A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. alpha-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic activation of bladder carcinogenic arylamines using a SOS/umu assay system

Human acetyltransferase genes NAT1 or NAT2 were expressed in a Salmonella typhimurium strain used to detect the genotoxicity of bladder carcinogens. To clarify whether the human and rodent bladder carcinogenic arylamines are activated via either NAT1 or NAT2 to cause genotoxicity, a SOS/umu genotoxicity assay was used, with the strains S. typhimurium NM6001 (NAT1-overexpressing strain), S. typhimurium NM6002 (NAT2-overexpressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain). Genotoxicity was measured by induction of SOS/umuC gene expression in the system, which contained both an umuC"lacZ fusion gene and NAT1 or NAT2 plasmids. 4-Aminobiphenyl, 2-acetylaminofluorene, beta-naphthylamine, o-tolidine, o-anisidine, and benzidine exhibited dose-dependent induction of the umuC gene in strain NM6001. Although the induction of umuC by these chemicals was observed in the NM6002 strain, the induction was considerably lower than in the NM6001 strain. In the parent strain, NM6000, none of these compounds induced umuC gene expression. We also determined activation of these chemicals by recombinant human cytochrome P450 (P450 or CYP) 1A2 enzyme in three S. typhimurium tester strains. The activation of the chemicals was stronger in the NM6001 strain than that in NM6002. The specific NAT1 inhibitor 5-iodosalicylic acid inhibited umuC gene expression induced by aromatic amines used. These results could provide evidence that the bladder carcinogenic aromatic amines are mainly activated by the NAT1 enzyme to produce DNA damage rather than NAT2. The NAT1-overexpressing strain can be used to determine the genotoxic activation of bladder carcinogenic arylamines in the umu test and could provide a tool for predicting the carcinogenic potential of arylamines.     

Mutagenicity assay for nitroarenes of air pollutants held in leaves of woody plants.

A new method was developed for the detection of mutagenic nitroarenes held in the leaves of woody plants using a small amount of leaves. This method consists of extraction of mutagen from fresh leaves (15-30 g) by ultrasonication with ethyl acetate and purification by elution with benzene on a silica gel column to remove such inhibitory components for mutagenesis as chlorophyll. The mutation assay uses the new Salmonella typhimurium strains YG1021 (a nitroreductase-overproducing strain of TA98) and YG1024 (an O-acetyltransferase-overproducing strain of TA98), which are very sensitive to some nitroarenes in the absence of S9, and standard strain YG1020 (TA98 containing pBR322-Aps) for comparison. This method was applied to woody plants growing on various sites in the Tokyo metropolitan area and the suburbs. It was shown that the leaves of all woody plants tested contained different amounts of mutagens, probably mutagenic nitroarenes, depending upon their growing sites.     

Study of promutagen biotransformation in the Ames test. III. The role of conjugation with glucuronic acid and sulfate in the modification of mutagenic effect of nitrosomorpholine, diethylnitrosamine and cyclophosphamide

The role of reactions of conjugation with uridine diphosphoglucuronic acid (UDPGA) and with 3-phosphoadenosine-5-phosphosulfate (PAPS) in modification of the mutagenic effect of diethyl nitrosamine (DENA), nitrosomorpholine (NM) and cyclophosphane (CP) was studied by the Ames test. It was shown that adding UDPGA to the activating mixture significantly decreased the level of the mutagenic effect of DENA, NM and CP on bacteria Salmonella typhimurium TA 1950, when S9 and microsomal fractions of rat liver homogenate were used. Adding PAPS to the activating mixture when S9 and cytosole fractions were used, did not affect mutagenic action of DENA on S. typhimurium TA 1950 and TA 1535, enhancing the mutagenic effect of CP on TA 1535, with no such influence on TA 1950. Introduction of PAPS into the activating mixture elevated the mutagenic effect of NM on both bacterial strains using S9 fraction but not cytosole fraction.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

On the direct effect of clouds and atmospheric particles on the productivity and structure of vegetation

