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1.
An XAD-2 resin concentrate of chlorination-stage pulp mill effluent was found to induce mutations in Salmonella typhimurium strains TA1535, TA100 and TA98 but not in strains TA1537 or TA1538. The presence of either S9 mix, S9 mix without cofactors, or heat-inactivated S9 mix, reduced the mutagenic effects. Dose-related increases in gene conversion, mitotic recombination and aberrant colony formation in Saccharomyces cerevisiae strain D7 also were found.  相似文献   

2.
Three pyrrolizidine alkaloids (PAs), monocrotaline, retrorsine and isatidine, were tested for their clastogenic activity under different conditions of metabolic activation in vitro. All three compounds exhibited a weak activity when V79 cells were treated at very high concentrations for 18 h in the absence of a metabolizing system. Short-term (2 h) treatment with rat liver S9 mix led to a strong and concentration-dependent increase in chromosomal aberrations for retrorsine. Isatidine was not mutagenic with S9 mix and monocrotaline was positive at high concentrations only. In contrast, a prolonged treatment (18 h) in vitro under activation conditions in the presence of primary hepatocytes led to clear concentration-dependent positive responses for all three PAs investigated. Particularly the results with isatidine demonstrate that in vitro tests using S9 mix for metabolization can generate misleading results. It is not clear whether the results could be attributed to a better activation of the test compounds by intact hepatocytes in comparison to S9 mix or if the fact that only hepatocytes allow a treatment for the whole culture period under activation conditions was more important. Owing to its strong cytotoxicity the exposure to S9 mix is generally limited to 2-4 h, limiting also the exposure of the target cells to a test chemical as well as its metabolites. The results presented show significant differences in mutagenic potency of PAs due to variations in the activation system. This underlines the usefulness of primary hepatocytes, e.g., for the detection of pre-mutagens. The PAs investigated are present in plants which are used for phytotherapeutic medicinal products. They do not contribute to their efficacy and are, therefore, not to be tolerated in amounts that may impose a risk for the user.  相似文献   

3.
A metabolic activation system with rat-liver microsome fraction plus cofactors (S9 mix) was applied to chromosomal aberration tests in vitro for the screening of chemical mutagens or carcinogens in the environment. Dialkylnitrosamines only induced chromosomal aberrations in Chinese hamster cells (CHL) when treated with S9 mix. The incidence of chromosomal aberrations in CHL varied with experimental conditions, e.g. incubation time, recovery time, components of S9 mic and inducers used for preparation of S9. For dimethylnitrosamine (DMN), the maximal incidence was obtained when the cells were incubated with S9 mix for 3 h and harvested 24 h after treatment. Therefore, this system (3 h incubation and 24 h recovery) was routinely applied to further screening of other chemicals with S9 prepared from PCB-pretreated rats. 10 carcinogens (e.g. 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, quinoline, etc.) out of 16 induced aberrations when they were treated with S9 mix, whereas the remaining 6 carcinogens (e.g., 3-methyl-cholanthrene, 4-o-tolylazo-o-toluidine, etc.) induced few or no aberrations even after activation. Two insecticides, allethrin and diazinon, were strongly positive at relatively low doses only when they were activated with the S9 mix. Medical drugs, such as ethenzamide, methyl p-hydroxybenzoate and nitrofurazone, and a food additive, sodium hypochlorite, were positive on activation. Chemicals used for industry, such as styrene monomer and tris-dichloropropylphosphate, were also positive in our activation system.  相似文献   

4.
Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.  相似文献   

5.
The mutagenic activity of quercetin for Salmonella typhimurium TA98 was inhibited by addition of metal salts. MnCl2 was a potent inhibitor, followed by CuCl2, FeSO4, and FeCl3, the probable mechanism being facilitated catalytic oxidation of quercetin. With quercetin incorporated at a level of 100 nmoles/plate, approximate doses (nmoles/plate) to give 50% inhibition of mutagenic activity were: MnCl2 less than 10 (-S9), 18 (+S9); CuCl2 65 (-S9), greater than 100 (+S9); FeSO4 190 (-S9), greater than 300 (+S9); or FeCl3 275 (-S9), greater than 300 (+S9). Ascorbate, superoxide dismutase, and, to a lesser extent, NADH and NADPH, all enhanced the mutagenic activity of quercetin in the absence of the mammalian-microsome (S9) system, but had no significant effect in the presence of the S9 mix. The maximum enhancement of activity by ascorbate or superoxide dismutase was approximately 87% of the increase achieved by addition of the S9 mix. Tyrosinase (catechol oxidase) substantially reduced the mutagenic activity of quercetin in the absence of the S9 mix. At lower levels of tyrosinase, activity was restored by incorporation of the S9 mix. It is proposed that the S9 mix enhances the mutagenic activity of quercetin by scavenging superoxide radicals, thus inhibiting the autoxidation of quercetin, and possibly by reducing quinone oxidation products of quercetin. The mutagenic activity of quercetin increased substantially when the pH of the media was decreased. This may be due in part to a decrease in ionization of quercetin at lower pH, thereby increasing its absorption by the tester strain, to a decrease in the rate of autoxidation of quercetin at lower pH, or to a combination of these.  相似文献   

