Classical Nitroreductase Research Articles

Crystal Structure of Reduced MsAcg, a Putative Nitroreductase from Mycobacterium smegmatis and a Close Homologue of Mycobacterium tuberculosis Acg

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria's adaptation to a dormancy state.

Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.

Identification of a Mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr aene within macrophages, in standing versus shaking cultures, and under low oxygen conditions.

Tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. This effort will benefit greatly from a better understanding of gene regulation in Mycobacterium tuberculosis, particularly with respect to this pathogen's response to its host environment. We examined the behavior of two promoters from the divergently transcribed M. tuberculosis genes acr/hspX/Rv2031c (alpha-crystallin homolog) and Rv2032/acg (acr-coregulated gene) by using a promoter-GFP fusion assay in Mycobacterium bovis BCG. We found that Rv2032 is a novel macrophage-induced gene whose expression is coregulated with that of acr. Relative levels of intracellular induction for both promoters were significantly affected by shallow standing versus shaking bacterial culture conditions prior to macrophage infection, and both promoters were strongly induced under low oxygen conditions. Deletion analyses showed that DNA sequences within a 43-bp region were required for expression of these promoters under all conditions. Multiple sequence alignment and database searches performed with PROBE indicated that Rv2032 is one of eight M. tuberculosis genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment. These findings show that mycobacterial culture conditions can greatly influence the results and interpretation of subsequent gene regulation experiments. We propose that these differences might be exploited for dissection of the regulatory factors that affect mycobacterial gene expression within the host.

Synthesis, metabolism and structure–mutagenicity relationships of novel 4-nitro-(imidazoles and pyrazoles) in Salmonella typhimurium

A new series of 4-nitro-(imidazoles and pyrazoles) were synthesized as novel antimycotics and tested for their activation to mutagenic forms using Salmonella typhimurium TA98 and TA100, in the presence and in the absence of metabolic activation. TA100NR, TA100/1,8-DNP 6, YG1026 and YG1029 strains were employed to identify a specific metabolic reaction which governs the mutagenic potency. Derivatives in the pyrazole group were generally found to be non mutagenic and active imidazoles were weak-direct-acting mutagens. For most of the compounds the mutagenic responses in TA98 were absent or 12- to 22-fold lower compared to TA100. The presence of a methyl or a benzylic group on the imidazole ring and substituents on the N 1 and N 3 positions were determinant for mutagenicity. Metabolism by bacterial enzyme systems was important to the expression of genotoxicity. Active compounds showed no mutagenicity toward the strain defective in classical nitroreductase and increased mutagenicity, from 2- to 7-fold depending on the test compound, toward the corresponding overproducing bacteria. On the other hand, compounds displayed reduced mutagenicity to the O-acetyltransferase strain without having increased activity in the corresponding overproducing bacteria, YG1029.

Targeted disruption of the gene encoding the classical nitroreductase enzyme in Salmonella typhimurium Ames test strains TA1535 and TA1538

The gene encoding the `classical nitroreductase' (CNR) of Salmonella typhimurium was disrupted. In this manner, cnr null mutant derivatives of strains TA1535 and TA1538 were constructed, and named YG7131 and YG7127, respectively. In both strain backgrounds, cnr gene disruption reduced nitrofurazone-reductase activity. This reduction almost completely eliminated the nitroreductase activity of strain TA1538. In contrast, the nitroreductase activity of strain TA1535 was much higher than that in TA1538. In this background, cnr gene disruption resulted in a reduction in nitroreductase activity by a similar absolute amount as in TA1538, but representing only about one-quarter of the original activity of TA1535. The results suggest that S. typhimurium has originally at least two distinct nitroreductases, one of which is already deficient in strain TA1538; the CNR is present in both TA1535 and TA1538. Also, these two strains (including their derivatives, TA98 and TA100) are not isogenic with regard to nitroreductase activity. After the introduction of plasmid pKM101, the sensitivities of the strains YG7132 and YG7128, the cnr-null mutants of TA98 and TA100, respectively, against several nitro compounds were compared with those of the conventional cnr-deficient strains TA98NR and TA100NR and the wild-type strains TA98 and TA100. The mutagenicities of 2-nitrofluorene and 1-nitropyrene in YG7132 or TA98NR were ten-fold lower than those of the compounds in TA98. Similarly, the mutagenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide in strain YG7128 or TA100NR was substantially lower than that of the compound in TA100. However, the mutagenicity of 2-nitronaphthalene in YG7128 was between those observed with TA100 and TA100NR, suggesting that a nitroreductase in S. typhimurium other than CNR is involved in the metabolic activation of this compound. The cnr gene of S. typhimurium positively hybridized with DNA at 13 min on the E. coli chromosome where the nfsB and nfnB genes of E. coli are mapped. These results suggest that the cnr gene of S. typhimurium is a counterpart of the nfsB and nfnB genes of E. coli, and that the newly constructed cnr-deletion strains are useful to assess the role of nitroreductases in the metabolic activation of mutagenic nitro compounds.

