Nitroreductase-deficient Strain Research Articles

Carcinogenic risk of food additive AF-2 banned in Japan: a case study on reassessment of genotoxicity

BackgroundCarcinogenic risk assessment studies have been repeatedly improved and are still being debated to find a goal. Evaluation might be changed if new approaches would be applied to some chemicals which means that new approaches may change the final assessment. In this paper, the risk assessment of a chemical, in particular the proper carcinogenicity, is examined using the long-banned food additive, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2, as a case study.ResultsFirst, Ames tests were carried out using strains TA1535, TA100, TA1538, and TA98 and their nitroreductase-deficient strains YG7127, YG7128, YG7129, and YG7130. The results showed that mutagenic activity was reduced by about 50% in the nitroreductase-deficient strains, indicating that part of the mutagenic activity shown in Ames test was due to bacterial metabolism. Second, in vivo genotoxicity tests were conducted, including the one that had not been developed in 1970’s. Both a micronucleus test and a gene mutation assay using transgenic mice were negative. Third, assuming it is a genotoxic carcinogen, the virtual safety dose of 550 μg/day was calculated from the TD50 in rats with a probability of 10−5.ConclusionAF-2 has been shown to be carcinogenic to rodents and has previously been indicated to be genotoxic in vitro. However, the present in vivo genotoxicity study, it was negative in the forestomach, a target organ for cancer, particularly in the gene mutation assay in transgenic mice. Considering the daily intake of AF-2 in the 1970s and its virtually safety dose, the carcinogenic risk of AF-2 could be considered acceptable.

CYP1A1 and Cnr nitroreductase bioactivated niclosamide in vitro

Niclosamide produces genotoxic effects, such as point mutations in Salmonella sp., sperm-head abnormalities in mice and clastogenic effects in human lymphocytes in vitro and in vivo. As cytochrome P450 could be involved in the bioactivation of niclosamide, we investigated which subfamily was involved. We used liver microsomal fractions from rats treated with phenobarbital/β-naphthoflavone (PB/β-NF), benzo[a]pyrene (BaP) or cyclohexanol, which are known to induce different cytochrome P450 subfamilies, such as CYP2B, CYP1A1, CYP1A2 and CYP2E1. We also inhibited CYP1A and CYP2E using α-NF and diethyldithiocarbamate to identify the cytochrome P450 involved. Liver-S9 fractions obtained from PB/β-NF- and BaP-treated rats significantly increased the number of revertants induced by niclosamide, while the CYP1A1 inhibitor α-NF decreased the number of revertants. The incubation of niclosamide with CYP1A1 Supersomes™ increased the number of revertants, suggesting that CYP1A1 is responsible for the bioactivation of niclosamide. Nitroreduction is also involved in niclosamide bioactivation, as the nitroreductase-deficient strain YG7132 did not respond to the niclosamide treatment. Our findings indicated that a metabolite, derived from the action of CYP1A1 and a nitroreduction-reaction process, has a key role in the bioactivation of niclosamide.

Effects of bacterial and presystemic nitroreductase metabolism of 2-chloro-5-nitro-N-phenylbenzamide on its mutagenicity and bioavailability

2-Chloro-5-nitro-N-phenylbenzamide (GW9662), a potent irreversible PPAR-γ antagonist, has shown promise as a cancer chemopreventive agent and is undergoing preclinical evaluations. Studies were initiated to assess its bacterial mutagenicity and pharmacokinetic profile in two animal species prior to subchronic oral toxicity evaluations and the results are reported here. GW9662 was mutagenic in both TA98 and TA100 bacterial strains with and without metabolic activation but was negative in the nitroreductase-deficient strains (TA98NR and TA100NR) also with and without metabolic activation, indicating that GW9662 mutagenicity is dependent on nitroreduction. The mutagenic activity was predominantly via a base-substitution mechanism. Following oral dosing in rats and dogs, the parent compound, GW9662, was virtually absent from plasma samples, but there was chromatographic evidence for the presence of metabolites in the plasma as a result of oral dosing. Metabolite identification studies showed that an amine metabolite ACPB (5-amino-2-chloro-N-phenylbenzamide), a product of nitro reduction, was the predominant species exhibiting large and persistent plasma levels. Thus systemic circulation of GW9662 has been attained largely in the form of its reduced metabolite, probably a product of gut bacterial metabolism. GW9662 was detectable in plasma of rats and dogs after intravenous dose albeit at low concentrations. Pharmacokinetic analysis following intravenous dosing in rats showed a rapid clearance and an extensive tissue distribution which could have accounted for the very low plasma levels. Of note, the amine metabolite was absent following intravenous dosing in both rats and dogs, confirming it being a product of presystemic metabolism. The potential utility of GW9662 as a chemopreventive agent, especially as an Estrogen Receptor-α (ER-α) inducer in an otherwise ER-α negative breast tissue, is of great interest. However, the results shown here suggest that additional animal toxicological and bioavailability studies are required to establish a role of GW9662 as a chemopreventive agent.