6.
Hydroxyurea treatment affects the G1 phase in next generation CHO cells   总被引:1,自引:0,他引:1  
DNA replication kinetics were studied in populations of synchronized CHO cells treated in the previous generation with hydroxyurea. These CHO cells were re-synchronized by selective detachment of mitotic cells after previously synchronized G1 traversing cultures were treated with 0.1 mM and 2 mM hydroxyurea for 9 and 13 h. Our results show that these cells exhibit a shortening of G1 of at least 1 h relative to cells selected in mitosis from untreated exponentially growing cultures. Survival studies indicated that the hydroxyurea treatments did not affect plating efficiencies. Cell viability was reduced when the initially synchronized populations were blocked with 2 mM, but not 0.1 mM hydroxyurea for greater than 13 h. DNA replication measurements after these blocks showed that all cultures treated with 2 mM hydroxyurea for either 9, 13 or 15 h were blocked at the same point near the G1/S boundary, and then progressed through S phase with similar kinetics. The observed shortening of G1 in the next generation of these cells was independent of both the concentration (0.1 or 2.0 mM) and the time (9 or 13 h) of the hydroxyurea block. These results suggest that specific events relating to the next cell generation can be uncoupled from DNA synthesis and can occur when hydroxyurea inhibits normal cell cycle traverse of G1 cells into and through S phase.  相似文献   

7.
The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.  相似文献   

8.
The caffeine derivative 8-ethoxycaffeine (EOC) was tested in 3 different test systems in vitro. Each experiment was carried out with and without S9 mix. Incubation temperatures were 20 and 37 degrees C. (1) In the Salmonella/microsome test, EOC behaved as a pro-mutagen in the Salmonella typhimurium strain TA1535. No mutagenic activity was found in experiments without S9 mix. The influence of temperature was negligible. The mutagenic activity of EOC depended mainly on the mammals used to prepare the S9 fraction and on the agents given to them to induce liver enzymes. (2) EOC did not induce sister-chromatid exchanges in cell cultures, either at 20 or at 37 degrees C. (3) On the other hand, EOC induced chromosomal aberrations when the cells were incubated at 37 degrees C without S9 mix.  相似文献   

9.
When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.  相似文献   

10.
3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.  相似文献   

11.
Liver S9 fractions were prepared from male Wistar rats, either non-induced or induced with Aroclor 1254 and from 5 human kidney transplant donors. The preparations were compared for their ability to metabolize the premutagens present in coal tar to mutagenic metabolites in the Salmonella mutagenicity assay towards strain TA98. Low levels of mutagenicity of coal tar were seen with human S9 preparations. The differences between the S9 mix of the 5 donors in capacity to activate premutagens were approximately 6-fold. The activation of coal tar by rat liver S9 preparations was higher than by the human S9 preparations. The metabolic conversion of pyrene in coal tar to 1-hydroxypyrene by the same human S9 preparations was determined in a parallel assay. 3 human preparations showed a high correlation between the formation of 1-hydroxypyrene and bioactivation of coal tar to mutagenic metabolites. The slope values of the individual regression lines were equal, suggesting that 1-hydroxypyrene is a good indicator for the activation of premutagens present in coal tar.  相似文献   

