Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.     

Mutagenicity of several derivatives of dipyrido[1,2-a:2'',3''-d]imidazoles

The mutagenicity of 2-aminofluorene, 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl towards Salmonella typhimurium was studied in the presence of microsomes from liver, kidney and small intestine of untreated and pretreated rats. The aim was to study a possible correlation between the organotropism of these amines and their activation into mutagenic intermediates by these three tissues. Pretreatment of the rats with phenobarbital, Aroclor 1254 and 3-methylcholanthrene injected intraperitoneally increased the liver microsomal-mediated mutagenic activity of the three amines but remained without effect on the activating capacity of microsomes from the kidney and small intestine. However, pretreatment with 3-methylcholanthrene administered intragastrically increased the small-intestine microsomal-mediated mutagenicity of 2-aminofluorene almost 3-fold but remained without effect on the mutagenicity of 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl. No mutagenic effect was observed with 4-aminobiphenyl in the presence of kidney microsomes or with 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl in the presence of small-intestine microsomes, obtained from either untreated or pretreated animals. It is concluded that no relationship exists between the mutagenic activities of the three amines, as detected in the Ames test, and their carcinogenic organotropisms.     

Mutagenic response of mouse lymphoma cells after activation of benzidine and 2-aminofluorene with purified prostaglandin H synthase

The mutagenic response of L5178Y mouse lymphoma cells to the model aromatic amine carcinogens, benzidine (BNZ) and 2-aminofluorene (2-AF) in the presence of the mammalian peroxidase prostaglandin H synthase (PHS) was examined. Standard incubation conditions for mouse lymphoma cells and the PHS system were developed. The cells were exposed to BNZ and 2-AF with purified PHS in the presence or absence of a peroxide, 5-phenyl-4-pentenyl hydroperoxide (PPHP) which is required for PHS-dependent amine oxidation. Incubations were carried out in a medium consisting of Hanks' balanced salt solution with calcium and magnesium and 0.1% pluronic F-68. BNZ by itself or in the presence of PPHP induced a weak mutagenic response in mouse lymphoma cells, but the addition of PHS or PHS and its co-factor PPHP increased the mutagenic response approximately 5-fold over that observed in the absence of PHS. A maximal mutagenic response for BNZ was observed after incubation with the complete activating system, PHS and PPHP. These data are in agreement with the fact that BNZ is an excellent substrate for PHS. When 2-AF was incubated with mouse lymphoma cells, only a minimal mutagenic response was observed. Incubation of 2-AF with either PPHP or PPHP and PHS (complete peroxidase system produced a significant enhancement in mutagenic response. Thus, the mutagenic response of the mouse lymphoma cells to 2-AF was dependent on the peroxide, PPHP but not the enzyme PHS. These data suggest that 2-AF, which is a poor PHS substrate, is oxidized by a different catalyst than PHS. This work demonstrates that BNZ and 2-AF are converted by peroxide-dependent mechanisms to mutagens that can be detected in mammalian cells.     

Activation of some aromatic amines to mutagenic products by human red blood cell cytosol.

The ability of human red blood cell cytosol to activate aromatic amines was evaluated with the Ames test using Salmonella typhimurium TA98 in the liquid preincubation condition. While negative results were obtained with 4-acetylaminofluorene (4AAF) and 1-naphtylamine (1NA), a slight response was observed for 4-aminobiphenyl (4ABP) and 2-naphthylamine (2NA). Human red blood cell cytosol was able to activate 2-aminofluorene (2AF), 2-acetylaminofluorene (2AAF) and 2-aminoanthracene (2AA) to mutagenic intermediates. Extracts of human red blood cell cytosol incubated with 2AF were analyzed by gas chromatography: N-hydroxy-2-aminofluorene was identified as a metabolite.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.     

Mutagenicity towards Salmonella typhimurium of some known genotoxic agents,activated by isolated hepatocytes of monkey (Macaca fascicularis): Comparison with isolated human hepatocytes

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium.Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N′-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.     

