Mammalian Metabolic Activation System Research Articles

Antioxidant and antimutagenic activity of Curcuma caesia Roxb. rhizome extracts

The rhizomes of Curcuma caesia Roxb. (zingiberacea) are traditionally used in treatment of various ailments and metabolic disorders like leukoderma, asthma, tumours, piles, bronchitis, etc. in Indian system of medicine. Considering the importance of natural products in modern phytomedicine, the antioxidant and antimutagenic activities of C. caesia Roxb. rhizome extract and its fractions were evaluated. The ethanolic fraction showed highest antioxidant activity by DPPH assay (86.91%) comparable to ascorbic acid (94.77%) with IC50 value of 418μg/ml for EECC followed by MECC (441.90μg/ml)>EAECC(561μg/ml)>AECC(591μg/ml). Based on the antioxidant activity, three of the rhizome extracts were evaluated for their antimutagenic properties against indirect acting mutagen cyclophosphamide (CP) using Salmonella typhimurium strains TA98 and TA100. The antimutagenic activity of the extracts against indirect acting mutagen cyclophosphamide in the presence of mammalian metabolic activation system was found to be significant (p<0.01, p<0.05). All the extracts showed similar antimutagenicity in dose dependent manner. The total phenolic content as well as reducing ability of the extracts was also determined.

Potential toxic effects of iron oxide nanoparticles in in vivo and in vitro experiments

The aim of this study was to determine the potential toxic effects of iron(II,III)oxide nanoparticles (IONPs). In in vivo experiments, the toxic effects of IONPs were monitored in adult male Wistar rats by morphological methods after a single intratracheal instillation. For the control group 1 ml of physiological saline per animal was given, and the treatment group received the same volume of a suspension containing 1 and 5 mg kg⁻¹ body weight IONPs. Lungs and internal organs underwent histopathological examination after 1, 3, 7, 14 and 30 days. The mutagenic effect of these nanoparticles was evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and on Escherichia coli WP2uvrA strain, in the presence and absence of the mammalian metabolic activation system S9. The in vitro cytotoxic effect of IONPs was also examined in Vero cells after short-term (4 h) and long-term (24 h) exposure. There were no pathological changes in examined internal organs, except a very weak pulmonary fibrosis developing by the end of the first month in the treated rats. While in vitro MTT assay showed a moderate cytotoxic effect, IONPs proved to be devoid of mutagenic effect in the bacterial systems tested. The results may be a useful extension of our knowledge on the safety of magnetite nanoparticles in view of their possible medical applications, such as in hyperthermia and magnetic resonance imaging.

Antimutagenic effect of Phellinus rimosus (Berk) Pilat against chemical induced mutations of histidine dependent Salmonella typhimurium strains

Mutations are one of the important factors contributing to oncogenesis. Somatic mutations have been detected in oncogenes and tumor suppressor genes in various types of cancers. In vitro antimutagenic activity of ethyl acetate extract of macro fungus, Phellinus rimosus was evaluated by Ames’ mutagenicity assay. The effect was evaluated against the direct acting mutagens (sodium azide, N-methyl-N′-nitro-N-nitrosoguanidine, doxorubicin and 4-nitro-o-phenylenediamine) and mutagen needing activation (2-acetyl aminofluorine, and benzo[a]pyrene). The extract was significantly (p<0.05) and dose dependently effective against direct acting mutagens and mutagen needing activation. Among the antimutagenic activity against directly acting mutagens, effect was found to be highest against doxorubicin-induced mutation. The antimutagenic effect of the extract against indirect acting mutagen in the presence of mammalian metabolic activation system was also found to be significant (p<0.01). The background bacterial growth and number of revertant colonies in the extract alone treated plate with or with out metabolic activator was almost same as that of spontaneous revertants. This indicated the non-toxic nature of the extract. The effect was partially ascribed to the antioxidant activity. The results of the study suggest the possible antitumor mechanisms of P. rimosus.

