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

Dibenzo-p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1:3, 1:6 and 1:18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP6 without S9 mix. In a gas chromatography/mass spectrometry study, 2-nitrodibenzo-p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1:18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo-p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs.     

Metabolism of 2,4-dinitrotoluene by Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, and mutagenicity of the metabolites of 2,4-dinitrotoluene and related compounds to strains TA98 and TA100

The products detected in the incubation of 2,4-dinitrotoluene (2,4-DNT) with Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 were nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes and dimethyl dinitroazoxybenzene. The capacity of TA98NR to reduce 2,4-DNT was much lower than that of TA98 and TA98/1,8-DNP6. The bacterial products showed no mutagenic activity in the Ames assay using TA98 and TA100. These results indicate that the lack of mutagenic activity of 2,4-DNT is not due to low reductive metabolism of 2,4-DNT by the bacteria, but to the lack of mutagenic activity of the bacterial reductive products of 2,4-DNT, including dimethyl dinitroazoxybenzene.     

Identification and quantification of highly mutagenic nitroacetoxypyrenes and nitrohydroxypyrenes in diesel-exhaust particles

Heavy-duty diesel-exhaust particles were collected, extracted 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-induced rat liver (S9 mix). The neutral and acidic fractions showed high mutagenicity with TA98 in the absence of S9 mix, the acidic fraction having the highest specific activity. In the absence of S9 mix, the mutagenicity of crude, neutral and acidic fractions was greater in TA98 than in TA98NR and TA98/1,8-DNP6. Chemically-synthesized nitroacetoxypyrenes and nitrohydroxypyrenes were fractionated into the neutral and acidic fractions, respectively. These nitroarenes were purified by high-performance liquid chromatography and their mutagenicity was measured with the 4 strains. With TA98 in the absence of S9 mix, 1-nitro-3-acetoxypyrene, 1-nitro-6/8-acetoxypyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6/8-hydroxypyrene induced 16 700, 336, 992, 94 His+ revertants per plate per nmole, respectively. In the absence of S9 mix, the level of mutagenicity of these nitroarenes was highest in TA98, lowest in TA98/1,8-DNP6 and intermediate in TA98NR. The neutral and acidic fractions of diesel-exhaust particles were analyzed by gas chromatography-mass spectrometry and gas chromatography-mass fragmentography. The neutral fraction was found to contain nitroacetoxypyrenes, 1-nitropyrene, 1,6-dinitropyrene, while nitrohydroxypyrenes were detected in the acidic fraction. The amounts of 1-nitro-3-acetoxypyrene, 1-nitropyrene, 1,6-dinitropyrene and 1-nitro-3-hydroxypyrene were 6.3, 62, 0.81, and 70 ng per mg of crude extract, and accounted for 12, 3.6, 8.0, and 9.0%, respectively, of mutagenicity of the crude extract in TA98 in the absence of S9 mix.     

Metabolic activation of 2,4-dinitrobiphenyl derivatives for their mutagenicity in Salmonella typhimurium TA98

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we tested the mutagenic potency of 18 kinds of nitroarenes including nitrated biphenyl, fluorene, phenanthrene and pyrene on Salmonella typhimurium TA98 in the absence and presence of S9 mix. The mutagenicities of 2,4-dinitrobiphenyl derivatives and 4-nitrobiphenyl were enhanced by the addition of S9. 2,4,6-Trinitrobiphenyl (3 net rev./10 micrograms without S9) was activated 60-fold by the mammalian metabolic system (181 net rev./10 micrograms with 10% S9). The mutagenic potency of 2,4,2',4'-tetranitrobiphenyl in TA98, TA98NR and TA98/1,8-DNP6 was also enhanced by the addition of 10% S9. But 1-nitropyrene and 1,3-dinitropyrene, which are well-known mutagens and carcinogens, were deactivated to 3% and 0.4%, respectively, by the addition of 10% S9. Separate addition of microsomal and cytosolic fractions slightly activated the mutagenicity of 2,4,6-trinitrobiphenyl, and 2,4,2',4'-tetranitrobiphenyl was activated not only by S9 but also by the cytosolic fraction.     

Mutagenicities of xanthone derivatives in Salmonella typhimurium TA100, TA98, TA97, and TA2637

The mutagenicities of naturally occurring xanthones were tested in Salmonella typhimurium TA100, TA98, TA97, and TA2637 by the preincubation method. Xanthydrol, gentisein, gentisin, isogentisin, 1-hydroxy-3,7-dimethoxyxanthone, 1,3,7,-trimethoxyxanthone, desmethylbellidifolin, bellidifolin and dimethylbellidifolin were mutagenic, but unsubstituted xanthone was not mutagenic to TA100, TA98, TA97 and TA2637 with or without a metabolic activation system. The β-O-glucosides, norswertianolin and swertianolin, were only mutagenic when a metabolic activation system containing β-glucosidase was used, and the C-glucoside mangiferin was not mutagenic even with this system.     

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98.

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.     