In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction).The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.

Genotoxicity of nitrosulfonic acids, nitrobenzoic acids, and nitrobenzylalcohols, pollutants commonly found in ground water near ammunition facilities

2-Amino-4,6-dinitrobenzoic acid (2-A-4,6-DNBA), 4-amino-2,6-dinitrobenzoic acid (4-A-2,6-DNBA), 2,4,6-trinitrobenzoic acid (2,4,6-TNBA), 2-amino-4, 6-dinitrobenzylalcohol (2-A-4,6-DNBAlc), 4-amino-2,6-dinitrobenzylalcohol (4-A-2,6-DNBAlc), 2,4-dinitrotoluol-5-sulfonic acid (2,4-DNT-5-SA), 2,4-dinitrotoluol-3-sulfonic acid (2,4-DNT-3-SA), and 2, 4-dinitrobenzoic acid (2,4-DNBA) are derivatives of nitro-explosives that have been detected in groundwater close to munitions facilities. In the present study, the genotoxicity of these compounds was evaluated in Salmonella/microsome assays (in strains TA100 and TA98, with and without S9 and in TA98NR without S9), in chromosomal aberration (CA) tests with Chinese hamster fibroblasts (V79), and in micronucleus (MN) assays with human hepatoma (HepG2) cells. All compounds except the sulfonic acids were positive in the bacterial mutagenicity tests, with 2,4,6-TNBA producing the strongest response (8023 revertants/micromol in TA98 without S9 activation). 2-A-4,6-DNBA was a direct acting mutagen in TA98, but negative in TA100. The other positive compounds were approximately 1-3 orders of magnitude less mutagenic than 2,4,6-TNBA in TA98 and in TA100; relatively strong effects ( approximately 50-400 revertants/micromol) were produced by the benzylacohols in the two indicator strains. With the exception of 2,4-DNBA, the mutagenic responses were lower in the nitroreductase-deficient strain TA98NR than in the parental strain. 2,4-DNBA produced a marginally positive response in the V79-cell CA assay; the other substances were devoid of activity. Only the benzoic acids were tested for MN induction in HepG2 cells, and all produced positive responses. As in the bacterial assays, the strongest effect was seen with 2,4,6-TNBA (significant induction at >or=1.9 microM). 4-A-2,6-DNBA was positive at 432 microM; the weakest effect was observed with 2,4,-DNBA (positive at >or=920 microM). The differences in the sensitivity of the indicator cells to these agents can be explained by differences in the activities of enzymes involved in the activation of the compounds. The strong responses produced by some of the compounds in the human-derived cells suggest that environmental exposure to these breakdown products of nitro-explosives may pose a cancer risk in man.

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24 h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320 mg/kg in mice and 2000 mg/kg in rats: Muta™Mouse assay in colon and liver (5×320 mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [ 14 C ]-AMP397; comet assay (1×2000 mg/kg) in jejunum and liver of rats, sampling times 3 and 24 h after administration; micronucleus test (2×320 mg/kg) in the bone marrow of mice, sampling 24 h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

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.

Nitroarene Reduction and Generation of Free Radicals by Cell-Free Extracts of Wild-Type, and Nitroreductase-Deficient and -EnrichedSalmonella typhimuriumStrains Used in theumuGene Induction Assay

