Metabolism of K-region derivatives of 1-nitropyrene by rat liver in vitro

The metabolism of K-region derivatives of 1-nitropyrene wasstudied in order to provide insight into factors that may contributeto their mutagenic activities and to obtain information on unknownmetabolites of 1-nitropyrene. Using 9000 g supernatant fromlivers of Aroclor-treated rats, 1-nitropyrene-4,5-diol, a mutagenicmetabolite of 1-nitropyrene, was metabolized to 1-aminopyrene-4,5-diol,a mixture of 1-nitropyrene-4,5,9,10-tetraols, 1-amino-4,5-pyrenedioneand 1-nitro-4,5-pyrenedione. 1-Nitro-5H-phenanthro[4,5-bcd]pyran-5-one,a highly mutagenic lactone, was not detected. The metabolismof 1-nitro-4,5-pyrenedione yielded only 1-amino-4,5-pyrenedione;the lactone was not observed. No metabolites were detected whenthe lactone was incubated under conditions identical to thoseemployed for 1-nitro-4,5-diol and 1-nitro-4,5-pyrenedione. Uponre-examination of the metabolism of 1-nitropyrene, we were ableto detect 1-nitropyrene-4,5,9,10-tetraol, 1-amino-4,5-pyrenedioneand 1-nitro-4,5-pyrenedione as minor metabolites in additionto the major metabolites reported previously. The results ofthis study, combined with the mutagenicity data for the K-regionderivatives of 1-nitropyrene, suggest that nitroreduction of1-nitropyrene-4,5-diol and 1-nitro-4,5-pyrenedione to the correspondinghydroxylamines is an important pathway for their metabolic activationin Salmonella typhimurium and possibly in mammalian systems.     

Synthesis and mutagenicity of 1-nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene, potential intermediates in the metabolic activation of 1,6- and 1,8-dinitropyrene

1,6-Dinitropyrene and 1,8-dinitropyrene are environmental contaminantswhich are mutagenic in bacteria and cultured mammalian cells.Since nitroreduction, and possibly O-acetylation, have beenimplicated in the metabolic activation of these compounds, thereduced intermediates, 1-nitro-6-nitroso-pyrene and 1-nitro-8-nitrosopyrene,were synthesized and their mutagenicity examined in Salmonellatyphimurium and Chinese hamster ovary (CHO) cells. Nitrationof 1-acetyl-aminopyrene yielded a mixture of 1-acetylamino-6-nitropy-reneand 1-acetylamino-8-nitropyrene, which was separated by flashchromatography. Following deacetylation, the amino-nitropyreneswere oxidized to the desired nitronitrosopyrenes with m-chloroperoxybenzoicacid. Both nitronitrosopyrenes showed similar levels of mutagenicityin S. typhimurium strain TA98 and a nitroreductase-deficientanalogue, TA98NR, but much lower activity in the esterificase-deficientstrain, TA98/1, 8-DNP₆, which suggested that reduced metabolitesrequire further activation by O-acetylation. In contrast, theanalogous compound, 1-nitrosopyrene, was equally mutagenic inall three strains while its parent compound, 1-nitropyrene,demonstrated a much reduced mutagenicity in strain TA98NR. InCHO cells, 1-nitropyrene was not mutagenic and the dinitropyreneswere only weakly active, while all three nitroso-pyrene derivativeswere highly mutagenic. These data support the hypothesis thatnitrated pyrenes are metabolized to muta-gens through nitroreduction.In Salmonella the limiting step in the metabolic activationof 1-nitropyrene appears to be the initial reduction to 1-nitrosopyrene,while with the dinitropy-renes subsequent esterification ofthe reduced intermediates seems critical. With CHO cells, theinitial reduction to nitroso derivatives is the limiting stepfor all nitropyrenes, and esterification does not appear tooccur in the activation sequence.     