Zebrafish teratogenicity test with metabolic activation (m DarT): Effects of phase I activation of acetaminophen on zebrafish Danio rerio embryos

The zebrafish Danio rerio embryo test with metabolic activation (m DarT) was developed to assess the teratogenic effects of proteratogens. In this study induced rat liver microsomes (RLM) were used as a mammalian metabolic activation system (MAS), since they contain various cytochrome P450 (CYP) isoforms at high concentrations. Acetaminophen (APAP) is considered not to be teratogenic in vivo, however, in vitro teratogenic effects were observed, e.g. in rat whole embryo culture. The CYP2E1 activation of APAP to the reactive metabolite N-acetyl- p-benzoquinone imine (NAPQI) mainly occurs, when the glucuronidation and sulfatation pathways are saturated. In vivo the soft electrophile NAPQI is usually inactivated by hepatic reduced glutathione (GSH), a soft nucleophile. In this study, we investigated the teratogenic and lethal effects of APAP after CYP activation in zebrafish embryos. In the test groups with APAP and metabolic activation 11.7 ± 7.6% (2 mM), 25.0 ± 8.7% (4 mM) and 50.0 ± 21.8% (6 mM) affected embryos were seen, reaching statistical significance at 4 mM APAP. When embryos were exposed to 6 mM APAP, MAS and 3 mM GSH the percentage of affected embryos decreased to 6.7 ± 5.8%. In contrast teratogenic and lethal effects of metabolically activated cyclophosphamide (CPA) could not be prevented by GSH addition, because the CPA metabolites are strong electrophiles, which preferentially bind to hard nucleophiles like DNA and RNA. The teratogenic and lethal effects of metabolically activated APAP observed in zebrafish embryos with our m DarT standard protocol could be explained by the lack of GSH as a detoxifying system. By adding GSH it was possible to mimic the situation in mammals and thus avoid teratogenic effects in zebrafish embryos.

In vitro teratogenicity testing with zebrafish embryos combined with a mammalian metabolic activation system: Pro's and con's

In vitro teratogenicity testing with zebrafish embryos combined with a mammalian metabolic activation system: Pro's and con's

Development of a New Screening Assay to Identify Proteratogenic Substances using Zebrafish Danio rerio Embryo Combined with an Exogenous Mammalian Metabolic Activation System (mDarT)

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

Teratogenicity of benzo[ a]pyrene in the fish embryo assay (DarT) combined with a mammalian metabolic activation system

Teratogenicity of benzo[ a]pyrene in the fish embryo assay (DarT) combined with a mammalian metabolic activation system

Mutagenic activity and quantification of nitroarenes in surface soil in the Kinki region of Japan

To clarify the mutagenic potential of surface soil in the Kinki region of Japan, particularly in Osaka and neighboring cities, 62 surface soil samples were collected and their organic extracts were examined by the Ames/ Salmonella assay. All of the samples were mutagenic toward TA98 in both the presence and absence of a mammalian metabolic activation system (S9 mix). While all of the samples showed mutagenicity toward TA100 with S9 mix, only 45/62 (73%) were mutagenic without S9 mix. Fifty (81%) of the samples showed higher activity toward TA98 than TA100. The mean values of the mutagenicities of soil samples collected in Osaka prefecture ( n=35) toward TA98 with and without S9 mix were 2315 and 1630 revertants per gram of soil, respectively, and these were 2.9 and 2.6 times as high as the values for samples from other prefectures ( n=27), respectively. Three dinitropyrene (DNP) isomers, i.e. 1,3-, 1,6- and 1,8-DNP, and 3-nitrobenzanthrone (NBA) in the surface soil samples were quantified by fluorometric detection of the corresponding amino compounds, i.e. diaminopyrene isomers and 3-aminobenzanthrone, using high-performance liquid chromatography (HPLC). The three DNP isomers were detected in all of the soil samples ( n=26) that were mainly collected in Osaka prefecture, and the amounts of 1,3-, 1,6- and 1,8-DNP were 6–1526, 11–1772 and 10–2092 pg/g of soil, respectively. The contribution ratios of 1,3-, 1,6- and 1,8-DNP to the mutagenicity of soil extracts toward TA98 without S9 mix were 0.2–12, 0.3–12 and 0.5–27%, respectively. The amount of 3-NBA in soil samples ( n=8) was 144–1158 pg/g of soil, and the contribution ratio of 3-NBA to the mutagenicity of soil extracts was 2–38%. These results suggest that the surface soils in the Kinki region were highly polluted with mutagens and the pollution levels in Osaka prefecture were higher than those in other areas. DNP isomers and 3-NBA may be major mutagens that contaminate surface soil in this region.