Studies of the enzymatic properties of cell-free extracts prepared from overnight cultures of the normal, and nitroreductase-deficient and -enriched strains ofSalmonella typhimurium,designed for use in theumugene induction assay of Odaet al.(1992), were undertaken in an effort to clarify the nature of nitroreductase deficiency in relation to mutagenicity. The ability of these strains to promote oxygen consumption and free radical intermediates of representative nitroarene substrates was measured, respectively, by oxygen polarography and electron spin resonance (ESR) spectroscopy. The substrates 4-nitropyridineN-oxide (4NPO) and 4-nitroquinolineN-oxide (4NQO) stimulated the rate and extent of NADH-dependent oxygen consumption catalyzed by cell-free extracts prepared from wild-type, and nitroreductase-deficient and -enriched strains. The extent of oxygen consumption was greater than stoichiometric with respect to the amount of nitroaromatic substrate, which implied one-electron reduction of 4NQO by these bacterial extracts and subsequent redox cycling with oxygen. ESR spectroscopy confirmed the production of free radical metabolites of the nitroarene substrates, which were inferred by the oxygen consumption studies. At equal protein concentrations the cell-free extracts of each strain catalyzed univalent reduction of 4NPO yielding the 59 line signal characteristic of the 4NPO nitro anion radical. This ESR signal was potently inhibited by the flavoprotein inhibitors CuSO4and PCMB, albeit a twofold or higher concentration of both inhibitors was required to inhibit the signal produced by extract from the nitroreductase-deficient strain than that produced by the other strains. The results indicate that the nitroreductase-deficient strain ofSalmonella typhimuriumdeveloped for use in theumugene induction assay is not deficient in either one-electron nitro group or quinone reductase activity.

Nitroreductase independent mutagenicity of 1-halogenated-2,4-dinitrobenzenes

The 1-halogen substituted 2,4-dinitrobenzenes have been found to be mutagenic in Salmonella TA98 with an activity order of 1-fluoro>1-chloro>1-bromo>1-iodo. This specific activity was not lowered in the nitroreductase deficient strain TA98NR and O-acetyltransferase deficient strain TA98/1,8-DNP 6, indicating that the mutagenicity of these compounds is not dependent upon –NO 2 reduction to –NHOH and its subsequent esterification. The activity was, rather, higher in the former and remained almost equal in the latter strain compared to TA98, suggesting these compounds to react directly with bacterial DNA. Further, the reaction of these halodinitrobenzenes with methionine, at physiological pH, resulting in the formation of 1- S-methyldinitrobenzene showed that these compounds have the ability to attack DNA directly through nucleophilic substitution of the halogen atom and the role of the bacterial nitroreductases for production of mutagenic lesions was not considered. It was, further, proposed that these compounds interact with DNA through S NAr mechanism instead of S N2 cited in literature.

Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6-trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6-week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and beta-glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse-phase high-performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions, were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5-18, 21, 22, and 24-26 contained mutagens detected by strain TA98. In the nitroreductase-deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9-11, 15, and 25 clearly contained direct-acting mutagens. Fewer fractions were positive in strain TA100 (9-16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks.

Mutagenicity of HPLC‐fractionated urinary metabolites from 2,4,6‐trinitrotoluene‐treated Fischer 344 rats

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6-trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6-week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and β-glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse-phase high-performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5–18, 21, 22, and 24–26 contained mutagens detected by strain TA98. In the nitroreductase-deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9–11, 15, and 25 clearly contained direct-acting mutagens. Fewer fractions were positive in strain TA100 (9–16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks. Environ. Mol. Mutagen. 30:298–302, 1997. © 1997 Wiley-Liss, Inc.

Genotoxicity of influents and effluents of the wastewater treatment plant

Wastewaters are treated through different processes of wastewater treatment procedures. Nevertheless they often contain mutagens especially when the proportion of industrial wastewater in comparison to municipal wastewater is high. In this study we evaluated mutagenic potential of influents and effluents from a wastewater treatment plant that is processing both industrial and domestic wastewater. The mutagenicity of XAD-2 extracts of influent and effluent was evaluated by means of Ames test using S. typhimurium strains TA100 and TA98 in the presence and in the absence of metabolic activation. Extracts that were mutagenic to strain TA98 without metabolic activation were suspected to contain nitropolyaromatic hydrocarbons (nitro-PAHs). To confirm this hypothesis they were tested with nitroreductase (TA98NR) and O-acetiltransferase (TA98/1,8DNP6) deficient derivatives of strain TA98, that are resistant to nitropolyaromatic hydrocarbons. The nitroreductase deficient strain TA98NR was less sensitive to the mutagenic extracts of influent than the parent strain TA98. The O-acetiltransferase deficient strain TA98/1,8DNP6 was resistant to the mutagenic extracts of influent. The mutagenic extracts of the effluent were nearly equally mutagenic to the parent strain TA98 and both deficient strains TA98NR and TA98/1,8DNP6. On the basis of the responses of nitroreductase deficient strains on the influent and effluent it was concluded that the influent contained nitro-PAHs. These were not removed or inactivated during the biological treatment of the wastewater, but activated to the final nucleophylic form able to induce mutations strain TA98 and in its nitroreductase deficient derivatives TA98NR and TA98/1,8DNP6.