CARCINOGENESIS:In vivo formation of mutagens by intraperitoneal administration of polycyclic aromatic hydrocarbons in animals during exposure to nitrogen dioxide

Consumption of fossil fuels has increased indoor and outdoorconcentrations of polycyclic aromatic hydrocarbons (PAHs) andnitrogen dioxide (NO₂). To study the combined effect of PAHadministration and NO₂ exposure on mutagenicity of urine fromanimals we injected 400 mg/kg body wt i.p. one of five kindsof PAH (pyrene, fluoranthene, fluorene, anthracene and chrysene)into ICR mice, Wistar rats, Syrian golden hamsters or Hartleyguinea pigs after exposure to 20 p.p.m. NO₂ gas for 24 h andthen exposed the animals to NO₂ gas for an additional 24 h.During the latter 24 h we collected the urine and assayed itsmutagenicity with the Ames Salmonella strains after treatmentwith ß-glucuronidase and arylsulfatase and extractionwith dichloromethane. The urine from mice treated with bothPAH and NO₂ showed high mutagenicity for Salmonella typhimuriumstrains TA98 and TA100, whereas the urine from mice treatedwith PAH and air showed almost no mutagenic activity. The mutagenicitywas decreased in nitroreductase- and acetyltransferase-deficientstrains TA98NR and TA98/1, 8-DNP6 respectively. Treatment witha mixture of 20% of each of the five kinds of PAH and NO₂ augmentedthe urinary mutagenicity of mice 1.5-fold. The urine from hamsterstreated with pyrene or fluoranthene and NO₂ was also highlymutagenic, but that from rats or guinea pigs was not very mutagenic.The mutagenicity was also decreased in strains TA98NR and TA98/1,8-DNP₆. These results suggest that the urine contains nitrocompounds and that the nitration of PAHs occurs in the bodyof animals under exposure to NO₂ gas. Actually, the nitratedmetabolites of pyrene, l-nitro-6/8-hydroxypyrene and l-nitro-3-hydroxypyrene,were detected in the urine from mice treated with pyrene underexposure to NO₂ gas. To elucidate the mechanism of in vivo nitration,NO₂ (20 p.p.m.) was bubbled through 50 mM TVis-HCl buffer (pH7.4) or dichloromethane solution containing pyrene or 1-hydroxypyrene(10 µg/ml). Pyrene was not nitrated by NO₂ in either aqueousor organic solutions. However, 1-hydroxypyrene was changed tonitrohydroxy-pyrenes by NO₂ in the Tris-HCl buffer, but notin the organic solution. Ascorbic acid,     

Mutagenicity of dibromodulcitol (DBD), an alkylating anticancer drug) and its mono- and bifunctional conversion products studied by the Salmonella/microsome assay

Dibromodulcitol (DBD) and one of its most important bifunctionaltransformation products, dianhydrogalactitol (DAG) with similarcytostatic effect, were tested by the Salmonella/microsome assayon strains TA 1535, TA 1538, TA 98 and TA 100 using the plateincorporation technique. Both drugs were direct mutagens instrains TA 1535 and TA 100 and non-mutagenic in other strains.Their mutagenic effect was not influenced by S-9 mix from ratliver. Mutagenicity of DAG was very limited because of its markedtoxicity. The other monofunctional alkylating derivatives, i.e.,1-bromo-3, 6-anhydrodulcitol and 1,2-epoxy-3,6-anhydrodulcitolwere highly mutagenic in strains TA 1535 and TA 100 with andwithout S-9 mix despite having no anticancer effect. Anticanceractivity was exerted only by the bifunctional alkylating hexitols(DBD, DAG) which showed moderate mutagenic activity comparedto the monofunctional derivatives. No correlation could be establishedbetween the mutagenic and anticancer effect of the four structurallyrelated hexitols. Mutagenicity of urine and bile from rats aftera single administration of the maximum tolerated (450 mg/kg)dose of DBD was also examined, and the hexitol components ofthe same urine sample were identified by t.l.c. DBD and itsmutagenic transformation products were excreted in urine andnot through the bile. The mutagenic effect of DBD observed cannotbe attributed exclusively to DBD itself, because the parentmolecule, like other alkylating agents, easily undergoes spontaneousdecomposition under in vitro and in vivo conditions to releaseboth bi- and monofunctional alkylating solvolysis products andthese highly reactive derivatives may play a role in this effect.No significant difference in the relative mutagenicity was detectedbetween DBD and cyclophosphamide, used as a reference substance.     

