Addition Of S9 Mix Research Articles

The mutagenicity analysis of imidapril hydrochloride and its degradant, diketopiperazine derivative, nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used. BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard. Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit. ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds. ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.

Genotoxicity evaluation of two kinds of smoke‐water and 3,7‐dimethyl‐2H‐furo[2,3‐c]pyran‐2‐one

Smoke, smoke-water and aerosols have a stimulatory effect on seed germination and growth vigour of many seedlings, making them potentially useful for different purposes, provided they do not pose a health risk. Therefore, the genotoxicity of two kinds of smoke-water and 3,7-dimethyl-2H-furo[2,3-c]pyran-2-one, a variant of the most active smoke compound (3-methyl-2H-furo[2,3-c]pyran-2-one) was evaluated using the Vitotox assay. Smoke-water extracts were obtained from burning leaves: Themeda triandra (smoke-water Tt) and a mix of Themeda triandra and Passerina vulgaris (smoke-water Kb). No genotoxic effect was observed for any of the three samples. However, the three samples are toxic at the highest concentrations (3,7-dimethyl-2H-furo[2,3-c]pyran-2-one, 2 ppm; smoke-water Tt, dilutions 1 : 1, 1 : 2, 1 : 4; smoke-water Kb, dilution 1 : 1) without addition of S9 mix. Both the butenolide 3,7-dimethyl-2H-furo[2,3-c]pyran-2-one and smoke-water Tt are also toxic at high doses in the presence of S9 (2 ppm and dilutions 1 : 1 and 1 : 2, respectively), but not smoke-water Kb. Thus, from these results, no genotoxicity of these three samples can be assumed, which is accordance with the previous tests performed with 3-methyl-2H-furo[2,3-c]pyran-2-one and a smoke-water.

Estrogenic activity of 37 components of commercial sunscreen lotions evaluated by in vitro assays

Thirty-seven chemical components of commercial sunscreen lotions were evaluated for estrogen agonistic and/or antagonistic activity using two in vitro assays, (1) an ELISA-based estrogen receptor competitive binding assay (ER-ELISA) and (2) a modified yeast two-hybrid estrogen assay, with and without addition of a rat liver preparation, S9 mix. Eleven compounds, most of which were benzophenone derivatives and parabens, showed binding affinity to ER by ER-ELISA without S9 mix. Although the activities of almost all of the compounds were attenuated by addition of S9 mix, 4-octylphenylsalicylate and 2,2′-dihydroxy-4,4′-dimethoxybenzophenone acquired estrogenic activity, suggesting metabolic activation of these compounds. Two benzophenones showed agonistic activity in the yeast two-hybrid assay without S9 mix. The activity of one of these was reduced by S9 treatment and a further two benzophenones was activated. Eight parabens were active in this assay without S9 exposure, but their activities were eliminated by S9 treatment. Benzophenones with para-phenolic hydroxyl groups and parabens with branched and/or longer linear chains were generally more potent in both bioassays. In addition, weak antagonistic activity of 4- t-butylphenyl-salicylate, 2-ethylhexyl 4-dimethylaminobenzoate and (±)-α-tocopherolacetate was observed with S9 treatment. In vivo testing of the compounds reported here to have estrogen agonistic and antagonistic activities is required to confirm their effects at an organismal level.

Evaluation of Genotoxicity of 3-Amino-, 3-Acetylamino- and 3-Nitrobenzanthrone Using the Ames/Salmonella Assay and the Comet Assay

3-Nitrobenzanthrone (3-NBA) is an extremely potent direct-acting bacterial mutagen that has been detected in diesel exhaust particles, airborne particles and so forth. Recently, 3-aminobenzanthrone (3-ABA) and 3acetylaminobenzanthrone (3-AABA) were identified as metabolites of 3-NBA in mammalian cells. In this study, to clarify the genotoxicity of 3-ABA, 3-AABA and 3-NBA in vitro and in vivo, the mutagenicity and DNA-damaging activity of these chemicals were investigated by the Ames assay and by alkaline single gel electrophoresis (Comet assay), respectively. 3-ABA and 3-AABA were mutagenic toward four Salmonella typhimurium strains, i.e. TA98, TA100, YG1024 and YG1029, in the presence of a mammalian metabolic system (S9 mix). Both chemicals showed the highest mutagenicity toward YG1024, and induced 2180000 revertants/nmol of 3-ABA and 131000 revertants/ nmol of 3-AABA. 3-NBA also showed mutagenicity toward these four strains with and without S9 mix, but the potencies of 3-NBA in these strains were decreased by the addition of S9 mix. In the presence of S9 mix, the mutagenic activities of 3-NBA in four strains were comparable to or lower than those on 3-ABA. 3-ABA, 3-AABA and 3-NBA produced statistically significant DNA damage, which was detected by an increase in the DNA tail moment, in vivo 3 hr after intraperitoneal injection at 160 mg/kg body weight. 3-ABA, 3-AABA and 3-NBA induced significant DNA damage in the liver, kidney, spleen and lung, spleen only, and liver, kidney, lung and bone marrow, respectively. At a lower dose (40 mg/kg), 3-NBA produced significant DNA damage in the lung. These results indicate that 3-ABA, 3-AABA and 3-NBA are not only mutagenic in vitro in bacteria but also genotoxic in vivo in mouse.

