activities against Trp-P-2 decreased in the order of: 5,7,3',4'-tetrasubstituted flavones (IC(50): <0.1 mmol/plate), 5,7,4'-trisubstituted flavones (IC(50): 0.120-0.260 mmol/plate), 5,6,7,3',4'-pentasubstituted flavones (IC(50): 0.440-0. 772 mmol/plate). The four isolated flavones were also studied regarding their antimutagenic mechanisms with preincubation methods of the modified Ames test and emission spectroscopic analysis. The results suggested that all isolated flavones were desmutagens which directly inactivated Trp-P-2 or inhibited its metabolic activation.

Mutagenicity and co-mutagenicity of static magnetic fields detected by bacterial mutation assay

Possible mutagenic and co-mutagenic effects of strong static magnetic fields were estimated using bacterial mutagenicity test. Mutagenic potential of static magnetic fields up to 5T (T:1T=10,000 G) was not detected by the bacterial mutagenicity test using four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA either in the pre-incubation method or in the plate incorporation method. In the co-mutagenicity test, E. coli WP2 uvrA cells were treated with various chemical mutagens and were simultaneously exposed to a 2T or a 5T static magnetic field. Mutation rate in the exposed group was significantly higher than that in the non-exposed group when cells were treated with N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), 4-nitroquinoline- N-oxide (4-NQO), 2-amino-3-methyl-3 H-imidazo[4,5-f]quinoline (IQ) or 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). The mutagenicity of 2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), N 4-aminocytidine and 2-acetoamidofluorene (2-AAF) was not affected by the magnetic field exposure. Possible mechanisms of the co-mutagenicity of magnetic fields are discussed.

Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline.

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.