Metabolic activation of promutagens in the yeast-microsome test. II. Activation of cyclophosphamide and 2-aminofluorene

The effect of recombinogenic and mutagenic activation of promutagens, cyclophosphamide and 2-aminofluorene was studied in S9 mouse liver preparations. Cyclophosphamide was activated to induce reversions of an ochre mutation and heteroallelic reversion in yeast tester strains r2089-15V-P4 and P3288, respectively. Metabolic activation of this chemical was greatly enhanced by pretreatment of mice with AROGLOR--1254. 2-aminofluorene was a potent recombinogen after metabolic activation, but proved a poor inducer of reversions of the ochre mutation. For the stationary yeast cells, activated 2-aminofluorene was shown to be not recombinogenic, while for logarithmic cells grown in galactose medium it was moderately recombinogenic, being highly active in this respect for logarithmic cells grown in glucose medium. We recommend to use these compounds as positive controls for exogenous activation in the yeast/microsome test.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.     

Activation of aromatic amines to mutagens by various animal species including man

2-Acetylaminofluorene, 2-aminofluorene, 4-aminobiphenyl, 2-naphthylamine, 2-aminoanthracene and benzidine were assayed for mutagenicity in the Ames test in the presence of hepatic microsomal preparations derived from mouse, hamster, rat, pig and man. Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenase and mixed-function amine oxidase activities. All compounds were metabolically activated to mutagens by all activation systems, but with markedly different efficiencies, hamster being the only species which readily activated all amines. The hamster also exhibited the highest ethoxyresorufin O-deethylase and dimethylaniline N-oxidase activities.     

Effects of oxygen radical scavengers and antioxidants on phagocyte-induced mutagenesis

Phagocytic leukocytes from normal humans can produce mutations in bacteria. To define further the role of oxygen radicals in this mutagenic process, we performed experiments in which scavengers or antioxidants were added to the incubation of phagocytes and bacteria. We found that 1) superoxide dismutase, catalase, mannitol, and benzoate were all capable of inhibiting mutation, 2) sulfhydryl compounds and vitamin E were also inhibitory, and 3) the presence of vitamin C in the incubations increased the mutation frequency. These data suggest an important role for hydroxyl radicals in mediating phagocyte-induced mutations.     

Mutagenicity of benzidine and 4-aminobiphenyl after metabolic activation with isolated hepatocytes and liver 9000 × g supernatant from rat,hamster and guinea pig

The mutagenicity of benzidine and 4-aminobiphenyl towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from rat, hamster and guinea pig. The mutagenic potency of these compounds was also assayed with S9 (9000 × g supernatant) prepared from disrupted hepatocytes of these aryl amines was investigated.For all 3 animal species it was found that the mutagenicity of benzidine is higher with intact hepatocytes than with S9 prepared from disrupted hepatocytes. Addition of acetyl coenzyme A to the S9 fraction increased the mutagenicity of benzidine. In contrast to benzidine, the mutagenicity of 4-aminobiphenyl appeared to be lower with hepatocytes than with S9. Addition of acetyl coenzyme A to the S9 fraction decreased the mutagenicity of 4-aminobiphenyl.The mutagenic potency of 4-aminobiphenyl was almost equal in the presence of the liver preparations from the 3 different species, whereas obvious species differences were seen with benzidine.     

Evaluation of the mutagenicity of 'pan masala', a chewing substitute widely used in India

Mutagenicity of polar and non-polar extracts of a popular brand of 'pan masala' was examined using the Salmonella/mammalian microsome test (Ames assay) and 2 tester strains of Salmonella typhimurium, TA98 and TA100. These extracts were also subjected to pretreatment with sodium nitrite at acidic pH, to simulate conditions for endogenous nitrosation. The aqueous, aqueous:ethanolic and chloroform extracts as well as their nitrosated mixtures were non-mutagenic in the Ames assay, in the presence and absence of metabolic activation. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation demonstrating the presence of direct-acting frameshift mutagens in 'pan masala'.     