Mechanisms of induction of chromosomal aberrations by hydroquinone in V79 cells.

Hydroquinone occurs naturally in bacteria and plants and it is also manufactured for commercial use. Human exposure to this compound can occur by environmental, occupational, dietary and cigarette smoke exposure and from exposure to benzene, which can be metabolized to this compound. However, the main source of exposure to this compound is dietary, since hydroquinone is a naturally occurring compound in many foods. Hydroquinone can be metabolized to benzoquinones, which are potent haematotoxic, genotoxic and carcinogenic compounds that can also induce the formation of radical species, predisposing cells to oxidative damage. In order to clarify the involvement of radical species in the genotoxicity of hydroquinone, the induction of chromosomal aberrations in V79 cells was studied along with the assessment of the production of hydroxyl radicals at different pH values (6.0, 7.4 and 8.0), as well as the effect of antioxidant enzymes [catalase and superoxide dismutase (SOD)] on the clastogenic effect of hydroquinone. The results obtained indicate that the clastogenic activity of hydroquinone is dependent on the pH, suggesting that deprotonation is a fundamental step leading to DNA lesions under the experimental conditions used. The addition of S9 mix, SOD or SOD and catalase significantly decreased the clastogenic activity, suggesting the involvement of superoxide anion and hydrogen peroxide in the genotoxicity of hydroquinone. However, other species generated in the auto-oxidation process of hydroquinone, such as the semiquinone radical or the quinone, also seem to play a role in its genotoxicity, since the addition of antioxidant enzymes (catalase and SOD) or S9 mix do not lead to a complete abolition of the observed genotoxic activity. These results suggest the existence of at least two mechanisms associated with the genotoxic activity of this compound.

環境水のエストロゲン様活性と内分泌撹乱化学物質との関連性

To clarify the contribution of endocrine-disrupting chemicals (EDCs) to the estrogenicity of environmental water, its XAD-2 resin column-concentrates, as well as steroid hormones, alkylphenols and phytoestrogens were assayed by the yeast two-hybrid system with and without S9mix. Lake water, river water, tap water and sewage treatment water exhibited estrogenicity that was enhanced by S9mix. The strong affinity for the estrogen receptor in yeast was observed with steroid hormones containing the 3-position phenolic and 17-position alcoholic hydroxyl groups, and alkylphenols. Among the estrogens, the magnitude of estrogenicity was in the order of: E2>E1>>E3. The estrogenicity of glucuronide or sulfateconjugated estrogens was less than that of free estrogens. However, the estrogenicity of the conjugates of E1, E2 and E3 was enhanced by S9mix. This suggests that the estrogenicity of environmental water enhanced by the addition of S9mix may be due to the estrogen conjugates in the water. The attenuation of E2's estrogenicity by S9mix was also shown that is predicted to be due to its hydroxylation by P450 induced specifically by phenobarbital. The E2-conjugate may be decomposed by some hydrolase expressed primarily in rat liver.

Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents: Part II: Alkylation far away from the amino function