Mutation spectra of chemical fractions of a complex mixture: role of nitroarenes in the mutagenic specificity of municipal waste incinerator emissions

Using an ion-exchange procedure coupled to a microsuspension Salmonella assay, we fractionated the dichloromethane-extractable particulate organics emitted by a municipal waste incinerator. Most (80–95%) of the mutagenic activity resided in the neutral/base fraction; however, the polar neutral fraction accounted for 12% of the direct-acting mutagenic activity. The mutagenic potencies of the whole extract and the various fractions were 4–15 times greater in the absence than in the presence of S9. Results with strains deficient in classical nitroreductase (TA98NR) and transacetylase (TA98/1,8-DNP 6) indicated that a majority of the direct-acting mutagenicity was due to nitroarenes. This was confirmed by bioassay-directed subfractionation of the neutral/base faction by a cyanopropyl/HPLC method. The mutations in −3,000 revertants (∼ 400 each induced in TA98 by the whole extract, the neutral/base and polar neutral fractions from the ion-exchange column and 3 of the neutral/base subfractions from the HPLC column; along with 200 revertants each induced by the model nitroarene 1-nitropyrene (1NP) in strains TA98, TA1538 and TA100) were analyzed by probe hybridization and PCR DNA sequence analysis. The results indicated that nitroarenes such as 1NP that eluted in the neutral/base fraction accounted for at least 50% of the direct-acting mutagenicity and induced only a hotspot 2-base deletion in the sequence (CG) 4 in TA98. In contrast, most of the complex frameshifts (a frameshift with a flanking base substitution) induced by the whole extract were induced by nitroarenes other than 1NP that were activated by transacetylation and that eluted in the polar neutral fraction. This study (1) identifies nitroarenes as an important contributor to the mutagenic activity of the emissions from municipal waste incinerators; (2) confirms our previous conclusion that the mutation spectrum of a complex mixture reflects the dominance of particular classes of chemical mutagens within the mixture; and (3) demonstrates the possibility of isolating certain chemical fractions of a complex mixture that induce certain classes of mutations produced by the whole, unfractionated mixture.

Detection of mutagenic activity in textiles with Salmonella typhimurium

A hundred and ninety-six textile samples were tested in a modified version of the Salmonella/microsome assay for release of mutagenic contaminants. As heat sterilization of the samples can result in reduction of mutagenic activity, tests were performed with streptomycin resistant derivatives of Salmonella tester strains TA98 and TA100. Textile samples were preincubated in buffered saline (PBS), DMSO or ethanol. Subsequently, the fabrics were placed on streptomycin supplemented selective agar plates. In total, 18 samples (9.2%) exerted mutagenic activity. DMSO was the most effective solvent (15 positives) followed by ethanol (9 positive samples) and PBS (7 positives). Most fabrics (16) caused mutagenic effects only upon metabolic activation with liver S9 mix. Chemical analysis indicates that the positive results obtained with PBS are not due to release of histidine or formaldehyde. Three directly active samples gave negative results in strain TA98NR which is devoid of classical nitroreductase. With one exception all other textiles were negative in strain TA98/1,8-DNP 6 (which lacks O-acetyltransferase). These findings indicate that nitroaromatics and amines might be responsible for the mutagenic effects of the textiles.

Mutagenicity of aristolochic acids (I, II) and aristolic acid I in new YG strains in Salmonella typhimurium highly sensitive to certain mutagenic nitroarenes

Aristolochic acid I and II, two naturally occurring nitroaromatics, were studied for their mutagenicity in Salmonella typhimurium tester strains YG1020, 1021, 1024, 1025, 1026 and 1029 without exogenous metabolic activation. These strains contain multicopy plasmids which carry the genes for the classical bacterial nitroreductase or O-acetyltransferase. The strains TA98, TA100 and TA1537 were included in the study for comparison. Aristolic acid I, the analogue lacking the nitro group, and its sodium salt were also tested. Both aristolochic acids revealed mutagenicity in the respective YG strains derived from TA100, but the effect was no stronger than with the parent strain. Only weak activity was observed in TA98 and some YG strains derived from it. Aristolochic acid II was generally the more active compound. Aristolic acid I and its sodium salt did not exhibit any mutagenicity in any tester strain. From the results the following conclusions were drawn. (i) Only the nitro group is important for the mutagenicity of the aristolochic acids in S. typhimurium. (ii) It is suggested that aristolochic acid II is so efficiently metabolized by the classical bacterial nitroreductase that the additional activity produced from the YG strains no longer affects the metabolic activation. (iii) The methoxy group is probably responsible for the lower activity of aristolochic acid I, producing steric hindrance for binding of the genetically active intermediate to DNA or for binding of the substrate to the active site of the enzyme(s).