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

The mutagenic potencies of plant extracts containing quercetin in Salmonella typhimurium TA98 and TA100

Four commercial ethanolic plant extracts, Tinctura Alchemillae, Extractum Crataegi, Extractum Myrtilli and Tinctura Hyperici, were tested for their mutagenicity in Salmonella typhimurium TA98 and TA100 with and without S9 mix obtained from rats pretreated with phenobarbital. The extracts studied differed greatly in their mutagenic potencies but exhibited a very similar mutation pattern in which the strongest effect was always seen in tester strain TA98 with S9 mix. Simultaneously we investigated the extracts for the presence of quercetin and kaempferol. Only quercetin was detected in small amounts by thin-layer chromatography (TLC). The fractions containing quercetin were separated and collected using a Sephadex LH-20 column. Two different methods were employed to estimate the amount of quercetin in the extracts: a colorimetric assay developed by Christ and Müller, and a complexometric method by Belikov. The quercetin concentrations ranged between 2 mg (Tinctura Alchemilla) and 89 mg (Tinctura Hyperici) per 100 g of extract. We suggest that the mutagenicity of the 4 plant extracts is mainly due to the presence of quercetin for the following reasons: (1) all the plant extracts exhibit a mutation pattern which is very similar to that of quercetin, (2) the mutagenic potential of the extracts correlates well with their quercetin content, considering the fact that plant extracts are very complex mixtures often containing toxic or antimutagenic compounds.     

Isolation of the mutagenic and DNA adduct-inducing components from a commercial preparation of HC blue 1 using Salmonella (TA98) bioassay-directed HPLC fractionation.

In the present study we report the separation of the mutagenic impurities from the nitrophenylenediamine hair dye HC Blue 1. This was accomplished by bioassay-directed HPLC fractionation, using Salmonella strain TA98 and reverse phase HPLC analysis. The mutagenic fraction eluted between 80 and 90% methanol, whereas the HPLC fraction containing the parent compound HC Blue 1 eluted with 30% methanol and was non-mutagenic. 100% of the mutagenic activity applied to the column was recovered in fractions that did not possess the blue color of HC Blue 1. Also, HPLC-purified HC Blue 1 did not form DNA adducts (32P-postlabeling) in Salmonella strain TA98. On the other hand, commercial HC Blue 1 and the mutagenic fraction derived from commercial HC Blue 1 (HPLC-isolated) gave similar DNA-adduct profiles that consisted of 7 adducts. DNA adduction was examined concomitantly with mutagenicity and toxicity studies on the HC Blue 1 samples in TA98. The data indicated that, in Salmonella, both the mutagenicity and DNA adduction of commercial HC Blue 1 are due to impurities and not the parent compound.     

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)     

Mutagenicity in Salmonella typhimurium mutants of the benzene-soluble organic matter derived from air-borne particulate matter and its five fractions.

Air-borne particulate matter was collected on a filter, then extracted with benzene. The benzene-soluble material was separated into 5 fractions, namely acidic, basic, alipathic, polyaromatic and oxygenated fractions. The mutagenic activities of these fractions were examined with a set of Salmonella typhimurium mutants. The 6 mutants were from the TA1535 series, deep rough strains without excision repair, namely TA100 and TA98 (having a resistance-transfer factor) and the standard strain TA1535, TA1536, TA1537 and TA1538. Linear dose-response curves were obtained for the benzene-soluble organic matter, and its acidic, polyaromatic and oxygenated fractions with strain TA98 and a 9000 X g liver supernatant from both phenobarbital(PB)- and dibenz(a,h)anthracene(DBA)-treated rats. Among the 5 fractions tested, 3 fractions, namely the acidic, polyaromatic and oxygenated, played an important role in the mutagenicity of the benzene-soluble organic matter derived from air-borne particulate matter. The 9000 X g rat-liver supernatant was not required to make the acidic fraction mutagenic.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

Mutagenicity and carcinogenicity of tea, Camellia sinensis

Aqueous, caffeine free and tannin fractions of commercial tea and tannic acid were tested for mutagenicity in Ames test. Tea fractions of tannic acid were non mutagenic in strains TA 100, TA 98, TA 1535 and TA 1538 of Salmonella typhimurium with or without metabolic activation (rat-S9 mix) at different doses tested. In strain TA 98 the above tea fractions and tannic acid inhibited the S9 mix mediated mutagenicity of tobacco in a dose dependent manner. The different tea fractions at 60 degrees C, did not increase the tumor incidence in Swiss mice by gavage feeding. They also failed to produce tumors when injected subcutaneously. Caffeine free tea extract decreased the tobacco induced liver tumors but had no effect on lung tumors. The same fraction was ineffective in hexachlorocyclohexane induced liver tumors in Swiss mice.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

Mutagenic activity of furfural in Salmonella typhimurium TA100.

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[alpha]pyrene in the presence of metabolic activation.     