Alkyl and trifluoromethyl derivatives of 4-aminobiphenyl ( 1) (4ABP) and 2-aminofluorene ( 7) (2AF) were synthesised and assayed for mutagenicity using Salmonella typhimurium tester strains TA98 and TA100 with and without the addition of S9 mix. Modification of 1 was achieved by attachment of alkyl groups (methyl, ethyl, iso-propyl, n-butyl, tert-butyl) and a trifluoromethyl group (CF 3) in the 4′-position, the 3′-position (Me, CF 3) and the 3′-, 5′-positions (DiMe, DiCF 3). Compound 7 was modified by introduction of alkyl groups (methyl, tert-butyl, adamantyl) and a trifluoromethyl group (CF 3) in the 7-position. The derivatives of 1 and 7 show for groups with growing steric demand decreased mutagenic activity. The bulkiest groups (CF 3, tert-butyl and adamantyl) induce the strongest effects on the mutagenicity. It was even possible to eliminate the mutagenicity of 1 and 7 by introduction of such substituents. In the last part of the work, we compared the experimental mutagenicities with calculated values derived from QSAR correlations. Our findings show that the predictions for aromatic amines with bulky substituents were generally too high. The strongest deviations were observed in the case of the CF 3-, tert-butyl- and the adamantyl-group. Only the parent compounds and derivatives with small alkyl groups were predicted well. These investigations show that “large” substituents have an influence on the mutagenicity caused by their steric demand. To predict the correct mutagenicities of such compounds, it is necessary to introduce steric parameters in the respective QSAR equations which will be done in a forthcoming paper.

Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour. The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group ( ortho-position) disturbs the enzymatic reduction of the nitro group, because 3- tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3- tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4′- tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group “far away” from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.

酵母Ｔｗｏ‐ｈｙｂｒｉｄ法を用いた環境試料のエストロゲン様活性評価に関する研究

A number of bioassays for screening the compounds for estrogenic activity have been proposed so far, however, a yeast two-hybrid system appears more simple and inexpensive assay with high repeatability. The biggest problem of this bioassay is that it cannot measure estrogenic activity in some of the environmental samples. At is was assumed that the co-existing compounds were responsible for these false negatives, two methods were tested:1. Separation of such co-existing chemical interferences by use of liquid chromatographic method.2. Masking the retarding effect of such co-existing chemicals interferences by the addition of S9mix.The results indicated that both methods were effective to identify the estrogenic activity, however, the addition of S9mix was much more simple to conduct and more effective to determine the estrogenic activity in environmental samples.

個体毒性の検証に基づくｉｎ ｖｉｔｒｏ簡易毒性評価系の確立

For In Vitro bioassay system to evaluate water environment, it may be important to correlate In Vitro data closely to whole body toxicity. In order to establish the evaluation system for In Vitro bioassay, we focused on oxidative stress and investigated toxicities of organic halogenated compound using both In Vitro micronucleus test and in vivo hepatic injuries test.Micronuclei formation of HeLa cells was induced by chlorophenols and the induction was enhanced with addition of S9mix. The enhancement was inhibited by scavengers of hydrogen peroxide, hydroxyl radical or superoxide radical, indicating that micronuclei formation of HeLa cells was induced by oxidative stress which was caused with metabolites of chlorophenols.Oral treatment of 2,4,6-trichlorophenol or carbon tetrachloride increased the amount of serum GOT. in vivo free radical reactions which were estimated with in vivo ESR technique were also enhanced specifically in upper abdomen, indicating that metabolites of these compounds caused liver injuries through oxidative stress.The present study demonstrated the evaluation system of In Vitro and in vivo bioassays concerning oxidative stress caused with organic halogens. In Vitro bioassay system will be more valuable and reliable, comparing In Vitro data with in vivo one using indices of same mechanism of toxicity.

Induction or suppression of SV40 amplification by genotoxic carcinogens, non-genotoxic carcinogens or tumor promoters

Carcinogens are generally classified into two groups: genotoxic and non-genotoxic. As the final product of genotoxic and non-genotoxic carcinogens is the same, i.e., a clone of genetically altered cells, it could be possible that non-genotoxic carcinogens may yield genotoxic events as a secondary result of cell toxicity having led to mitogenesis/cellular proliferation, or that genetic alterations are induced that are normally neglected in genotoxicity tests. A genetic effect with possible relevance for the ultimate mechanism of carcinogenicity is the amplification of oncogenes. In the present experiments, amplification of SV40-virus DNA in transformed CO631-cells has been measured. In this system, two genotoxic carcinogens (positive control), two non-carcinogens (negative control), and eight non-genotoxic carcinogens have been tested. With the exception of testosterone and BVDU, all substances were tested when given alone. Testosterone and BVDU were tested in combination with either MTX or AMP in the presence or absence of S9-mix. The genotoxic carcinogens TEM and 4-NQO as well as the tumor promoters TPA, coumarin, mezerein and chrysarobin were able to induce SV40 amplification when given alone. The same was for acetamide and thioacetamide. The non-carcinogens acetone and dimethylformamide were ineffective. In case of testosterone, a co-amplification effect was seen in the absence of S9-mix, and an anti-amplification effect in presence of S9-mix. The anti-recombinogen BVDU reduced the SV40 amplification effect of AMP in experiments without addition of S9-mix. In the presence of activating S9-mix, this substance was effective at lower concentrations than without S9-mix, and showed an enhanced anti-amplification effect. Similar effects of testosterone and BVDU had been observed before in respect to stimulation or suppression of recombination. These results can be used as supportive evidence for the assumption that gene amplification is mechanistically related to recombination.

