Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro.

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02–0.8 μ g/plate (38–1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in “classical” nitroreductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 μg NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP₆, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.     

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Characterization of organic extracts from standard reference materials 1649, 'urban dust/organics,' and 1650, 'diesel particulate matter', using a microsuspension assay. A WHO/IPCS/CSCM study.

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 'urban dust/organics' (air particles), and 1650, 'diesel particulate matter' (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation assay. In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98-S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/micrograms, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/microgram, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 -S9 but the TA98-1,8-DNP6 levels were.     

Characterization of organic extracts from standard reference materials 1649, ‘urban dust/organics,’ and 1650, ‘diesel particulate matter’, using a microsuspension assay. A WHO/IPCS/CSCM study

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 ‘urban dust/organics’ (air particles), and 1650, ‘diesel particulate matter’ (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation asssay.In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98 - S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/μg, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/μg, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 - S9 but the TA98-1,8-DNP6 levels were.     

Dependence on Salmonella typhimurium enzymes of mutagenicities of nitrobenzene and its derivatives in the presence of rat-liver S9 and norharman

Dependence on S. typhimurium enzymes of mutagenicities of nitrobenzene (NB) and o-, p-chloronitrobenzenes (o-, p-CNBs), which are only mutagenic in the presence of S9 and norharman (NOH), was investigated using a nitroreductase-deficient strain TA98NR and an esterifying enzyme-deficient strain TA98/1,8-DNP6. NB exhibited mutagenicity towards TA98 but did not towards TA98NR strain in spite of the presence of S9 in the assay system. The mutagenicity of o-CNB towards TA98NR was significantly lower than that of o-CNB towards TA98. In contrast to NB and o-CNB, synthesized phenylhydroxylamine (PHA) and o-chlorophenylhydroxylamine (o-CPHA) exhibited approximately the same mutagenicity towards both tester strains. These results indicate that the nitroreduction required for the appearance of mutagenicity of the nitrobenzene derivatives in the presence of S9 and NOH is dependent on the nitroreductase of the tester strain. In addition, the mutagenicities of PHA and p-CPHA were significantly higher towards TA98/1,8-DNP6 than towards TA98, suggesting that the esterification of their hydroxylamines produced inactivation rather than activation. From these results, it was concluded that S9 and NOH play a role in metabolic activation other than the reduction of the nitro group to hydroxylamine and subsequent esterification for the mutagenesis of NB and its derivatives.     

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.     

Relationships between structure of nitrated arenes and their mutagenicity in Salmonella typhimurium; 2- and 2,7-nitro substituted fluorene, phenanthrene and pyrene

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we studied the relationships between the mutagenic potency and chemical structure of 2-nitro- and 2,7-dinitro-arenes including nitrated fluorene (Fl), dihydrophenanthrene (DHPh), phenanthrene (Ph), tetrahydropyrene (THPy), dihydropyrene (DHPy) and pyrene (Py) together with 9-NO2-Ph, 1-NO2-Py and 1.3-diNO2-Py. The mutagenicity tests were carried out on Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6 in the absence of S9 mix. The order of mutability of mononitro- and dinitro-arenes in TA98 is as given below: 2-NO2-THPy less than 2-NO2-Fl less than 2-NO2-DHPh less than 9-NO2-Ph less than 2-NO2-Ph less than 2-NO2-DHPy less than 1-NO2-Py less than 2-NO2-Py, and 2,7-diNO2-DHPh less than 2,7-diNO2-Fl less than 2,7-diNO2-THPy less than 2,7-diNO2-Ph less than 2,7-diNO2-DHPy less than 2,7-diNO2-Py less than 1,3-diNO2-Py. 9-NO2-Ph and 1-NO2-Py, which have been detected in environmental samples, are not as potent mutagens as 2-nitrated phenanthrene and pyrene, respectively. 2-NO2THPy (37.7 rev/nmole) was a weak mutagen, but 2,7-diNO2-THPy (3197 rev/nmole) was as potent a mutagen as 2,7-diNO2 (3925 rev/nmole). Tetrahydropyrene has a twisted form in its structure. 1,3-diNO2-Py (99660 rev/nmole) was more mutagenic than 2,7-diNO2-Py (37960.0 rev/nmole), and their mutagenicities were correlated with the behavior of the K-band in their UV spectra by the introduction of nitro groups on pyrene.     

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.     

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.     