Metabolism of 1-nitro[U-4,5,9,10-14C]pyrene in the F344 rat

1-Nitro[U-4,5,9,10-14C]pyrene was synthesized and administered to male F344 rats by intragastric gavage at a dose of 100 mg/kg of body weight. During the first 48 hr, 41% of the dose was eliminated in the feces, and 16% was eliminated in the urine. The corresponding figures after 120 hr were 51 and 19%. In rats with bile cannulae, 37% of the dose was excreted in the bile after 72 hr, and 6% was excreted in the urine. Fecal metabolites included 1-aminopyrene (isolated amount, 11.7% of the dose), 1-amino-6-hydroxypyrene and 1-amino-8-hydroxypyrene (4.6%), and unchanged 1-nitropyrene (6.6%). 1-Aminopyrene and the 1-aminohydroxypyrenes were identified as their acetyl-derivatives by comparison of their chromatographic retention times, mass spectra, and UV spectra to those of synthetic standards. Biliary metabolites included 1-aminopyrene, 1-amino-6-hydroxypyrene, 1-amino-8-hydroxypyrene, 1-nitro-6(8)-hydroxypyrene, and 1-nitro-3-hydroxypyrene, as well as their glucuronide and sulfate conjugates. The isolated amounts of these metabolites accounted for approximately 5% of the dose. 1-Amino-6-hydroxypyrene and 1-amino-8-hydroxypyrene and their glucuronide and sulfate conjugates were also tentatively identified in the urine and accounted for about 3% of the dose. Significant quantities of unidentified water soluble metabolites were present in the urine and bile. The results of this study indicate that metabolic reduction of the highly mutagenic 1-nitrohydroxypyrenes occurs in vivo in the rat and suggest that this is a possible activation pathway in 1-nitropyrene carcinogenesis.     

Mutagenicity of gastric juice: the importance of controlling histidine concentration when using Salmonella tester strains

The mutagenic activity of gastric juice has been assessed usingbacterial tester strains that undergo reverse mutation (Salmonellatyphimurium:his^– his⁺). Free histidine, a known sourceof inaccuracy in this mutation test system, was detected in42 of 73 juice samples (concentration range 3.5–992.4µg/ml); high histidine concentrations were significantlycorrelated with hypochlorhydria. The effect of histidine wascontrolled by using a pre-incubation modification of the Salmonellafluctuation test in which juke samples and their correspondingcontrol cultures containing equivalent amounts of histidinewere incubated with the tester bacteria prior to plating out.Significant mutagenic activity was found in a high proportionof samples (18 of 20). The histidine content in gastric juicewhich can affect in vitro mutagenicity testing must be adequatelycontrolled before positive or negative results can be equatedwith the presence or absence of intragastric carcinogens.     

Effect of the nitro group conformation on the rat liver microsomal metabolism and bacterial mutagenicity of 2- and 9-nitroanthracene