Unexpected genetic toxicity to rodents of the N′, N′-dimethyl analogues of MNU andENU

Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU [N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity. Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the -NH2 group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained.

Genotoxicity of Some Metal-Based Antineoplastics, Evaluated by SOS Chromotest and Cytogenetic Analysis

The paper reports the screening results of two metal-based antineoplastic drugs with mutagenic potential, such as Romcis (trademark of Cisplatinum, produced in Romania) and diphenylantimony(III) diisopropyldithiophosphate (PADTF). Their effects were compared with those induced by Cyclophosphamide. Two mutagenicity tests, the SOS Chromotest and cytogenetic analysis were applied. The tests were carried out with or without metabolic activation (addition of S9-mix), either in E. coli PQ 37 cultures, using four doses (0.3, 3, 30 and 300 pmol compound/assay) for the SOS Chromotest or in leukocyte cultures using 0.3 mM from each compound, for cytogenetics. The dose- response relationships and SOSIP values revealed an indirect mutagenic potential for Cyclophosphamide, amplified by S9 mix in bacterial cultures and an antiproliferative, clastogenic effect on lymphocytes. For Romcis and diphenylantimony(III) diisopropyldithiophosphate, a significant positive response by SOS Chromotest was recorded, which correlated with increased frequencies of chromosomal aberrations.

The use of the Salmonella BA9 forward mutation assay in sediment quality assessment: mutagenicity of freshly deposited sediments of the River Elbe

The aim of the present study was to investigate whether bacterial mutagenicity assays can be applied in sediment quality assessment. The arabinose-sensitive forward mutation strain Salmonella typhimurium BA 9 was added to freshly deposited sediments of the River Elbe, collected during March, April, and May 1994. Up to twelve locations were sampled each month and mutagenicity was determined employing the solid phase and sequentially prepared toluene- and methanol-extracts of the sediments. Mutagenicity was detected at all sites within the solid sediments without addition of S9-mix; however, the addition of mammalian enzymes (S9) enhanced the mutagenic effect. In contrast, mutagenicity of toluene extracts containing the lipophilic fraction of the sediment samples was higher in the absence of S9-mix; peaks of mutagenic activity in these samples were observed at Dessau (mouth of the River Mulde) and close to the city of Hamburg (Bunthaus). Similar results were obtained with methanolic extracts of the sediments, although the effects were usually lower in comparison to the corresponding toluene extracts. These results show that the mutagenicity assays are capable of assessing water/sediment contamination and reveal that the mutagenicity detected in sediments reflects local industrial activities as well as hydrologic conditions.

Mutagenicity of nitrodibenzopyranones in the Salmonella plate-incorporation and microsuspension assays

The mutagenicity of three isomers of nitro-6 H-dibenzo[ b, d]pyran-6-one (NDBP), 2-NDBP, 3-NDBP and 4-NDBP, was characterized in the plate-incorporation (PI) and microsuspension (MS) assay using Salmonella typhimurium tester strains in the presence or absence of S9 mix. In both assays, all of the NDBPs showed mutagenicity in every strain. In the absence of S9 mix, TA98 was the strain most sensitive to the mutagenicity of NDBPs. The activity of NDBPs was reduced in TA98NR and TA98/1,8-DNP 6 strains relative to TA98, suggesting that NDBPs cause frameshift mutation and that nitroreduction by ‘classical’ nitroreductase and acetylation are significant steps for their metabolic activation. Mutagenic potency of NDBPs in TA98 without S9 mix in the MS assay (2-NDBP 104 300 rev./μg, 3-NDBP 23 500 rev./μg, and 4-NDBP 15 300 rev./μg) was much higher than that in the PI assay (2-NDBP 38 rev./μg, 3-NDBP 162 rev./μg, and 4-NDBP 7 rev./μg). Although additional S9 mix increased the mutagenicity of NDBPs in the PI assay, the mutagenic potency of NDBPs in the MS assay using strains TA98 and TA100 was decreased by the addition of S9 mix. In the PI assay, frameshift and base-substitution activities of both isomers were enhanced by the addition of the pKM101 plasmid, suggesting the induction by these isomers of complex frameshifts (frameshifts with associated base substitutions) in strain TA98. In the PI assay, 2-NDBP generally exhibited more base-substitution than frameshift activity; however, the reverse was true for 3-NDBP. In the MS assay, both isomers exhibited more frameshift than base-substitution activity.