12.
Fourteen new quinoline derivatives were synthesised and their mutagenicity compared in the Ames test using Salmonella typhimurium TA100 as indicator strain with and without (Aroclor-induced) S9 mix. None of the synthesised quinoline derivatives had to our knowledge been examined before in the Ames test. Quinoline and the monohydroxyquinolines were included as reference compounds. Three of the new derivatives, i.e., quinoline 7,8-oxide, N-methyl-quinoline 5,6-oxide and trans-quinoline-5,6,7,8-dioxide appeared to be mutagenic. Quinoline 7,8-oxide was positive only in the presence of S9 mix, the specific mutagenicity amounting to 2498 +/- 96 and 1289 +/- 120 revertants per mumole with 20 and 10% S9 in the mix, respectively. Both N-methyl-quinoline 5,6-oxide and trans-quinoline-5,6,7,8-dioxide were weakly positive, the former only in the presence of the S9 mix, and the latter irrespective of the presence of S9 mix, the specific mutagenicity amounting to 134 +/- 6 and 123 +/- 10 revertants per mumole, respectively. The mutagenic potency of quinoline 7,8-oxide was of the same order as that of quinoline itself and was distinctly lower than that of 8-hydroxyquinoline. Inconclusive results were obtained with trans-7,8-dihydroxy-7,8-dihydroquinoline, 5,6-dihydroxy-7,8-epoxy-5,6,7,8-tetrahydroquinoline and 8-hydroxyquinoline-N-oxide; if these compounds are mutagenic their mutagenic potency would be at least 20-30 times lower than that of the parent compounds. None of the other chemically synthesised quinoline derivatives showed mutagenic activity with TA100 either in the presence or in the absence of S9 mix. The results obtained with the reference compounds were in accordance with literature data.  相似文献   

13.
The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.  相似文献   

14.
Zhang Z  Fu J  Yao B  Zhang X  Zhao P  Zhou Z 《Mutation research》2011,722(1):39-43
To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.  相似文献   

15.
Cyclophosphamide (CPA) and its main metabolites were analyzed with respect to their mutagenic activities in Salmonella, human peripheral lymphocytes (PL), and Chinese hamster ovary (CHO) cells. In Salmonella, the compounds were activated with S9 mix from rat livers, which were unstimulated or stimulated with Aroclor 1254 or phenobarbital. For the enzyme inducers the following order of efficiency was found for all test compounds except carboxyphosphamide: phenobarbital greater than Aroclor 1254 greater than non-induced. The most potent mutagens in all 3 test systems were 4-OH-CPA, PAM and nor-HN2. S9 mix transforms 4-OH-CPA to strong mutagenic compounds in the Salmonella assay. All metabolites tested in the Salmonella assay were activated by S9 mix to higher mutagenic potential.  相似文献   

16.
The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase (hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.  相似文献   

17.
In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2',4'-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO2 and Al2O3 column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2',4'-tetranitrobiphenyl with S9 mix at 37 degrees C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4'-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4'-diamino-2',4-dinitrobiphenyl by its chemical and physico-chemical properties.  相似文献   

18.
Sediments in estuaries are of important environmental concern because they may act as pollution sinks and sources to the overlying water body. These sediments can be accumulated by benthic organisms. This study assessed the mutagenic potential of sediment extracts from the Yangtze River estuary by using the Ames fluctuation assay with the Salmonella typhimurium his (−) strain TA98 (frameshift mutagen indicator) and TA100 (baseshift mutagen indicator). Most of the sediment samples were mutagenic to the strain TA98, regardless of the presence or absence of exogenous metabolic activation (S9 induction by β-naphthoflavone/phenobarbital). However, none of the samples were mutagenic to the strain TA100. Thus, the mutagenicity pattern was mainly frameshift mutation, and the responsible toxicants were both direct (without S9 mix) and indirect (with S9 mix) mutagens. The mutagenicity of the sediment extracts increased when S9 was added. Chemical analysis showed a poor correlation between the content of priority polycyclic aromatic hydrocarbons and the detected mutagenicity in each sample. The concept of effect-directed analysis was used to analyze possible compounds responsible for the detected mutagenic effects. With regard to the mutagenicity of sediment fractions, non-polar compounds as well as weakly and moderately polar compounds played a main role. Further investigations should be conducted to identify the responsible components.  相似文献   

19.
Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.  相似文献   

20.
Frequencies of sister-chromatid exchanges (SCE) were determined in second-division metaphases of human lymphocytes, exposed for 1 h during the G0 phase to mitomycin C (MMC) alone or to cyclophosphamide (CP) in the presence of S9 mix. The cells were then cultured for 72 h in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA) or Lens culinaris (LcH-A) extracts. Large differences in mitotic indices (MI) and cell-cycle kinetics were observed among cells subjected to the various treatments. However, in the controls as well as in the cultures submitted to a G0 mutagenic exposure, the yield of SCE was not influenced by the mitogenic agent and was, therefore, independent of the proliferation properties of the cultured lymphocyte population.  相似文献   

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