Metabolism of thioamides by Ralstonia pickettii TA

Information on bacterial thioamide metabolism has focused on transformation of the antituberculosis drug ethionamide and related compounds by Mycobacterium tuberculosis. To study this metabolism more generally, a bacterium that grew using thioacetamide as the sole nitrogen source was isolated via enrichment culture. The bacterium was identified as Ralstonia pickettii and designated strain TA. Cells grown on thioacetamide also transformed other thioamide compounds. Transformation of the thioamides tested was dependent on oxygen. During thioamide degradation, sulfur was detected in the medium at the oxidation level of sulfite, further suggesting an oxygenase mechanism. R. pickettii TA did not grow on thiobenzamide as a nitrogen source, but resting cells converted thiobenzamide to benzamide, with thiobenzamide S-oxide and benzonitrile detected as intermediates. Thioacetamide S-oxide was detected as an intermediate during thioacetamide degradation, but the only accumulating metabolite of thioacetamide was identified as 3,5-dimethyl-1,2,4-thiadiazole, a compound shown to derive from spontaneous reaction of thioacetamide and oxygenated thioacetamide species. This dead-end metabolite accounted for only ca. 12% of the metabolized thioacetamide. Neither acetonitrile nor acetamide was detected during thioacetamide degradation, but R. pickettii grew on both compounds as nitrogen and carbon sources. It is proposed that R. pickettii TA degrades thioamides via a mechanism involving consecutive oxygenations of the thioamide sulfur atom.     

The involvement of dimethyl sulfoxide in a bacteriotoxic response of the Ames assay tester strains TA98 and TA100

Dimethyl sulfoxide is a widely accepted and recommended solvent in which to dissolve compounds to be tested for mutagenicity via the Ames Salmonella/mammalian microsome assay. Using tester strains TA98 and TA100, we observed a bacteriotoxic response with various fractions isolated from beer when dissolved in DMSO but not when dissolved in water. Further characterization of the role of solvent in simple model systems consisting of butanol, DMSO and bacteria strongly suggests a chemical reaction occurs between dimethyl sulfoxide and specific chemical constituents of the test substance, nutrient broth, or the Ames bacterial strains. The result of such an interaction could be misinterpreted as a toxic response to the test substance when, in fact, the bacteriotoxicity could be due to another compound, chemically distinct from the test substance.     

Mutagenicity of nitrofurans in Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6

8 representative 2-substituted 5-nitrofurans were assayed for mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6. The tested compounds were: 5-nitro-2-furanacrylic N-(5-nitro-2-furfurylidene)hydrazide (1); furazolidone (2); 5-nitro-2-furanacrolein (3); 5-nitro-2-furaldehyde semicarbazone (4); 5-nitro-2-furaldehyde (5); nitrofurantoin (6); 5-nitro-2-furaldehyde diacetate (7); and 5-nitro-2-furoic acid (8). These compounds exhibited markedly different mutagenic activities in TA98, and these mutagenicities were similar both in the presence and the absence of rat-liver hepatic S9 activation enzymes. The mutagenic responses ranged from potent (90-300 revertants/nmole, compounds 1-3), to medium (about 10 revertants/nmole, compounds 4 and 6), to weak (0-4 revertants/nmole, compounds 5, 7 and 8). The mutagenicity of 3 was similar in all 3 tester strains, while compound 8 was essentially inactive. The mutagenicities of 1, 4, 5 and 7 were decreased 30-75% in TA98NR, while 2 and 6 showed an even greater depression of activity in this strain. Compound 6 with S9 was about equally mutagenic in TA98 and TA98/1,8-DNP6, while the activities of 6 without S9 and 2 and 7 both with and without S9 were 50-75% lower in TA98/1,8-DNP6. Compounds 1, 4 and 5 were only about 5-10% as mutagenic in TA98/1,8-DNP6 as in TA98. These results suggest that: (i) nitrofurans and their S9-mediated metabolites have similar mutagenic potencies; (ii) with the possible exception of No. 3, nitroreduction is the major route of mutagenic activation for these nitrofurans; and (iii) for compounds 2, 6 and 7, both the presumed N-hydroxy and N,O-ester derivatives of the corresponding aminofuran metabolites appear to lead to mutations.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Mutagenicity of smoked, dried bonito products

Mutagens have been found in smoked, dried bonito products, popular items in Japanese foods. The mutagens were isolated by means of blue cotton, an absorbent cotton preparation with covalently bound trisulfo-copper-phthalocyanine residues, and by means of XAD-2 resin. The mutagenicity was positive in Salmonella typhimurium strain TA98 with metabolic activation. The mutagens are produced during the process of smoking-and-drying bonito (a process called baikan). The activity was much higher than that expected from the content of benzo[a]pyrene. In contrast to benzo[a]pyrene, the mutagens were not inhibited by ellagic acid. The mutagenicity was not abolished by treatment with nitrite. Thin-layer and high-performance liquid chromatographic analysis gave two mutagenic fractions, both of which were distinguishable from benzo[a]pyrene and from the pyrolysis products Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, A alpha C and MeA alpha C. The major mutagenic component was not chromatographically distinguishable from IQ and MeIQx, and the minor one was very similar to MeIQ. The smoked, dried bonito products contained free fatty acids, which were inhibitory to the mutagenicity of the bonito products.