The aerobic and hypoxic metabolism of 2-nitroanthracene (2-NA)and 9-nitroanthracene (9-NA), two components of diesel exhaust,was studied and the mutagenicities of the parent compounds andtheir metabolites were compared. 2-NA was metabolized by 3-methyIcholanthrene-inducedrat liver microsomes under aerobic conditions to 2-NA trans-5,6-dihydrodiol, 2-NA trans-7, 8-dihydrodioI, 2-NA 7-keto-5, 6,7, 8-tetrahydro-trans-5, 6-diol, 2-NA 6-keto-5, 6, 7, 8-tetrahydro-trans-7,8-diol, 2-nitro-9, 10-anthraquinone and 2-NA 5, 6, 7, 8-tetrahydrotetrol.When incubations were conducted under hypoxic conditions, 2-aminoanthracenewas produced faci-ly. N.m.r. spectral analysis indicated thatthe nitro-substituent of 2-NA and all of its ring-oxidized metabolitespreferentially adopted an orientation in which the nitro groupwas co-planar or nearly co-planar with the aromatic ring system.2-NA and its two trans-dihydrodiol metabolites were mutagenicin Salmonella typhimurium strain TA98, both in the presenceand in the absence of S9 enzymes while the two tetrahydrodiol-ketoneswere much less mutagenic. When assayed in strains TA98NR andTA98/1, 8-DNP₆, the mutagenic activities of 2-NA and the trans-7,8-dihydrodioI were decreased. 2-Aminoanthracene was mutagenicin strain TA98 only in the presence of S9 enzymes. When 2-aminoanthracenewas metabolized aerobically, the corresponding trans-5, 6- and7, 8-dihydrodiols were not detected. These results suggest that2-NA can be metabolized to mutagenic products by (i) nitroreductionand (ii) ring-oxidation followed by nitroreduction, but notnitroreduction followed by ring-oxidation. Aerobic metabolismof 9-NA produced 9-NA trans-1, 2- and 3, 4-dihydrodiols, whilemetabolism was not detected under anaerobic conditions. Previousstudies indicated that 9-NA and its two metabolites were notmutagenic in TA98. The differences in the orientation of thenitro substi-tuents in 2-NA and its ring-oxidized metabolitesand in 9-NA and its metabolites can be employed to explain thestrong mutagenicity of 2-NA and weak mutagenicity of 9-NA whenassayed both in the absence and in the presence of S9 activationenzymes.     

Mutagenicity of hexachloro-1,3-butadiene and its S-conjugates in the Ames test--role of activation by the mercapturic acid pathway in its nephrocarcinogenicity

The mutagenicity of hexachloro-1,3-butadiene and its S-conjugates1-(glutathion-S-yl)-1 ,2,3,4,4-pentachloro-1,3-butadiene (GTB),1,4-(bis-glutathion-S-yl-1,2,3,4-tetrachloro-1,3-buta-diene(BGTB) and 1,4-(bis-cystein-S-yl)-1,3-but adiene (BGTB) wasinvestigated in Salnwnella typhimurium TA100 using a modifiedpreincubation assay. GTB was a direct-acting mutagen; the mutagenicpotency of GTB was markedly enhanced by rat kidney nucrosomesor mitochondria and less so by cytosol. The ba-conjugates BGTBand ECFB were not mutagenic in the strains TA100, TA2638 andTA98. Purified HCBD was not mutagenic either without exogenousmetabolic activation or with rat liver microsomes fortifiedwith NADPH. Preincubation with rat liver micro somes and glutathioneresulted in an unequivocal mutagenic activity of HCBD whichwas increased by additional inclu sion of rat kidney microsomes.The cysteine conjugate ß-lyase inhibitor aminooxyaceticacid decreased the mutagenicity of HCBD and its S-conjugates.These results provide strong evidence that formation of theconesponding monoglutathione S-conjugate from HCBD and subsequentcleavage of this conjugate by -glutamyltranspeptidase and ß-lyasemay be responsible for the nephrocarcinogenicity of the parentcompound in vivo, whereas formation of the bis-glutathione S-conjugateprobably plays no role in the organ specific effects of HCBD.     

Identification of ring oxidized metabolites of 1-nitropyrene in the feces and urine of germfree F344 rats

1-Nitro[U-4,5,9,10-¹⁴C]pyrene in trioctanoin was administeredby gavage to male germfree F344 rats. Extracts of the 120 hfeces and urine were analyzed by h.p.l.c., with or without acetylation.4,5-Dihydro-4,5-dihydroxy-1-nitropyrene, 1-nitro-3-hydroxypyrene,1-nitro-6-hydroxypyrene and 1-nitro-8-hydroxypyrene but not1-aminopyrene, 1-amino-6-hydroxypyrene or 1-amino-8-hydroxypyrenewere identified by co-chromatography with standards. These resultsare in contrast to those obtained in the conventional F344 ratin which 1-aminopyrene, 1-amino-6-hydroxypyrene, and 1-amino-8-Miydroxypyrenewere fecal and urinary metabolites, indicating that intestinalmicroflora are involved in their formation.     

