Mutagenicity of fractions of cigarette smoke condensate in Neurospora crassa and Salmonella typhimurium

The mutagenicities of selected fractions of cigarette smoke condensate (CSC) were studied in Neurospora crassa for the presence of direct-acting mutagens. CSCs from the University of kentucky Reference Cigarette 1R1 were assayed in a forward-mutation test at the adenine-3 (ad-3) region in resting conidia of a 2-component heterokaryon. Direct-acting mutagenic activity was found in an enriched polycyclic aromatic hydrocarbons (EPAH) fraction and in a basic fraction (Swain 5). No direct-acting mutagenic activity was detected in an acidic fraction (Swain 8), although it was highly toxic to resting conidia. The EPAH fraction also was tested in the presence of S9 mix prepared from Aroclor-1254-induced rat liver. It was found to be mutagenic, but higher doses were required than in the absence of S9 mix. In addition, the mutagenicities of CSC and 10 fractions of CSC were investigated in Salmonella typhimurium TA1538 by the incorporation and preincubation methods. In general, preincubation did not enhance the mutagenicities of the fractions, and the two rankings of mutagenic potency of the condensates that were obtained by the two methods were not significantly different. This is the first report of the presence of potent direct-acting mutagenicity in the EPAH and Swain 5 fractions of CSC.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

Structure-mutagenicity relationship among aminoquinolines, aza-analogues of naphthylamine, and their N-acetyl derivatives

The mutagenicity of 7 positional isomers of aminoquinolines (AQ) and their N-acetyl derivatives (AcAQ) was tested in Salmonella typhimurium TA100 and TA98 in the presence and absence of S9 mix. In a series of aminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 5-AQ greater than 8-AQ greater than 7-AQ greater than 3-AQ greater than 2-AQ much greater than 4-AQ, 6-AQ. The alpha-positional isomers, 5-AQ and 8-AQ, are more mutagenic than the beta-isomer, 2-, 3-, 6-, 7-AQ's. These results are in contrast to the finding that beta-naphthylamine is more mutagenic than alpha-naphthylamine. In a series of N-acetylaminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 7-AcAQ greater than 6-AcAQ greater than 8-AcAQ much greater than all the others. It is suggested that the AQ and AcAQ series might exert their mutagenicity through different molecular mechanisms (i.e., metabolic activation) from each other. The rate of metabolic activation does not seem to be correlated with the mutagenic potency of the compounds. It is noteworthy that 7-AQ and 8-AQ are mutagenic in both the strains tested in the absence of S9 mix.     

Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix

Small amounts of seminal fluid strongly enhanced the mutagenicity of the precursor mutagen benzo[a]pyrene (BP) in the Salmonella/microsome test. This previously unreported effect was found only in the presence of S9 mix for metabolic activation. The increase far exceeded the additive effect expected from experiments where seminal fluid and BP were tested separately with S9 mix. Testing of the direct-acting mutagen 4-nitro-o-phenylene-diamine (NPD) together with seminal fluid resulted in a lower mutagenic activity than that of NPD alone. Seminal fluid had a bactericidal effect on the Salmonella bacteria, thus only volumes up to 40 microliter could be used per plate. The mutagenic effect of only seminal fluid and S9 mix was slightly increased over controls in a standard Ames test, but was equal to the spontaneous mutation rate with a preincubation test modified according to Kado and coworkers. There were no significant differences between seminal plasma from smokers and non-smokers in any experimental series. Seminal fluid concentrated 20-fold by extraction with the mutagen-removing adsorbant Mutasorb did not have any enhancing effect on the mutagenicity of BP, nor did it exhibit any mutagenic activity in itself with or without S9 mix.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Comparison of the mutagenicity of quinoline and all monohydroxyquinolines with a series of arene oxide, trans-dihydrodiol, diol epoxide, N-oxide and arene hydrate derivatives of quinoline in the Ames/Salmonella microsome test.

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.     

Mutagenicity of the coccidiostat diaveridine in the Salmonella/mammalian microsome assay

The coccidiostat diaveridine was tested for mutagenicity in the Salmonella/microsome assay with tester strains TA100 and TA98. This compound was not mutagenic in either tester strain in the presence and absence of rat S9 mix, but was found to be mutagenic in strain TA100 after metabolic activation with hamster S9 mix.     