The Mutagenicity Study of Surface Water from Meiliang Bay, Taihu Lake in Winter By E.coli UC1121 L-arabinose Resistance Forward Mutation Test

The L-arabinose resistance forward mutation test (ara test), together with Ames test, was performed for evaluation of mutagenicity of the surface water samples collected from Meiliang Bay, Taihu Lake in winter. The five of the nine surface water samples were detected as mutagen positive by ara test, most of them were collected from the estuary which flow into the lake or from the edge of shore area. The same tendency was observed with the involvement of mammalian S9 metabolic activation system. The roughly, coincident results were confirmed by Ames test when the sample doses applied were enhanced to two and a half times compared with that of ara test. The data suggest that detection sensitivity of ara test is higher if intricate environmental samples are concerned. Thus, prospect for ara test to be the primary choice is prompted when the genotoxicity screening of environmental mixtures is required.

Mutagenic activity of surface soil and quantification of 1,3-, 1,6-, and 1,8-dinitropyrene isomers in soil in Japan.

To clarify the mutagenic potential of nonagricultural surface soil in Japan, 110 soil samples were collected from five geographically different areas between November 1996 and March 1997, and organic extracts of the soil samples were examined by the Ames/Salmonella assay. Most of the soil extracts showed mutagenicity toward both strains TA98 and TA100 in the presence and/or absence of a mammalian metabolic activation system (S9 mix), suggesting that surface soil is largely contaminated with environmental mutagens. Soil samples collected at Hekinan, Kobe, and Osaka were highly mutagenic toward both strains, and their potencies toward TA98 without S9 mix were extremely high, inducing more than 12 000 revertants per gram of soil. On the other hand, soil samples from Muroran showed strong mutagenicity toward TA100 with S9 mix. Furthermore, 1, 3-dinitropyrene (DNP), 1,6-DNP, and 1,8-DNP in soil samples collected at 10 sampling sites in three metropolitan areas were quantified by fluorometric detection of the corresponding diaminopyrene isomers using high-performance liquid chromatography (HPLC). Three DNP isomers were detected in all soil samples, and the amounts of 1,3-, 1,6-, and 1,8-DNP isomers in the soil samples were 12-3270, 14-5587, and 13-6809 pg/g, respectively. The gross amount of three DNP isomers in surface soil collected at Hekinan was more than 10 ng per gram of soil. The highest contribution ratios of DNP isomers to the mutagenicity of soil extracts were observed for the samples collected at Osaka, and the total of the contribution ratios of three DNP isomers was about 50%. These results suggest that surface soil is largely contaminated with mutagenic compounds and that DNP isomers are one class of major mutagenic and carcinogenic compounds contaminating surface soil.

Isolation and chemical--structural identification of a novel aromatic amine mutagen in an ozonized solution of m-phenylenediamine.

The mutagenicity of a m-phenylenediamine (m-PD) solution was markedly enhanced by oxidation with ozone. The ethyl acetate extracts from a m-PD solution ozonized at pH 10.7 were fractionated by normal-phase and reversed-phase column chromatography to isolate mutagens by monitoring mutagenic activities on Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system (S9 mix). From fraction 5-3-2, which exhibited the strongest mutagenicity (308000 revertants/mg), a major mutagenic compound was isolated. On the basis of the high-resolution EI-mass, (1)H NMR and (13)C NMR spectral, and X-ray crystallography data, the structure of this compound was determined to be 2-amino-5-[(3-aminophenyl)amino]-4-[(3-aminophenyl)imino]-2, 5-cyclohexadien-1-one (PDT-1), which is a novel compound. PDT-1 is a newly identified frame-shift type mutagen, inducing 65400 revertants and 295000 revertants of S. typhimurium TA98 and YG1024 per micromole, respectively, in the presence of S9 mix. When a m-PD solution was oxidized with 1 or 2 mol of ozone at pH 4.0, 7.0, and 10.7, the contribution of PDT-1 to the mutagenicity of ethyl acetate extracts from the ozonized m-PD solution was 5-23%.