Interaction between polycyclic aromatic hydrocarbons, crude oil and oil dispersants in the Salmonella mutagenesis assay

Mutagenicity assays were carried out in the Salmonella/microsometest, using five S. typhimurium his strains (TA1535, TA1537,TA1538, TA98 and TA100), both in the presence and absence ofpost-mitochondrial preparations from Aroclor-induced rat liversand suitable co-factors. Seven oil dispersants showed a widerange of toxicity towards the bacterial strains, without elicitingany mutagenic response at sub-lethal concentrations. One sampleof crude oil and its dimethylsulphoxide (DMSO) extract werealso negative, and no mutagenic effect could be detected bychecking mixtures of crude oil with each of the seven dispersantstested. Two polycyclic aromatic hydrocarbons, benzo(a)pyrene(BP) and benz(a) anthracene (BA), which are generally consideredto be the most documented carcinogenic components of crude oil,were mutagenic with a frameshift mechanism, requiring metabolicactivation. BP mutagenicity was not affected by oil dispersantsnor by seawater. Conversely, the mutagenicity of BP DMSO-solutionswas abolished in the presence either of whole crude oil, ofits DMSO extract, or of crude oil/dispersant mixtures. Theselosses of mutagenicity could be mainly ascribed to a mechanicaltrapping of BP by oil components.     

Mutagenic activation of carcinogenic N-nitrosopropylamines by liver S9 fractions from mice, rats and hamsters: evidence for a cytochrome P-450-dependent reaction

The mutagenic potential of nine carcinogenic N-nitrosopropylamineswas examined by Ames preincubation assay using liver 9000 gsupernatant (S9) fractions from female rats and male hamstersand mice for metabolic activation. N-Nitrosobis(2-hydroxypropyl)amine,N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, N-nitrosobis(2-oxopropyl)amine,N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine,N-nitrosomethyl(2-hydroxypropyl)amine, N-nitrosomethyl(2-oxopropyl)amine,N-nitroso(2,3-dihydroxypropyl)(2-hydroxypropyl)amine and N-nitrosomethyl(2,3-dihydroxypropyl)amineall showed positive mutagenicity in strain TA100 in the presenceof liver S9 from three animal species pretreated with polychlorinatedbiphenyls or phenobarbital (PB). The S9-mediated mutagenicityof these N-nitrosamines was almost completely diminished bythe removal of NADP⁺ from the assay system. All the activitieswere considerably decreased by preincubation in an atmosphereof carbon monoxide or adding cytochrome c to the S9 mixture.Metyrapone considerably inhibited mutagenicity, whereas 7,8-benzoflavonewas totally lacking this effect. These results demonstrate acorrelation between the mutagenicity of nine N-nitrosopropylaminesmediated by liver S9 from three animal species and their knowncarcinogenicity in rodent in vivo experiments, and that thePB-inducible major cytochrome P-450 is selectively involvedin the mutagenic activation. A relationship between mutagenicpotencies of the N-nitrosamines and their known carcinogenicpotencies in rats and hamsters is discussed     

Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material

Ram seminal vesicle (RSV) microsomal preparations activate benzidineand other arylamines to mutagenic species in a modified Amesassay. We have examined the mechanism of this activation processin more detail. The mutagenic effect was neither arachidonicacid-dependent nor indomethacin in hibitable. The mutagenicspecies was stable for at least 30 min in experiments in whichaddition of bacteria was delayed. Acetylbenzidine was a muchmore potent mutagen than benzidine in this system. Substitutionof the acetylase-deficient tester strain TA98/1,8-DNP₆ for strainTA98 markedly reduced the mutagenicity of acetylbenzidine andcompletely eliminated the mutagenicity of benzidine. Benzidineanalogues 3,3'-dimethoxybenzidine (o-dianisidine), o-tolidineand 3,3',- 5,5'-tetramethylbenzidine were not mutagenic in theRSV activation system. RSV-dependent activation of all radiolabeledcongeners examined resulted in covalent binding to calfthymusDNA. The rank order of binding was: 3,3'-dichloro-benzidine> henzidine > o-dianisidine > acetylbenzidine >tetramethylbenzidine. This binding required active enzyme andarachidonic acid or hydrogen peroxide. The reactive specieswas short-lived: delayed addition of DNA reduced the level ofbinding nearly to zero. Binding was inhibitable by indomethacin,but this inhibition was incomplete in the cases of dichlorobenzidineand acetylbenizidine. We conclude that the extracellular generationof peroxidase-catalyzed oxidation products does not explainthe RSV microsome-dependent mutagenicity observed with thesecompounds.     