Comparison of mutagenicity and theoretical reactivity of 2,4-dinitrobenzaldehyde and 2,6-dinitrobenzaldehyde in bacterial mutation assay and molecular orbital method

The mutagenicities and theoretical reactivity indices of 2,4-dinitrobenzaldehyde (2,4-DNBAl) and 2,6-dinitrobenzaldehyde (2,6-DNBAl) were investigated using Salmonella typhimurium strains TA98, TA98NR, TA98/1,8-DNP6, and TA100, TA100NR and TA100/1,8-DNP6, by means of the modified intermediate neglect of differential overlap/3 (MINDO)/3) method. The mutagenic activities of 2,4-DNBAl in TA98NR and TA98/1,8-DNP6 were lower than in TA98, whereas the activity in TA100NR was higher than in TA100 and TA100/1,8-DNP6. The mutagenic activity of 2,6-DNBAl in TA100 and that in TA100 and TA100/1,8-DNP6 decreased. These results suggest that the mutagenicities of 2,4-DNBAl and 2,6-DNBAl are dependent either on the microbial nitroreduction and subsequent acetylation or the presence of an aldehyde group. Among the reactivity indices examined, the frontier electron density values were correlated to the mutagenicities of 2,4-DNBAl and 2,6-DNBAl in TA100, TA100NR and TA100/1,8-DNP6 and the values of energy of the lowest unoccupied molecular orbit were correlated to the mutagenicities of several substituted dinitrobenzenes.     

Mutagenicity of the phenolic microsomal metabolites of 3-nitrofluoranthene and 1-nitropyrene in strains of Salmonella typhimurium

The environmental pollutant 3-nitrofluoranthene is metabolized in vitro and in vivo to several products including the phenolic metabolites 3-nitrofluoranthen-6-ol (3NF-6-ol), 3-nitrofluoranthen-8-ol (3NF-8-ol), and 3-nitrofluoranthen-9-ol (3NF-9-ol). Similarly, 1-nitropyrene is metabolized to the phenolic metabolites 1-nitropyren-3-ol (1NP-3-ol), 1-nitropyren-6-ol (1NP-6-ol), and 1-nitropyren-8-ol (1NP-8-ol). The mutagenicity of these compounds was investigated using strains of Salmonella typhimurium deficient in either certain nitroreductase or the aryl hydroxylamine O-esterificase. In TA98, 3-nitrofluoranthene and 3NF-8-ol were equally mutagenic at approximately 10³ revertants/nmole while 3NF-6-ol and 3NF-9-ol were 10-fold less mutagenic. 1-Nitropyrene and 1NP-3-ol likewise were equally mutagenic at approximately 700 revertants/nmole and 1NP-6-ol and 1NP-8-ol were 100-fold less mutagenic. The mutagenicity of 1-nitropyrene was dependent on the ‘classical nitroreductase’ which is absent in TA98NR, and that of 3-nitrofluoranthene, 3NF-8-ol, and 1NP-3-ol was less dependent on this nitroreductase. Using TA98/1,8DNP₆, it was determined that the mutagenicity of 3-nitrofluoranthene, 3NF-8-ol, and 1NP-3-ol but not 1-nitropyrene was dependent on the presence of the O-esterificase. 3-Nitrofluoranthene and 3NF-8-ol were mutagenic in TA100, while 3NF-6-ol and 3NF-9-ol were considerably less mutagenic. 3-Nitrofluoranthene was not mutagenic in TA100NR nor in TA100-Tn5-1,8-DNP1012. None of the phenolic metabolites of 3-nitrofluoranthene were mutagenic in TA100-Tn5-1,8DNP1012 indicating a strong dependence for mutagenicity of the O-esterificase of the 1,8-dinitropyrene nitroreductase which is absent in this strain. These results are discussed in view of possible mechanisms for the differences in the mutagenicity of the phenolic metabolites of these two nitrated arenes.     

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.     

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)     

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.     

Mutagenic potency of some conjugated nitroaromatic compounds and its relationship to structure

The mutagenicities of 12 conjugated non-fused nitroaromatic compounds and 1 amino analogue were determined in strains TA100 and TA98 of Salmonella typhimurium. Reversions by p-nitroaromatics increased in the order of the acetophenone, benzaldehyde, styrene, chalcone, cinnamic acid and stilbene indicating the importance for mutagenic potency of extended conjugation to the p-nitrophenyl substituent. Highest mutagenicity was found with alpha-substituted 4-nitrostyryl derivatives of which the phenyl derivative (31 revertants per nmole in TA100) was the most active. Generally, the TA100 strain was more sensitive than TA98 to these mutagens and S9 treatment was unnecessary for activity, although 4-nitrochalcone required S9 activation. Para-nitro isomers of the cinnamic acids and chalcones were much more active than the corresponding ortho and meta isomers. The 4-aminocinnamic acid analogue was inactive suggesting that complete reduction in Salmonella of 4-nitrocinnamic acid to an active amino derivative is not response for the high mutagenicity of the former. Mutagenicity of these p-nitrostyryl compounds may be explained by the covalent interaction of the electrophilic benzylic carbon with Salmonella DNA.     

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.