龈下菌斑涂片两种染色分类方法的比较

目的:用革兰染色和刚果红负染两种不同的染色方法对同一龈下菌斑样本进行分类计数,比较两种分类方法的优缺点,提出应用中的问题以及解决方法.方法:随机抽样采集40例牙周病患者的80个位点龈下菌斑,同一标本进行生理盐水涂片革兰染色和刚果红负染.光学显微镜油镜(15×100)镜检共计数200个细菌,包括5种不同形状细菌.采用SPSS 10.01统计软件,对数据进行分析.结果:两种染色分类方法计数统计结果为螺旋体、弯曲菌、梭形菌3种菌数值P＞0.05无统计学意义,而球菌、杆菌2种菌数值经统计P＜0.01有高度统计学意义.结论:刚果红负染简便易行,但龈下菌斑球菌、杆菌进行百分计数时,不适宜用刚果红负染法,应根据实验目的选择恰当的方法.     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

Analysis of Congo red-induced changes in the cell surface and macrocolony structure of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Adsorption of the vital dye Congo red suppresses swarming of Azospirillum brasilense in a semiliquid medium, and the bacteria become able to spread with the formation of microcolonies. By using direct and stereoscopic light microscopy, the patterns of the front of Azospirillum spreading in a semiliquid medium containing the dye were analyzed. It was found that in a medium with Congo red, small motile colonies were formed among the individual cells, and once formed, they left the boundaries of the swarming front. The microcolonies produced by azospirilla in the presence of the dye were ordered bacterial structures, rather than random cell aggregates. Transmission electron microscopy revealed that the cells grown without the dye had polar flagella, whereas the cells from the medium with Congo red had no flagella and were covered with a layer of fibrillike material. Immunochemical data for the cell surface changes resulting from interaction with the dye make it possible to consider Azospirillum lipopolysaccharide as a probable Congo red receptor.     

The importance of 2-aminofluorene in the mutagenic activation of 2-acetylaminofluorene

The mutagenic activation of 2-acetylaminofluorene (AAF) and its derivatives N-hydroxy-AAF and 2-aminofluorene (AF) by pulmonary and hepatic microsomal fractions from untreated rabbits was investigated using Salmonella strain TA98. The mutagenicity of AAF in the presence of hepatic microsomes followed typical saturation kinetics. However, in the presence of pulmonary microsomes, the mutagenic activity increased linearly with increasing substrate concentration and approximated that obtained with low concentrations of AF. N-Hydroxy-AAF was 1/10th as mutagenic as AF in the presence of pulmonary microsomes, but 2-2.5 times more mutagenic than AF in the presence of hepatic microsomes. The activation of AAF by both fractions was completely inhibited by the deacetylase inhibitor paraoxon. Although AAF does not appear to be a substrate for cytochrome P450 form 5, antibodies to this form inhibited the activation of AAF by pulmonary and hepatic microsomes by 90% and 60%, respectively. These results indicate that the mutagenic activation of AAF by these fractions primarily involves deacetylation to AF, followed by cytochrome P450 form 5-mediated activation of AF.     

Quantitative evaluation of intracellular degradation in Entamoeba invadens

A quantitative study on digestion of erythrocytes by Entamoeba invadens was attempted. Trophozoites of the IP-1 strain were fed red blood cells for 30 min, and subsequently phagocytosis was stopped by means of osmotic shock; post-phagocytosis incubations for up to 15 h were made in order to evaluate intracellular digestion, after staining the red blood cells with benzidine. Eighty-two per cent of trophozoites were capable of phagocytosing erythrocytes, containing an average of 5.5 erythrocytes per amoeba. Erythrocyte digestion within amoebae was shown by loss of benzidine-stainable material and proceeded with a first-order kinetics, with a t1/2 approximately 7 h. Within 15 h there were no amoebae containing erythrocytes. The procedure described may be useful for the evaluation of intracellular digestion in other Entamoeba species.     

Decolorization and biodegradation of the azo dye Congo red by an isolated <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> YNWH 226

A strict aerobic Acinetobacter baumannii YNWH 226, isolated from an activated sludge reactor treating textile wastewater, was able to grow on Congo red as the sole carbon source under aerobic conditions. The decolorization and TOC reduction efficiency were 99.1 and 93.72%, respectively. The effects of the Congo red concentration were studied. The environmental factors (i.e., pH, temperature and agitation speed) on the biodegradation of Congo red in aqueous phase were studied and evaluated using response surface methodology. The results indicated that when the Congo red concentration was 100 mg/L, the optimal decolorization conditions were as follows: 37°C, pH 7.0 and 180 rpm. The single A. baumannii YNWH 226 was able to form aromatic amines by reductive breakage of the azo bond and then oxidize them into non-toxic metabolites.     

Understanding the degradation of Congo red and bacterial diversity in an air–cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation

We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air–cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.