Mutagenic substances in red and white wine in Chile, a high risk area for gastric cancer

Chilean home-made and commercial wines were analyzed for the presence of mutagenic substances using the Salmonella mutagenicity test with preincubation. Strains TA98 and TA100 were used in the absence and in the presence of S9 mix. 90% of red wines from a total of 30 samples and 54% of white wines from a total of 22 were found to be mutagenic. In all cases, S9 mix did not affect the mutagenicity of the samples. At least in one case, more than one mutagen was present, since the mutagenicity with TA98 could be selectively inactivated without affecting that with TA100. This study supports the hypothesis that wine consumption may be an important risk factor for upper gastrointestinal cancer, particularly for adenocarcinoma of the stomach, which is highly prevalent in Chile.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

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.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

Nitroarenes in Suimon River sediment

Mutagenic activity was observed in sediments of the Suimon River bed with and without S9 mix. The direct-acting mutagens in the sediment were investigated. The sediment was extracted with methanol and fractionated on a Silica gel column. The benzene fraction from the Silica gel column exhibited mutagenic activity without S9 mix in strain TA98, while it failed to show mutagenic activity in nitroreductase-deficient strain TA98NR. This observation led to the suspicion that nitro compounds were the direct-acting mutagens of these samples. The benzene fraction was treated by heptafluorobutyric anhydride (HFBA) and investigated with gas chromatography equipped with an electron capture detector (GC-ECD). 2-Nitrofluorene, 4,4'-dinitrobiphenyl, 2,7-dinitrofluorene and 1-nitropyrene were detected and measured quantitatively. The mutagenic activity of a mixture of these compounds was compared with that of the original fraction and the direct-acting mutagenicity of Suimon River sediment can be explained by these nitroarenes, especially 1-nitropyrene.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

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.     

Detection of 1-nitropyrene in yakitori (grilled chicken)

Pieces of raw chicken with or without a marinating sauce were grilled over a city gas flame, extracted with benzene-ethanol (4:1) by ultrasonication and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor 1254-treated Sprague-Dawley rat liver (S9 mix). The basic fraction of yakitori without the sauce was more mutagenic than the other fractions for S. typhimurium strain TA98 in the presence of S9 mix. This is probably due to the presence of amino acid or protein pyrolysates. However, when the chicken was grilled with the sauce, the basic fraction showed lower mutagenicity for strain TA98 in the presence of S9 mix than did the same fraction without the sauce. The neutral fraction of yakitori with sauce showed high mutagenicity for strain TA98 in the absence of S9 mix, but low mutagenicity for strains TA98NR and TA98/1,8-DNP6, suggesting that this fraction might contain nitropyrenes (NPs). The neutral fraction of yakitori was analyzed by high-performance liquid chromatography (HPLC). The neutral fraction of the chicken grilled with the sauce for 3, 5 and 7 min contained 3.8, 19 and 43 ng, respectively, of 1-NP per gram of yakitori accounting for 3.0, 2.7 and 1.3%, respectively, of the total mutagenicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of nifurtimox (nfx) and 8 nfx analogues has been investigated with the L-arabinose forward-mutation assay of Salmonella typhimurium. The nfx analogues tested were obtained by replacing the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1); pyrazol-1-yl (2); benzimidazol-1-yl (3); 1,2,4-triazol-4-yl (4); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (5); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (6); 1-adamantyl (7); 4,6-diphenylpyridin-1-yl-2-one (8). The mutagenic activity of each chemical was determined by the standard plate-incorporation test, in the presence or absence of the S9 activation mixture. The 9 compounds were mutagenic and exhibited linear dose-mutagenic response relationships. They were direct-acting mutagens and showed a nearly 1000-fold range in mutagenic potency from chemical 1 to nfx. In most cases, the addition of S9 mixture to the test plates decreased the mutagenicity of compounds. This effect was particularly noticeable in the case of chemicals 1-3, 5 and 7 where a more than 70% decrease in mutagenic activity was observed in the presence of the S9 mixture. The mutagenic potency of compounds in the Ara test showed a negative linear correlation with previously reported antitrypanosomal activity. Thus, chemicals 6 and 8 with in vitro activities against Trypanosoma cruzi clearly superior to that of nfx showed 2 of the lowest mutagenic potencies in the Ara test and these were only somewhat higher than the mutagenicity of the reference drug.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

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.     

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.     

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

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.     

Assessment of the Mutagenicity of Sediments from Yangtze River Estuary Using Salmonella Typhimurium/Microsome Assay

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.