The volume of shade within vegetation canopies is reduced by more than an order of magnitude on cloudy and/or very hazy days compared to clear sunny days because of an increase in the diffuse fraction of the solar radiance. Here we show that vegetation is directly sensitive to changes in the diffuse fraction and we conclude that the productivity and structure of vegetation is strongly influenced by clouds and other atmospheric particles. We also propose that the unexpected decline in atmospheric [CO₂] which was observed following the Mt. Pinatubo eruption was in part caused by increased vegetation uptake following an anomalous enhancement of the diffuse fraction by volcanic aerosols that would have reduced the volume of shade within vegetation canopies. These results have important implications for both understanding and modelling the productivity and structure of terrestrial vegetation as well as the global carbon cycle and the climate system.     

Study of promutagen biotransformation in the Ames test. IV. The effect of transplanted tumors on the biotransformation of various antineoplastic agents

The effect of transplantation of rat tumours Jensen sarcoma, sarcoma 45, sarcoma M-1, as well as of inoculation of rat normal connective tissue on the processes of biotransformation of antitumour preparations cyclophosphane (CP), thiophosphamide, prospidine and of model compound nitrosomorpholine (NM) was studied. The study was accomplished by means of the Ames test with indicator bacterial strain Salmonella typhimurium TA 1950 in relation to the reactions of the 1st and the 2nd phases of xenobiotics metabolism. It was shown that the presence of tumours leads to inhibition of both metabolic activation processes of the promutagens NM and CP and the conjugation reactions of genetically active metabolites of these compounds with reduced glutathione. Genetic danger is supposed to be increased during application of antitumour preparations, the mutagenic activity of which is due to the activity of their metabolites. It is noted that the most essential effect on biotransformation processes of NM and CP was exhibited by sarcoma M-1, the most important changes of the biotransformation processes of promutagens being observed in the initial period of pathologic process, i.e. on the 3rd day after inoculation. Transplantation of the normal connective tissue of rats had no effect on reactions of both the 1st and the 2nd phase of metabolism of the promutagens studied.     

Effect of mutagenic metabolic activation of N-nitrosomorpholine on microorganisms]

Almost similar sensitivity is demonstrated of different methods of studying mutagenic metabolic activation of N-nitrosomorpholine (NM) in Salmonella typhimurium TA 1950 (host-mediated assay and the system of metabolic activation with rat liver homogenate). The role of correlation of certain ingredients of NM metabolic activation system with homogenates (homogenate and cofactor NADPH concentrations) is studied. The fact of influence of microsome protein activator (phenobarbital) on the NM mutagen activation effect is established.     

Toxic genetic effects of flunitrazepam

The mutagenic activity of Flunitrazepam, the active ingredient of the drug Rohypnol, has been investigated by using the Salmonella/microsome mutagenicity test. A dose-related mutagenic effect was observed on Salmonella typhimurium strain TA 100 either in the absence or in the presence of a rat liver microsomal fraction (S9) as in vitro metabolic activation system. By adopting a modification of the Salmonella test, the mutagenicity of urines from rats or patients treated with the drug was evaluated. In these cases mutagenic activity was detected toward the Salmonella strains TA 98 and TA 100 both in presence and in absence of the metabolic activation system. The data indicate that Flunitrazepam and/or its urinary metabolites can induce both base-pair substitutions or frame-shift point mutations.     

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Abstract Escherichia coli F-18, a normal human fecal isolate, and Salmonella typhimurium SL5319, an avirulent strain, are known to colonize the streptomycin-treated CD-1 mouse large intestine by utilizing nutrients present in intestinal mucus for growth. Moreover, previous experiments suggested the possibility that E. coli F-18 and S. typhimurium SL5319 utilized different mucus nutrients. Therefore, mouse cecal mucus was fractionated into high and low molecular weight components, and each fraction was inoculated either simultaneously or separately with E. coli F-18 and S. typhimurium SL5319. A 50 kd fraction was found in which the growth of S. typhimurium SL5319 suppressed growth of E. coli F-18. Evidence is presented that in this fraction S. typhimurium SL5319 utilizes peptides, presumably generated by mucus proteases, as a source of amino acids for growth. Furthermore, it is shown that S. typhimurium SL5319 grows in this 50 kd fraction with a generation time of 27 min in the presence of at most 7 μg of carbohydrate per ml and 2.2 μg of peptide per ml, and that S. typhimurium SL5319 suppresses E. coli F-18 growth in this fraction by sequestering iron. The data are discussed with respect to the role of peptide utilization and iron sequestration in the ability of S. typhimurium SL5319 to colonize the mouse large intestine.     