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I: Purification and properties of a nitroreductase enzyme from Escherichia coli—A potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the “classical nitroreductase” of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active against CB1954 ( k cat=360 min −) han Walker DT diaphorase ( K cat = 4 min −1) and also has a lower K m for NADH (6 vs 75 μM).

Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae

The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.

Effect of rat liver cytosolic enzymes and cofactors on mutagenicity of 1-amino-8-nitropyrene

1-Amino-8-nitropyrene (1,8-ANP), a product of 1,8-dinitropyrene metabolism by either bacterial or mammalian enzymes, is weakly mutagenic to the 'classical nitroreductase'-deficient Salmonella tester strain TA98NR. The addition to the test system of rat liver cytosol without cofactors did not produce any effect on the 1,8-ANP mutagenic response toward TA98NR strain. Conversely, when both rat hepatic cytosol and NADPH (1 mM) were added to the mutagenicity assay, a 10-fold increase in 1,8-ANP mutagenic activity was observed. This suggests the involvement of rat hepatic cytosolic NADPH-dependent nitroreductase(s) in 1,8-ANP mutagenic activation. The addition to the mutagenesis assay of pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, produced a dose-dependent decrease of 1,8-ANP mutagenic activation, whereas 2,6-dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O-acetyltransferase, did not affect the activation of 1,8-ANP to a mutagen at concentrations that selectively inhibit only bacterial sulfotransferase. This indicates that bacterial O-acetyltransferase but not sulfotransferase plays a role in the mutagenic activation of 1,8-ANP. Addition of acetyl co-enzyme A (AcCoA) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), cofactors for O-acetyl-transferase and sulfotransferase respectively, to the test system caused a dose-dependent inhibition of 1,8-ANP mutagenic activation by rat liver cytosol and NADPH, probably due to the formation of highly reactive O-acetoxy and N-sulfate ester derivatives of 1,8-ANP, which react with nucleophilic sites before reaching bacterial DNA. This hypothesis was confirmed by DNA covalent binding in in vitro experiments showing that both the cofactors AcCoA and PAPS enhanced the NADPH/rat liver cytosol-mediated covalent binding of 1,8-ANP to DNA from calf thymus 10- and 3-fold respectively. It seems likely that rat hepatic cytosolic nitroreductases activate 1,8-ANP to an N-hydroxyarylamine derivative which can be further metabolized to mutagenic species by either bacterial or mammalian O-acetyltransferase.

Comparative mutagenicity of nitrofluoranthenes in salmonella typhimurium TA98, TA98NR, and TA98/1,8DNP6

The nitration of fluoranthene, one of the most abundant polycyclic aromatic hydrocarbons (PAH) in diesel fuels, occurs in the laboratory under either electrophilic or free-radical conditions to give nitro-PAH. 3-Nitrofluoranthene (3-NF) is the major product under electrophilic ionic conditions while 2-nitrofluoranthene (2-NF) is the major product under free-radical nitration conditions. The free-radical nitration of fluoranthene also yields 1,2- and 1,3-dinitrofluoranthene (1,2-DNF and 1,3-DNF). Nitration on the 3-position of fluoranthene enhances the mutagenic potency more strongly than on the 2-position. Thus, 3-NF is a more potent mutagen than 2-NF and 1,3-DNF is more potent than 1,2-DNF, an isomer with one near coplanar nitro group and one nitro group substantially out of plane with the fluoranthene skeleton, when tested against Salmonella typhimurium TA98, TA98NR, and TA98/1,8-DNP6. In addition, the activation of these dinitro-PAH to mutagens does not depend on the "classical nitroreductase" and/or O-acetylase, suggesting that they are activated via different pathways. Despite the fact that 3-NF and 1-phenyl-4-nitronaphthalene (1-P-4NN), a non-planar analog of 3-NF, have virtually identical reduction potentials, their mutagenic potencies differ by three orders of magnitude. This finding suggests that when nitro-PAH of varying steric requirements are compared, the reduction potential may not predict mutagenic potency as well as had been previously suggested.