Mutagenic activity of dopamine after nitrosation

Dopamine hydrochloride is reported to be a new mutagen precursor in this study. After treatment with nitrite under acidic conditions, dopamine hydrochloride showed direct-acting mutagenicity on Salmonella typhimurium TA100, TA98 and Escherichia coli WP2uvra. The addition of S9 mix did not affect the mutagenicity of nitrosated dopamine significantly in these three strains. Meanwhile, a comparison of the mutagenicity of nitrosated dopamine with nitrosated tyramine was carried out.

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.

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.

Bacterial mutagenicity of 2-chloro-1,3-butadiene (chloroprene) caused by decomposition products.

Since the literature on genotoxicity of 2-chloro-1,3-butadiene (chloroprene) is controversial, the mutagenicity of this compound was reinvestigated with respect to its chemical stability. Because of the volatility of chloroprene, Ames tests with S. typhimurium TA 100 were carried out with gas-tight preincubation. Propylene oxide, a volatile direct mutagen, served as a positive control. Benzo[a]pyrene was used as a control for an indirect mutagen. Using this experimental regimen, freshly distilled chloroprene was not mutagenic. However, a mutagenic effect occurred linearly with increasing age of the chloroprene distillates. Aged chloroprene gave the same positive results whether preincubation was gas-tight or not. Analysis by gas chromatography (GC) revealed several decomposition products in aged chloroprene distillates. The direct mutagenicity towards TA 100 correlated with the integrated amounts of four of these substances; these substances always occurred in the same relative ratio. When chloroprene was kept under anaerobic conditions, products occurred with time which were partly different from those obtained under aerobic conditions. The direct mutagenicity of anaerobically aged chloroprene was only weak, but the mutagenic effect was enhanced about two- to threefold by addition of S9 mix. Partial identification of chloroprene decomposition products was done by gas chromatography-mass spectrometry (GC-MS): major byproducts of chloroprene, probably responsible for mutagenic properties of aged chloroprene samples, were cyclic chloroprene dimers.

Effects of 2-chlorodibenzofuran on fetal development in mice.

2-Chlorodibenzofuran (2-MCDF), a monochlorinated derivative of dibenzofuran, has been detected as a contaminant in chlorinated tap water in Japan. Shiraishi et al. (1985) analyzed polynuclear aromatic hydrocarbons in Tsukuba tap water and detected 0.04-1.4 ng/L of 2-MCDF. This means that unintended human exposure to 2-MCDF has occurred, and therefore safety evaluation of 2-MCDF is needed. Toxicological information on 2-MCDF is limited and available only on mutagenicity and metabolic fate. In the Ames test, 2-MCDF showed weak mutagenic activity on Salmonella typhimurium strain TA 98 above 0.4 {mu}mol/plate, but not on strain TA 100 even at 10 {mu}mol/plate, and this activity was diminished by the addition of S9 mix. After intravenous or oral administration to rats, 2-MCDF is rapidly metabolized and excreted in bile and urine. Major metabolites in the rate bile fluid were 2- and/or 7-hydroxylated forms of 2-MCDF. For developmental toxicity, we previously examined the embryotoxicity of 2-MCDF by using post-implantation rat embryo culture. When day 9 embryos were continuously exposed throughout 48 hr culture period either in the presence or in the absence of a metabolic activation system, 2-MCDF at 1 mM was embryotoxic and caused morphological abnormalities of the embryos. However, dosing of up to 1000 mg/kg/daymore » of 2-MCDF to pregnant rats during days 9 to 11 of gestation, corresponding to the culture period, had no effects on the embryo-fetal growth. It was tentatively concluded from these results that 2-MCDF had weak-inactive teratogenicity in rats. In the present study, we further examined fetal effects of 2-MCDF by a teratogenicity test in which the dosing period covers the major organogenic period. We used mice, since they have been used for the evaluation of teratogenicity of polychlorinated congeners of 2-MCDF, because of their higher susceptibility than that of rats. 16 refs., 1 fig., 3 tabs.« less