Comparison of nucleic acid hybridisation and other tests for detecting tobacco rattle virus in narcissus plants and potato tubers

All isolates of tobacco rattle virus (TRV) found in naturally infected narcissus leaves produced nucleoprotein particles, mostly in large concentrations but, because of antigenic diversity, less than half of the isolates were identified by immunosorbent electron microscopy (ISEM) and still fewer by enzyme-linked immunosorbent assay. All were identified by a nucleic acid hybridisation test in which DNA complementary to RNA-1 of strain PRN of TRV was allowed to react with nucleic acid extracted from leaf tissue. Spraing-affected tubers in some potato stocks yielded only NM isolates of TRV. These isolates do not produce virus particles and they were therefore not detected by ISEM. The infectivity of nucleic acid extracts from recently harvested tubers with spraing symptoms was much greater than that of extracts prepared from tubers after 8 months' storage. In other potato stocks, some spraing-affected tubers contained NM isolates and the rest contained particle-producing isolates (M isolates) of TRV. The infectivity of sap and of nucleic acid, extracted 7 months after harvest from tubers infected with M isolates, was much greater than that of nucleic acid extracted from comparable tubers infected with NM isolates. TRV was detected by nucleic acid hybridisation in extracts of almost all tubers containing either M or NM isolates, even when the tubers were not tested until 7–8 months after harvest. The probable sequence of events occurring after tubers are infected with TRV is outlined, and it is suggested that the virus will rarely become established in fields as a result of planting infected tubers.     

Mutagenic activation of 2-acetylaminofluorene by guinea-pig liver homogenates: essential involvement of cytochrome P-450 mixed-function oxidases

2-Acetylaminofluorene (AAF) was highly mutagenic to Salmonella typhimurium strain TA98, when activated by a liver post-mitochondrial supernatant fraction (S9 fraction) from guinea-pigs, in spite of the resistance of this species to AAF carcinogenesis and the low capacity of the liver of this species for N-hydroxylation of AAF. The mutagenicity was comparable to or higher than that resulting from activation by mouse- or rat-liver S9 fraction, and was not enchanced by treatment with cytochrome P-450 inducers, a combination of phenobarbital and 5,6-benzoflavone. In an attempt to understand this unexpected result we examined whether a cytochrome P-450 mixed-function oxidase system participated in the mutagenic activation of AAF by guinea-pig liver, as it does in the case of mouse liver. The mutagenic activation was: (1) completely dependent on the addition of a co-factor, NADPH, to the mutation assay system, (2) completely suppressed by antiserum against NADPH--cytochrome c reductase, and (3) sensitive to a cytochrome P-450 inhibitor, 7,8-benzoflavone. These results indicate that the cytochrome P-450 enzyme system is essentially involved even in the mutagenic activation of AAF by guinea-pig-liver S9 fraction. Based on both the present and other data, the mechanism of the mutagenic activation is discussed to explain the observed high mutagenic potential of AAF in the presence of guinea-pig-liver S9 fraction.     

Hydroperoxidase I catalyzes peroxidative activation of 3,3'-dichlorobenzidine to a mutagen in Salmonella typhimurium

Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester strains. Mutagenicity is observed when an S. typhimurium strain which is sensitive to frame-shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. In this paper, we show that the bacterial enzyme, hydroperoxidase I, is responsible for much of this activation. We constructed isogenic tester strains which lack hydroperoxidase I or II, due to Tn10 insertions in the structural genes encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was measured in each strain. A tester strain lacking hydroperoxidase I activity was much less sensitive than was the parent strain. When hydroperoxidase I activity was restored in this strain, via a plasmid-borne copy of the gene encoding the Escherichia coli protein, sensitivity to peroxide-dependent dichlorobenzidine mutagenicity was enhanced.     