Identification of 1,6- and 1,8-dinitropyrene isomers as major mutagens in organic extracts of soil from Osaka, Japan.

The organic extracts of soil collected at parks in residential areas in Osaka and neighboring cities in the Kansai area, Japan, showed mutagenicity in Salmonella typhimurium strain TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The soil extracts from Ibaraki and two different sites in Osaka, i.e., Sumiyoshi-ku and Minato-ku, were mutagenic in strain TA100 as well as in strain TA98. Direct-acting mutagenicity of soil extracts from Sumiyoshi-ku and Minato-ku toward strain TA98 were 66 or more times higher than that of the other cities. Both extracts exerted stronger mutagenicity in strains YG1021 and YG1024 than TA98 and TA100, and the potency was especially high in strain YG1024: Sumiyoshi-ku, 153 000 revertants/g of soil; and Minato-ku, 246 000 revertants/g of soil. Two mutagenic compounds (I and II) were isolated from the Soxhlet extract of soil from the park in Sumiyoshi-ku by repetitive separation using normal-phase and reversed-phase column chromatography. By comparing the mass and UV spectra and retention times for HPLC on two individual ODS columns of compounds I and II with those of authentic chemicals, we identified these two compounds as 1,6- and 1,8-dinitropyrene (DNPy) isomers. Amounts of DNPy isomers in soil from Sumiyoshi-ku and Minato-ku were 1.7-2.2 ng/g. Forty-three percent and 40% of the mutagenicity of soil from Sumiyoshi-ku and Minato-ku could be attributed to these DNPy isomers, respectively.

Mutagenicity of an Ozonized Solution of Phenylenediamine Derivatives in the Salmonella Test.

o-Phenylenediamine (o-PD), m-phenylenediamine (m-PD), 2, 4-diaminotoluene (DAT) or 2, 4-diaminoanisole (DAA) in aqueous solutions were oxidized with ozone at pH 10.7, and the ozonized solutions were tested for their mutagenicity in Salmonella typhimurium TA 98 in the presence or absence of a mammalian metabolic activation system (S 9 mix). The mutagenicity of m-PD and DAA aqueous solutions with S 9 mix was markedly enhanced by oxidation with 1-to 4-fold mol of ozone, but vanished with sufficient ozonation. An aqueous solution of m-PD and DAA was ozonized at pH 4.0, 7.0 and 10.7, and the mutagenicity of the ozonized solution and extract with ethyl acetate (AcOEt) was tested in S. typhimurium TA 98 with S 9 mix. At all pH values of the solution, mutagenicity was markedly enhanced by ozonation, and that of the AcOEt extract was equal to or greater than that of the aqueous solution. Aminophenazines were observed in the AcOEt extract from the ozonized solution of m-PD and DAA. The contribution of the aminophenazines to mutagenicity, however, was low, and it is suggested that other major mutagenic compounds were formed.

Antimutagenic effect of plant flavonoids in the Salmonella assay system.

The antimutagenic effects of 27 kinds of plant flavonoids on the mutagenicity of aflatoxin B1(AFB1) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in Salmonella typhimurium TA100 were investigated. In the mixed applications of AFB1 (1 microgram/plate) with the flavonoids (300 micrograms/plate) in the presence of a mammalian metabolic activation system (S9 mix), chrysin, apigenin, luteolin and its glucoside, kaempferol, fisetin, morin, naringenin, hesperetin, persicogenin, (+)-catechin and (-)-epicatechin showed the antimutagenic effect against AFB1 with more than 70% inhibition rate. A little or no antimutagenicities except flavone against MNNG (0.5 microgram/plate) were observed. For the antimutagenicity of the flavonoids on AFB1, the flavonoid structure that contains the free 5-, 7-hydroxyl group seemed to be essential. However, saturation of the 2,3-double bond or elimination of the 4-keto group did not affect the activity.