SHORT COMMUNICATION: Effects of 6-nitro substitution on 5-methylchrysene tumorigenicity, mutagenicity and metabolism

6-Nitro-5-methylchrysene was prepared by nitration of 5-methylchryseneand the mutagenic and tumorigenic activities of the two compoundswere compared. Whereas 5-methylchrysene was a strong tumor initiatoron mouse skin, no tumors were observed in the mice treated with6-nitro-5-methylchrysene. In Salmonella typhimurium TA100, bothcompounds were mutagenic in the presence, but not in the absence,of rat liver 9000 g supernatant. The major metabolite of 6-nitro-5-methylchrysenein rat liver in vitro was trans-1,2- dihydro-1,2- dihydroxy-6-nitro-5-methylchrysene.In view of the ready conversion of 6-nitro-5-methylchryseneto a 1,2-dihydrodiol, its apparent lack of tumorigenicity inmouse skin was intriguing.     

Comparison of the mutagenic activity of dialkylnitrosamines in animal-mediated and in vitro assays using an Escherichia coil indicator

An intrasanguineous host-mediated assay was used to establishthe mutagenic potential of a series of dialkylnitrosamines toE. coli K12/343/113 in the liver and spleen of mice. For calibratingpurposes, dose-and time-dependent kinetics of mutation inductionby the model compound diethylnitrosamine in this assay was determined.Comparison with the results of the same compound in vitro revealsthat activation in the intact liver of living mice is more efficientand proceeds for a longer period of time than during incubationin the presence of a liver homogenate. The mutagenicity of fiveother dialkylnitrosamines (dimethyl-, diethanol-, diisopropyl-,methylethyl-, and methyl-n-propylnitro-samine) was also determined.The results of host-mediated assays in livers and spleens ofmice indicate a good correlation with the carcinogenic propertiesof these compounds as far as the effect on the liver is concerned.The mutagenic activity in vitro shows, however, a poor correlationwith carcinogenicity data, mainly because some of the carcinogenicnitrosamines are not detectable in those tests. It is concludedthat, under the present experimental conditions, intrasanguineoushost-mediated assays are more sensitive and more representativeof the carcinogenic activity of dialkylnitrosamines than invitro assays using S-9 liver fractions.     

Effect of methyl substitution on mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline isolated from broiled sardine

2-Amino-3-methylimldazo [4,5-f]quinoline [I] is a potent mutagenisolated from broiled sun-dried sardine. [I] and its seven ofderivatives, (two isomers, one demethylated derivative and fourmethyl-substituted derivatives), were tested for mutagenicityon Salmonella typhimurium TA98 and TA100 in the presence ofS9 mix. 2-Amino-1,4-dlmethylimldazo [4,5-f]quinoline was thestrongest mutagen of these 8 compounds on TA98, giving 159,000revertants/nmol (750,000 revertants/µg). The demethylatedderivative, 2-aminoimidazo [4,5-f]quinoline, had very weak mutagenicity,inducing only 55 revertants/nmol (200 revertants/µg).Compounds having a methyl group at position N-1 or N-3 of 2-aminoimidazo[4,5-f]quinoline were strong mutagens. The 1,5-dimethyl-derivativewas more mutagenic than 3,5-dimethyl-derivative. Introductionof a methyl group at position 4 and position 5 enhanced andreduced the mutagenicity, respectively. All the compounds testedwere more mutagenic to TA98 than to TA100, but their relativeorders of mutgenlcity with TA98 and TA100 were the same.     