Gliding Motility Mutants of Myxococcus xanthus

Two gliding motility mutants of Myxococcus xanthus are described. The semimotile mutant (SM) originated by high-frequency segregation from the motile FB(t) strain. Segregation was enhanced by acridine dye treatment. SM cells glide only when apposed to other cells in a swarm. The nonmotile strain (NM) originated by mutation from SM. NM cells neither glide individually nor cooperatively. FB(t), SM, and NM are indistinguishable with respect to fine structure, vegetative growth rate, glycerol-induced microcyst formation, spheroplasting, bacteriophage sensitivity, and responses to light. The motility mutants are more resistant to penicillin and more sensitive to actinomycin D than is the gliding wild type. The NM mutant is also a morphogenetic mutant; it is unable to form fruiting bodies.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.     

Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles

The adhesion of Salmonella typhimurium to the mineral particles quartz, albite, feldspar, and magnetite was shown to correlate with the hydrophobicity of the cell surface as measured by hydrophobic interaction chromatography. The same effects were also seen for seven other selected test strains, including Streptococcus faecalis, Streptococcus faecium, Escherichia coli, Citrobacter freundii, Shigella sonnei, and Shigella boydii. When the test strain of Salmonella typhimurium, was repeatedly cultivated in Luria broth, thus selecting for different degrees of fimbriation and roughness of the cell surface, varied cell hydrophobicity but constant negative and positive charge values were obtained. High hydrophobicity values always coincided with enhanced adhesion to the mineral particles. The negative charge of the bacterial surface as measured by electrostatic interaction chromatography appeared to play no role in the adhesion event. However, the positive charges on the cell surface contributed to the adhesion process. This was especially evident for cells exhibiting a high degree of hydrophobicity. Alteration of the pH between 4 and 9 did not significantly affect the adhesion process.     

Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles.

The adhesion of Salmonella typhimurium to the mineral particles quartz, albite, feldspar, and magnetite was shown to correlate with the hydrophobicity of the cell surface as measured by hydrophobic interaction chromatography. The same effects were also seen for seven other selected test strains, including Streptococcus faecalis, Streptococcus faecium, Escherichia coli, Citrobacter freundii, Shigella sonnei, and Shigella boydii. When the test strain of Salmonella typhimurium, was repeatedly cultivated in Luria broth, thus selecting for different degrees of fimbriation and roughness of the cell surface, varied cell hydrophobicity but constant negative and positive charge values were obtained. High hydrophobicity values always coincided with enhanced adhesion to the mineral particles. The negative charge of the bacterial surface as measured by electrostatic interaction chromatography appeared to play no role in the adhesion event. However, the positive charges on the cell surface contributed to the adhesion process. This was especially evident for cells exhibiting a high degree of hydrophobicity. Alteration of the pH between 4 and 9 did not significantly affect the adhesion process.     

The cda GenoTox assay: a new and sensitive method for detection of environmental genotoxins, including nitroarenes and aromatic amines

A new bacterial test system for detection of genotoxic compounds was developed, based on two new Salmonella typhimurium tester strains, TGO1 and TGO2. Both strains contain a gene fusion between a strong SOS-promotor, P(cda), and the gfp gene, which allows detection of genotoxic compounds that induce the SOS response. SOS induction was detected by means of flow cytometry. TGO1 showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine compared with a previously developed strain, which had an Escherichia coli strain as host instead of S. typhimurium. S9 mix was introduced into the assay, making the test system suitable for detection of indirect mutagens. Furthermore, the genes for bacterial nitro-reductase (NR) and o-acetyl transferase (o-AT) were inserted into TGO2, making it an NR- and o-AT-over-expressing strain. This resulted in an assay that was able to detect the nitroarene 1-nitropyrene and the aromatic amine 2-aminoanthracene with high sensitivity.