Genotoxicity of influents and effluents of the wastewater treatment plant

Wastewaters are treated through different processes of wastewater treatment procedures. Nevertheless they often contain mutagens especially when the proportion of industrial wastewater in comparison to municipal wastewater is high.In this study we evaluated mutagenic potential of influents and effluents from a wastewater treatment plant that is processing both industrial and domestic wastewater. The mutagenicity of XAD-2 extracts of influent and effluent was evaluated by means of Ames test using S. typhimurium strains TA1OO and TA98 in the presence and in the absence of metabolic activation. Extracts that were mutagenic to strain TA98 without metabolic activation were suspected to contain nitropolyaromatic hydrocarbons (nitro-PAHs). To confirm this hypothesis they were tested with nitroreductase (TA98NR) and O-acetiltransferase (TA98/1,8DNP6) deficient derivatives of strain TA98, that are resistant to nitropolyaromatic hydrocarbons. The nitroreductase deficient strain TA98NR was less sensitive to the mutagenic extracts of influent than the parent strain TA98. The O-acetiltransferase deficient strain TA98/1,8DNP6 was resistant to the mutagenic extracts of influent. The mutagenic extracts of the effluent were nearly equally mutagenic to the parent strain TA98 and both deficient strains TA98NR and TA98/1,8DNP6.On the basis of the responses of nitroreductase deficient strains on the influent and effluent it was concluded that the influent contained nitro-PAHs. These were not removed or inactivated during the biological treatment of the wastewater, but activated to the final nucleophylic form able to induce mutations strain TA98 and in its nitroreductase deficient derivatives TA98NR and TA98/1,8DNP6.

Genotoxicity of potential metabolites of nitroscanate — an antischistosomal drug

The potential metabolites of nitroscanate(4-isothiocyanato-4′-nitrodiphenyl ether) such as 4-amino-4′-nitrodiphenyl ether (ANDE), 4-acetamido -4″-nitrodiphenyl ether (AcNDE), 4-acetamido-4′-nitrosodiphenyl ether (4-N = 0), 4-acetamido-4′- hydroxylaminodiphenyl ether (4-NHOH), 4-acetamido-4′-acetohydroxamicdiphenyl ether [4-N(OH)AC], 4-acetamido-4′-for-mohydroxamicdiphenyl ether [4-N(OH)CHO] and 4-acetamido-4′-acetylaceto-hydroxamicdiphenyl ether [4-N(OAc)Ac] were synthesized and investigated in the standard Salmonella mutagenicity test using TA98, TA98NR, TA98/1,8-DNP 6, TA100 and TA100NR as indicator strains, in the presence and absence of hepatic S9. The relative order of activity among nitro and its reduction products, 4-N =0 and 4-NHOH in TA98 and TA100 was 4-N - 0 > 4-NHOH > AcNDE. In nitroreductase deficient strain TA98NR, AcNDE was inactive, but expressed a slight activity in TA100NR while 4-N =0 and 4-NHOH showed a large increase in specific activity in both the strains. In O-acetyltransferase deficient strain TA98/1,8-DNP 6, AcNDE was inactive, while 4-N = 0 and 4-NHOH showed a sharp fall inactivity. The hydroxylamine derived products with an activity order 4-N(OAc)Ac > 4-N(OH)CHO > 4-N(OH)Ac in both TA98 and TA100, showed 3–6 times increase in the specific activity for the latter two compounds in the presence of S9 mix, which was inhibited in the presence of paraoxan, indicating N-deacylation as an important metabolic activation pathway. Except the 4-NO in TA100, the observed mutagenicity of nitroscante (NSC) was higher than those of potential metabolites and the nor-isothiocyanato derivative 4′-nitrodiphenyl ether, thereby showing that -NCS function has a potentiating effect on the mutagenicity of this drug.