Mechanism of N-acetylcysteine (NAC) and other thiols as both positive and negative modifiers of MNNG-induced mutagenicity in V79 Chinese hamster cells

Carcinogenic xenobiotics can be detoxified by nucleophilic thiols,which interact directly or through enzyme catalyzed reactionswith electrophilic metabolites/compounds or metabolically producedoxidants. Formation of such conjugates is assumed to be a dominatingcompetitive pathway reducing the mutagenic and carcinogeniceffects of several known carcinogens. In the case of the potentcarcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) the situationis different since this carcinogen is transformed to reactiveintermediates by nucleophilic agents such as thiols. As a consequenceof this, the modulating effect of thiols has to be discussedin relation to the reaction kinetics of this nitroso compound.In this report we present data on the mutagenicity of MNNG whichwas enhanced by intracellular excess of glutathione, achievedby pre-treatment with N-acetylcysteine, cysteine and glutathione.Depletion of GSH by treatment with diethylmaleate or buthioninesulfoximineresulted in a decreased mutagenic effect of MNNG. A decreasedeffect of MNNG was also obtained by extracellular excess ofthiols. The modification of MNNG-mutagenicity was compared tothe effect of thiols on the mutagenicity of N-methyl-N'-nitrosourea(MNU) in V79 Chinese hamster cells. No effect of the thiolscould be detected on the mutagenicity of MNU, indicating thatthe intermediates of MNU and MNNG react, in agreement with thereaction kinetics, in favour of water and thiols become lessimportant. The results indicate that the activation of MNNGto mutagenic intermediates is occurring within the cells. Thisactivation is mediated by reaction of MJ with thiols (and amines)and subsequent release of short-lived alkylating intermediates.The mutagenic effect of MNNG can be reduced or enhanced respectivelyby decreas ing or increasing the intracellular thiol levels.As demonstra ted with MNU, intracellular thiol concentrations(in milli/molar range), which is high enough to bring aboutthe activation of MNNG, are not sufficiently high to protectDNA from damage by the alkylating intermediate. Extracellularlevels of thiols ‘protect’ the cells simply by increasingthe decomposition of MNNG in the treatment solution.     

Studies on the antimutagenic activity of ascorbic acid in vitro and in vivo

The possibility that ascorbic acid, as a nucleophile, may inhibit mutagenicity induced by electrophilic metabolites of N-nitrosocompounds was examined. In vitro data are presented to showthat ascorbic acid does not decrease the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a modified Ames bacterialmutagenicity system if deionized water is used to prepare theincubation medium. However, ascorbic acid prevents the mutagenicityof MNNG in vitro if added to bacteria in a medium prepared witheither sterile tap water or deionized water and Cu²⁺ ions andthat this an-timutagenic response is blocked by EDTA. Additionalin vitro experiments suggest that when ascorbic acid and Cu²⁺ions are mixed in aqueous solution, H₂O₂ and free radicals derivedfrom H₂O₂ are formed and these compounds may deactivate N-nitrosocompounds. In vivo data are presented to show that ascorbicacid supplementation to guinea pigs (2000 mg/kg body weight/day)has no effect on the mutagenicity of N-nitrosodimethylamine,MNNG, N-methyl-nitrosourea and streptozotocin using the intrahepatichost-mediated bacterial mutagenicity assay. Additional in vivostudies demonstrate that simultaneous oral administration ofascorbic acid prevents the mutagenicity that follows the in-tragastricnitrosation of aminopyrine by nitrite while dietary pre-treatmentwith ascorbic acid does not. These findings suggest that ascorbicacid can block the intragastric formation of mutagenic N-nitrosocompounds but that ascorbic acid has no effect on mutagenicityof N-nitroso compounds once they are formed.     

Microsomal metabolism of 1-nitrobenzo[e] to a highly mutagenic K-region dihydrodiol