Mutagenicity tests of tazobactam/piperacillin, tazobactam and piperacillin

As a part of safety tests of tazobactam/piperacillin (TAZ/PIPC), the reverse mutation tests using bacteria, the chromosomal aberration tests using cultured cells and the micronucleus tests using male mice were conducted in order to evaluate the in vitro and in vivo mutagenicity of TAZ, PIPC, TAZ/PIPC. 1. The reverse mutation tests were carried out on TAZ, PIPC and TAZ/PIPC at dose ranges, where few antibacterial effects could be detected, using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA. All of three test articles showed that no significant increases were observed in the number of colonies in all tester strains in both systems, with and without mammalian metabolic activation (S9 Mix), as compared with solvent controls. 2. The chromosomal aberration tests were carried out on these test articles using cultured Chinese hamster lung cells (CHL). The cells were treated with TAZ, PIPC or TAZ/PIPC at the doses of 2.5, 5.0 and 10 mM with and without S9 Mix. In the test of PIPC with S9 Mix, the dose of 1.25 mM was set in addition to the three doses. The incidences of structural- and numeral-aberration were 0-3% in the absence or presence of mammalian metabolic activation system, and no significant increases were observed in the incidence of chromosomal aberrations as compared with solvent controls. 3. The micronucleus tests were carried out at doses of 625-5000 mg/kg of TAZ or TAZ/PIPC, or at 625-2500 mg/kg of PIPC. The femoral marrow cells were 48 h after administering intravenously to CD-1 male mice. The frequencies of polychromatic erythrocyte with micronuclei were 0.02-0.17%, 0.02-0.10% and 0.03-0.07% in the groups treated with TAZ, PIPC and TAZ/PIPC, respectively, and no significant increases were observed with dose dependence. The results indicated that these test articles were negative in the assessment standard using the background data. 4. The present study indicates that TAZ, PIPC and TAZ/PIPC have no in vitro and in vivo mutagenic potential.

In vitro system for detecting non-genotoxic carcinogens.

Chemical risk assessment has been limited by the inability of in vitro short-term assays to identify the true carcinogenic potential of many substances. Numerous methods exist for identifying mutagenic and clastogenic agents, but a practical means of identifying non-genotoxic carcinogens has remained elusive. Experiments described here suggest that some chemicals may participate in carcinogenesis by modulating the enzymatic processes of drug metabolism. The tumor promoters butylated hydroxyanisole, butylated hydroxytoluene, deoxycholic acid, reserpine, trypan blue, and 12,-O-tetradecanoyl phorbol-13-acetate were chosen as model non-genotoxic carcinogens. The enzyme-modulating action of these chemicals was measured using a modified Ames plate incorporation assay whereby the known tumor promoters were plated with a promutagen in the presence of a mammalian metabolic activation system (S9). Each of the non-genotoxic carcinogens significantly increased the mutagenic response of metabolically activated promutagen(s). These experiments suggest that the carcinogenic role of some chemicals may be attributed to their ability to modify the biochemical pathways of drug metabolism. By enhancing or inhibiting the activity of various enzymes, some tumor promoters may create an environment that increases a cell's mutational burden, thereby contributing to neoplastic transformation.

Mutagenicity of nitro derivatives produced by exposure of dibenzofuran to nitrogen oxides

Dibenzofuran (DF) was reacted with various concentrations of nitrogen oxides (NOx) under light irradiation. The mutagenicities of the reaction mixtures were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP 6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DF-Nox (molar ratios 1:3, 1:6, and 1:18) reaction mixtures exhibited mutagenic potency in strain TA98 without S9 mix, and their direct-acting mutagenicity was reduced in strains TA98NR and TA98/1.8-DNP 6. Four mononitrodibenzofurans and 2 dinitrodiobenzofurans, i.e., 1-nitrodibenzofuran (NDF), 2-NDF, 3-NDF, 4-NDF, 2,7-dinitrodibenzofuran (DNDF) and 2,8-DNDF, were identified with authentic samples in the DF-NOx (1:18) reaction mixture by HPLC cochromatography and a gas The order of mutagenicity of nitrodibenzofurans in strain TA98 without S9 mix was as follows: strains TA98 and TA100 was enhanced by the addition of S9 mix. Since these nitrodibenzofurans were less mutagenic in strains TA98NR and TA98/1,8-DNP 6 than in strain TA98 without S9 mix, it was presumed that their mutagenicity was dependent on their activation by the ‘classical’ bacterial nitroreductase and/pr transacetylase, which are absent in strains TA98NR and TA98/1,8-DNP 6 but present in strain TA98, respectively by 3-NDF and 4-NDF were mutagenic in strain TA100 without S9 mix. 2-NDF and 3-NDF were determined as corresponding amino derivatives in DF-NOx (1:3), (1:6) and (1:18) reaction mixtures. They contributed about 30–65% of the direct-acting mutagenicity of reaction mixtures in strain TA98.