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenicities of river water in Bangkok, Tokyo and Kyoto

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 mutagenecity 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 of TA98. In contrast to NB and o-CNB, synthetized phenylhydorxylamine (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 conculded that S9 and NOH play a role in metabolic activation other than the nitro group to hydroxylamine and subsequent esterification for the mutagenesis of NB and its derivatives.

Bioassay-directed chemical analysis of genotoxic components in urban airborne particulate matter from Barcelona (Spain)

Organic extracts of airborne particulate matter, collected in the city of Barcelona, were subjected to three-level, bioassay-directed, chemical fractionation, including gel permeation chromatography (GPC) and normal-phase (NP) and reversed-phase (RP) liquid chromatography (LC). The chemical characterization, directed by the Salmonella microsome mutagenicity assay (TA98, TA98NR and TA98/1,8DNP 6+/-S9), was carried out by capillary GC (CGC) coupled to selective detection systems, and by GC-MS techniques. The results obtained with the nitroreductase deficient strains show the important contribution of nitroaromatic compounds. Detailed chemical analysis of the mutagenic fractions led to the identification of 82 aromatic compounds and revealed the large contribution of chemical classes that are more polar than polycyclic aromatic hydrocarbons such as aromatic ketones, quinones and aldehydes.

Substituent effects on the genotoxicity of 4-nitrostilbene derivatives

4-Nitrostilbene and twelve of its derivatives (eleven E-stilbenes and two Z-stilbenes) were examined for possible quantitative structure-activity relationships of their in vitro and in vivo genotoxicity. Relative mutagenicity was studied with and without S9 activation in Salmonella strains TA98 and TA100, as well as in the nitroreductase deficient strains TA98/NR and TA100/NR. Chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of the nitrostilbenes were observed as an indicator of in vivo genotoxicity. All of the compounds were active in TA98 and TA100 without S9 activation, with the exception of 4-amino-4′-nitrostilbene in TA100. Mutagenic activity was greatly reduced or eliminated in the NR strains, which is consistent with metabolic activation of the compounds by bacterial reductase. The presence of S9 lowered the activity of most of the nitrostilbenes presumedly by enzymatic detoxication. Hammet values of substituents, partition coefficients and frontier orbital energies ( E LUMO and E HOMO) were studied for correlations with mutagenicity of the eleven E-stilbenes. Correlations could be established between mutagenicity in TA98 without S9 activation and the Hammet values. The same mutagenicity could also be correlated to E LUMO. Rationales for these correlations include the concept that electron-withdrawing groups which lower E LUMO should facilitate the reduction of the nitro group, leading to the proximate mutagen hydroxylamine. The correlations are also explained by the concept that electron-withdrawing groups should help stabilize the hydroxylamine intermediate and make the ultimate mutagenic species, the nitrenium ions, more reactive toward DNA. The relationship between mutagenicity and electronic effects of substituent groups found in vitro could not be extended to the in vivo results. However, except for the dinitrostilbenes, where insolubility prevented their testing, all the nitrostilbenes produced a statistically significant increase in chromosomal aberrations compared to the negative solvent control.

Polar organic matter in airborne particles: chemical characterization and mutagenic activity

Polar organic matter (extracted from inhalable particles collected in Elizabeth, NJ, and from National Institute of Standards and Technology Standard Reference Material SRM 1649) has been characterized by determining elemental and ionic composition, chemical classes, and mutagenic activity. The acetone extracts of SRM 1649 and Elizabeth, NJ, samples were 46 and 40% carbon, respectively. When compared to the NJ extract, the SRM extract was enriched in aldehydes and ketones and deficient in carboxylic acids. Significant amounts of organic nitrogen were found in both extracts. Infrared spectra and class tests suggested the presence of nitro compounds, organic nitrates or nitrites, amines, and amides. Fluorescence suggested the presence of polycyclic aromatic species. For SRM 1649 the acetone extracts accounted for 36% (-S9) and 40% (+S9) of the mutagenic activity in the Ames assay with TA98 (specific mutagenic activity). The acetone extract of SRM 1649 had about four times greater mutagenic activity than that from the NJ particles. Both extracts showed substantial decreases in mutagenic activity when tested with nitroreductase-deficient strains of TA98. A simple resolubilization of the NJ extract concentrated the most mutagenic components into the least polar of the three fractions. 29 refs., 3 figs., 5 tabs.

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 β-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 activvity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaptthalene, 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.