Aerobic metabolism of 1-nitrobenzo [e](1-nitro-BeP) by rat livermicrosomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP,and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichioropropylene 1,2-oxidewas incorporated into the metabolism, 1-nitro-BeP 4,5-oxidewas the predominant metabolite, and 1-nitro-BeP trans-4,5-dlhydrodiolwas not detected. All of the metabolites were purified by bothreversed- and normal-phase HPLC and characterized by analysisof their mass and 500 MHz proton NMR spectral data. 1-Nitro-BePwas not metabolized under hypoxic conditions. 1-Nitro-BeP andits four metabolites were assayed in Salmonella typhimuriumtester strains TA98, TA98NR and TA98/1,8-DNP₆, both in the presenceand absence of S9 activation. As predicted, 1-nitro-BeP wasa weak mutagen without S9 (2 revertants/µg in TA98); theaddition of S9 resulted in {small tilde}18, 17 and 4 revertants/µin TA98, TA98NR and TA98/1,8-DNP₆ respectively. The two phenolicmetabolites were mutagenic both in the presence and absenceof S9, producing moderate responses (19–84 revertants/µg)In addition, while the 1-nitro-BeP 4,5-oxide was only weaklymutagenic in TA98 (6–14 revertants/µg), 1-nitro-BePtrans 4,5-dihydrodiol was unexpectedly potent ({small tilde}300revertants/µg both with and without S9). These resultsindicate that microsomal epoxidation of 1-nitro-BeP followedby epoxide hydrolase-catalyzed hydrolysis of the resulting epoxideto the 1-nitro-BeP trans-4,5-dihydrodiol results in the mostpotent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic thanthe corresponding parent nitro-PAHs. Also, the lower S9-mediatedmutagenicity of 1-nitro-BeP in TA98/1,8-DNP₆ compared with TA98indicates that the mutagenicity of 1-nitro-BeP is dependentupon nitroreduction and transesterification. Finally, we previouslyhypothesized that nitrated PAHs with their nitro substituentsperpendicular or nearly perpendicular to the aromatic ringsare very weak or non-direct-acting mutagens in Salmonella typhimuriumtester strains. The results reported in this communication demonstratethat ring-oxidized derivatives of nitro-PAHs do not always followthis structure— mutagenicity correlation.     

Mutation in mammalian cells by isomers of 5-methylchrysene diolepoxide

The two pairs of diastereomeric anti- and syn-diolepoxide derivativesof 5-methylchrysene in both bay regions were tested for cytotoxicityand for mutagenicity at the hprt locus of Chinese hamster V79cells as determined by the ability of the cells to form coloniesin medium containing 6-thioguanine. The concentration of compoundin the cell media required to achieve 37% survival ranged from0.3 to 4.5 µg/ml. Although the mutagenic effectiveness,i.e. the induced mutation frequency per unit concentration ofcompounds, varied over a 30-fold range, the mutagenic efficiency,i.e. the induced mutation frequency at an equivalent level ofcell survival, showed only a 3-fold variation. The anti-1,2-diol-3,4-epoxideisomer (anti-5MCDE-I) was found to be the most mutagenic ofthe 5-methylchrysene diolepoxide isomers. This finding is consistentwith previous observations on the tumorigenicity of these diolepoxides.     

Correlation between induction of unscheduled DNA synthesis in the liver and excretion of mutagenic metabolites in the urine of rats exposed to the carcinogenic air pollutant 2-nitrofluorene

The genotoxic effects of 2-nitrofluorene (NF) have been studiedin vivo by measuring the induction of DNA repair, i.e. unscheduledDNA synthesis (UDS), in hepatocytes from male rats pretreatedby oral gavage with NF. During the NF exposure, Urine was collectedfor 24 h, and its mutagenicity was investigated in the plateincorporation assay, using Salmonella TA98 as tester strain.The urine samples were also used for the identification of excretedmetabolites of NF. Rats treated with 2-acetylaminofluorene (AAF)were studied simultaneously. A positive UDS response was observed12 and 24 h following a single gavage exposure to 12.5, 25 and50 mg/kg NF, with the response returning to near control levelsby 36 h. The positive control AAF induced approximately twicethe response observed with NF, and both compounds gave a UDSresponse that was 2–3 times higher in Wistar rats relativeto Sprague-Dawley rats. A potent directacting mutagenic effectwas observed in urine samples after NF treatment, while AAFexposure only gave rise to a weak mutagenic effect, the NF/AAFratio being 10/1. The stronger urinary mutagenicity after NFtreatment relative to AAF treatment was associated with thepresence of hydroxylated NFs. The genotoxic effect observedin the liver after NF treatement is, on the other hand, morelikely due to the same AAF metabolites that are also formedafterin vivo treatment with AAF.