Mutagenicity of the reaction products of dibenzo- p-dioxin with nitrogen oxides

Dibenzo- p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP 6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1: 3, 1: 6 and 1: 18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP 6 without S9 mix. In a gas chromatography/mass spectrometry study, 2nitrodibenzo- p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1: 18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo- p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP 6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs

Studies on the Mutagenicity of o-Dinitrobenzene-nucleic Base Adducts in Salmonella typhimurium Strains and the Structural Elucidation of the Purine Adducts.

o-Dinitrobenzene (DNBz) and each nucleic base were made to react with NaOH in dimethylsulfoxide at room temperature for 3-60 h. All of the synthesized compounds were the 2-nitrophenyl adducts of nucleic bases. The mutagenicity test of the synthesized compounds and of o-DNBz were then carried out against Salmonella typhimurium TA98, TA98NR and TA100 without a mammalian metabolic activation system. o-DNBz has no mutagenic potency against all the strains at a dose up to 100 μg. In the compounds with the adducts of nucleic bases, the mutagenic potencies of the adducts of adenine and guanine in TA98 were 50 revertants/100 μg and 255 revertants/10 μg, respectively and the most potent mutagen in TA100 was an adduct of guanine (382 revertants/100μg). Finally, the structures of the adducts of adenine and guanine could be elucidated as N1-(2-nitrophenyl)-adenine and N1-(2-nitrophenyl)-guanine, respectively, by the acid and alkaline degradation and by the isopentyl nitrite treatment of these adducts.

The Inhibition of Mutagenicity Ascribed to Oxidized Products from Hairdye Component: m-Phenylenediamine with Hydrogen Peroxide by Iron II Sulfate, Phenol Derivatives or Metal Chelates.

2, 7-Diaminophenazine (2, 7-diNH2-Pz) formed from m-phenylenediamine (PD) by hydrogen peroxide (H2O2) oxidation was extremely mutagenic in Salmonella typhimurium strain TA98 with a mammalian metabolic activation system (S9 mix). In order to evaluate the modulating effect of phenol derivatives and iron (II) sulfate on H2O2 oxidation of m-PD, m-PD and mixtures of m-PD and phenol derivatives (m-PD/phenols) were treated with 3% H2O2 in the presence or absence of iron (II) sulfate and their mutagenicity was tested using strain TA98 with S9 mix. The total mutagenicity, which was calculated from the yields of oxidized mixtures (i.e. ethyl acetate extract) and their mutagenicity, decreased remarkably by the addition of iron (II) sulfate into the H2O2 oxidation system. Since the yields of 2, 7-diNH2-Pz and ethyl acetate extract decreased more in a H2O2-iron (II) sulfate oxidation system than those in a H2O2 oxidation system, the reduction of the total mutagenicity was presumed to be due to the accelerated polymerization of m-PD by iron (II) sulfate added. Furthermore, the modulating effect of 5 kinds of metal chelates on the mutagenicity of 2, 7-diNH2-Pz and on the H2O2 oxidation of m-PD was evaluated. All tested metal chelates reduced the mutagenic response of 2, 7-diNH2-Pz in strain TA98 with S9 mix, and the strongest inhibitory effect was observed in the case using hemin. The amount of free 2, 7-diNH2-Pz in the mixture solution of 2, 7-diNH2-Pz and metal chelates was correlated to the mutagenic potency of the mixture. It was suggested that the mutagenic inhibitory effect of metal chelates depend on the complex formation of metal chelates with 2, 7-diNH2-Pz. The addition of metal chelates into the H2O2 oxidation system of m-PD significantly decreased the amount of free 2, 7-diNH2-Pz in the reaction mixture. The reduction of the yield of 2, 7-diNH2-Pz was assumed to be based on the polymerization of m-PD by metal chelates as catalyst and formation of complex with 2, 7-diNH2-Pz and metal chelates.