Nucleotide sequence ofSalmonella typhimuriumnitroreductase gene

'Classical nitroreductase' is an enzyme involved in theintracellular metabolic activation ofmutagenic nitroarenes (1).Wehave already cloned the nitroreductase gene ofSalmonellatyphimuriumTA1538(2) anddeterminedthenucleotide sequenceby dideoxy sequence analysis ofboth strands. Below is shownthe nucleotide sequence of 1690base fragment which containsanopenreadingframeof651 nucleotides withpotential to encodethe nitroreductase. Themaxicell technique wasusedto identifythe nitroreductase and its molecular weight was estimated as28KDa, which is close to the calculated molecular weight of23,955. Possible sequence of -35, -10, S.D. and rho-independent transcriptional termination signal are indicated.

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

‘Classical nitroreductase’ is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.

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 10 3 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 TA98/1,8DNP 6, 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 nitracrine analogues in Salmonella typhimurium: Mutational specificity and activation by bacterial enzymes and rat-liver S9

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-(nitracrine), 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium, using a plate-incorporation assay. In view of the potential importance of DNA binding and nitro group reduction, binding constants and redox potentials were determined. The parent compound was mutagenic only in the frameshift tester strain TA1537, and this effect was not enhanced by the plasmid pKM101. Each of the nitroarenes showed significant activity in S. typhimurium strains TA1537 and TA1538, and this mutagenicity was enhanced by the presence of plasmid pKM101. All caused reversion of the base-pair substitution allele in his G46 in strain TA100 and this effect was either largely or totally dependent upon the plasmid. The frameshift mutagenic effects of the 3-nitro and 4-nitro compounds appeared to be little dependent upon the classical nitroreductase which is deficient in strain TA98NR, or transacetylase enzyme lacking in strain TA98/1,8-DNP 6, whereas the activity of the 1-nitro compound depended partly on that nitroreductase enzyme, and that of the 2-nitro compound on having both activities present. In the latter two cases, the mutagenic effects of the compounds could not be restored by the addition of an S9 mix of mammalian enzymes. Mutagenicity data were compared with physicochemical parameters. The results do not distinguish between the view that the orientation of the nitro group with respect to the aromatic plane dictates mutagenic potential, and the earlier view that the nitro group redox potential is important. However, the extraordinary mutagenic potency of the 1-NO 2 derivative is not explainable by its physicochemical properties alone.

The antimutagenic effect of hydroxyurea

Using an ion-exchange procedure coupled to a microsuspension Salmonella assay, we fractionated the dichloromethane-extractable particulate organics emitted by a municipal waste incinerator. Most (80–95%) of the mutagenic activity resided in the neutral/base fraction; however, the polar neutral fraction accounted for 12% of the direct-acting mutagenic activity. The mutagenic potencies of the whole extract and the various fractions were 4–15 times greater in the absence than in the presence of S9. Results with strains deficient in classical nitroreductase (TA98NR) and transacetylase (TA98/1,8-DNP6) indicated that a majority of the direct-acting mutagenicity was due to nitroarenes. This was confirmed by bioassay-directed subfractionation of the neutral/base faction by a cyanopropyl/HPLC method. The mutations in −3,000 revertants (∼ 400 each induced in TA98 by the whole extract, the neutral/base and polar neutral fractions from the ion-exchange column and 3 of the neutral/base subfractions from the HPLC column; along with 200 revertants each induced by the model nitroarene 1-nitropyrene (1NP) in strains TA98, TA1538 and TA100) were analyzed by probe hybridization and PCRDNA sequence analysis. The results indicated that nitroarenes such as 1NP that eluted in the neutral/base fraction accounted for at least 50% of the direct-acting mutagenicity and induced only a hotspot 2-base deletion in the sequence (CG)4 in TA98. In contrast, most of the complex frameshifts (a frameshift with a flanking base substitution) induced by the whole extract were induced by nitroarenes other than 1NP that were activated by transacetylation and that eluted in the polar neutral fraction. This study (1) identifies nitroarenes as an important contributor to the mutagenic activity of the emissions from municipal waste incinerators; (2) confirms our previous conclusion that the mutation spectrum of a complex mixture reflects the dominance of particular classes of chemical mutagens within the mixture; and (3) demonstrates the possibility of isolating certain chemical fractions of a complex mixture that induce certain classes of mutations produced by the whole, unfractionated mixture.