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.     

Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium

Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains. With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153. With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP₆. The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele. Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S. typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

High mutageniticity of metabolically activated chrysene 1,2 dihydrodiol: evidence for bay region activation of chrysene.

Chrysene and the 3 metabolically possible vicinal $\text{trans}$ dihydrodiols of chrysene were tested for mutagenicity towards $\text{S. typhimurium}$ strain TA100 in the presence of hepatic microsomes or a highly purified hepatic microsomal monooxygenase system. The products formed during the metabolic activation of chrysene 1,2-dihydrodiol were more than 20 times as mutagenic to the bacteria than the metabolites formed from chrysene, chrysene 3,4-dihydrodiol or chrysene 5,6-dihydrodiol. When the double bond in the 3,4-position of chrysene 1,2-dihydrodiol was saturated, the resulting tetrahydrodiol could not be metabolically activated. These results, which strongly suggest that chrysene 1,2-dihydrodiol is activated by metabolism to either or both of the diastereomeric chrysene 1,2-diol-3,4-epoxides, provide additional support for the bay region theory of polycyclic hydrocarbon carcinogenicity.     

Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Mutagenicity of 3a,8a-dihydrofuro[2,3-b]benzofuran, a model of aflatoxin B1, for Salmonella typhimurium TA100.

Racemic 3a,8a-dihydrofuro[2,3-b]benzofuran has been chemically synthesized as a model of the vinyl ether structure of aflatoxin B₁ (AFB₁) and tested for mutagenicity. In the presence of 9000g rat liver supernatant fraction the compound induced his⁺ revertant colonies in $\text{S. typhimurium}$ TA 100 but with only one five-thousandth the activity of AFB₁. No mutagenicity was found when strain TA98 was used. Omission of the rat liver preparation abolished mutagenic activity. The reduced compound, tetrahydrofurobenzofuran, was inactive as a mutagen either in the presence or absence of the rat liver supernatant.     

Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates

Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC, ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP₆ with deficient O-acetyl-transferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the ³²P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC.     

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.     

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.     

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.     

Dinitropyrene-resistant Salmonella typhimurium are deficient in an acetyl-CoA acetyltransferase

Salmonella typhimurium strain TA98NR which is sensitive to 1,8-dinitropyrene mutagenesis possesses acetyl-CoA dependent acetyltransferase activity, while a strain selected for resistance to 1,8-dinitropyrene (TA98/1, 8-DNP6) is deficient in this activity. Acetyltransferase competent strains can acetylate 1,8-diaminopyrene, forming 1-N-acetylamino-8-aminopyrene and 1,8-N,N'-diacetyldiaminopyrene. The coincidence of dinitropyrene resistance and acetyltransferase deficiency implicates acetylation as an important process in the metabolic activation of dinitropyrene to a mutagenic intermediate.     

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.     

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.     

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.     

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.     

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.     

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.     

Metabolism of 1,8-dinitropyrene by Salmonella typhimurium

Earlier work has shown that many nitroaromatic and nitroheterocyclic compounds are directly 'activated' to their ultimate mutagenic forms through the action of bacterial nitroreductase enzymes. However, in the case of 1,8-dinitropyrene (DNP) and certain other nitroarenes the pathway of activation is more complex and neither the identity of the ultimate mutagens nor the nature of the DNA adducts formed are known. We now show that Salmonella typhimurium strains TA98 and TA1538, which are sensitive to DNP and have wild type nitroreductase complements, do metabolize DNP to 1-amino-8-nitropyrene (ANP) and 1,8- diaminopyrene (DAP) but that these compounds are much weaker mutagens than DNP. These two strains (TA98 and TA1538) contain two separable components of nitroreductase activity as determined using nitrofurazone as the substrate. The major component, at least, is capable of reducing both 1-nitropyrene (NP) and DNP although the rates are much lower than with nitrofurazone. TA98NR , a mutant of TA98 that is resistant to nitrofurazone and NP but not to DNP, lacked the major nitroreductase but retained two minor components. In contrast, a mutant ( DNP6 ) which is resistant to DNP (but not to NP) contained a full complement of nitroreductases. When the metabolism of [3H]DNP by crude extracts of TA98 was re-examined, previously undetected metabolites were found. These were more polar than DAP and ANP and were also seen when TA98NR was used as the source of enzyme. These metabolites were not formed when enzymes from TA98DNP6 or TA98NR / DNP6 were used. This work supports the notion that some enzymic activity other than (or in addition to) nitroreductase is required for the activation of DNP and that the new polar metabolites may be related to this process.     

Effects of l-cysteine and O-acetyl-l-serine in the synthesis and mutagenicity of azide metabolite

The ability of L-cysteine to inhibit azide-metabolite synthesis and mutagenecity is investigated in Salmonella typhimurium TA1530 and cys E₆ strains. L-cysteine specifically inhibits the synthesis of the mutagenic azide metabolite as other compounds containing SH group did not affect the production of this metabolite. Azide mutagenicity is completely inhibited by L-cysteine at a concentration (5 μmoles/plate) where the metabolite mutagenicity was not affected. $\text{O-}\text{Acetyl-}\text{L}\text{-serine}$ can reverse the L-cysteine mediated inhibition of the metabolite synthesis and thus mutagenicity in the same strains. These results suggest that $\text{O-}\text{acetyl-}\text{L}\text{-serine}$ may be required to synthesize the azide metabolite or its precursor.     

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.