Potent Direct-acting Mutagen Research Articles

Abstract 3099: Hydrocarbon carcinogenicity reexamined: An extremly potent direct-acting mutagen is common to smoke, soot and ash from a variety of combustion sources, including cigarette tobacco

Abstract Percivall Pott (1714-1788), an English surgeon, is generally credited with being the first to suggest that cancer may be induced by exposure to environmental carcinogens – in his case, cancer of the scrotum in London chimney sweeps occupationally exposed to soot. It is perhaps not surprising, therefore, that combustion products became a primary focus of later research into the mechanisms of chemical carcinogenesis. Early on, those studies pointed toward a particular class of compounds as being primarily responsible for cancer induction by such materials, a group of environmentally ubiquitous compounds called polycyclic aromatic hydrocarbons, or PAHs. One such PAH, benzo[a]pyrene (BaP), is perhaps the most studied compound in all of cancer research. While there is little doubt that BaP and its congeners are the primary carcinogens in incomplete combustion products like coal tar, their concentrations in other hydrocarbon mixtures are too low to fully account for their overall carcinogenic potency. It is possible that other factors such as tumor promotion contribute to the additional potency, but it might also be expected that there are significant contributions from nitro-PAH derivatives, in particular the isomers of dinitropyrene (DNP) found in carbon black, diesel exhaust and other combustion products. One such isomer, 1,6-dinitropyrene was shown by Iwagawa et al. to have three-times the carcinogenic potency of BaP when injected directly into the lungs of rats. In order to indirectly test nitroaromatic contribution to combustion product carcinogenicity, extracts of smoke soot, ash and food products exposed to smoke during processing were fractionated by HPLC, and the fractions tested in the Ames Test without metabolic activation. All showed a common peak of extremely potent direct-acting mutagenicity at a retention time similar to that of dinitropyrene. Efforts are currently underway to fully characterize the mutagen by a combination of standard analytical means. Citation Format: Gary R. Blackburn. Hydrocarbon carcinogenicity reexamined: An extremly potent direct-acting mutagen is common to smoke, soot and ash from a variety of combustion sources, including cigarette tobacco [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3099.

DNA adduct and tumor formations in rats after intratracheal administration of the urban air pollutant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and identified as a potent direct-acting mutagen in vitro and genotoxic agent in vivo. In order to evaluate the in vivo toxicity and carcinogenicity of 3-NBA in a situation corresponding to inhalation, a combined short-term and lifetime study with intratracheal (i.t.) instillation in female F344 rats was performed. DNA adduct formation, as a marker for the primary effect and analyzed by 32P-HPLC after single instillation, showed a few major DNA adducts and a rapid increase with a peak after 2 days, followed by a decline. No DNA adducts above the background level were observed after 16 days. The highest DNA adduct formation was observed in lung [approximately 250 DNA adducts/10(8) normal nucleotides (NN)] closely followed by kidney (approximately 200 DNA adducts/10(8) NN), whereas liver contained only 12% (approximately 30 DNA adducts/10(8) NN) of the levels of DNA adducts found in lung. In the tumor study, squamous cell carcinomas were found after 7-9 months in the high-dose group (total dose of 2.5 mg 3-NBA) and after 10-12 months in the low-dose group (total dose of 1.5 mg 3-NBA). The fraction of squamous cell carcinoma out of the total amount of tumors observed at the end of experiment at 18 months, corresponded to 3/16 and 11/16 in the low- and high-dose group, respectively. A single case of adenocarcinoma was also observed in each group. In the control group, no tumors were observed during the entire study of 18 months. In addition, a few cases of squamous metaplasia were also observed in the lung in both dose groups but not in the controls. In conclusion, 3-NBA forms DNA adducts in the lung immediately after i.t. administration but almost all DNA adducts were eliminated after 16 days. Tumor formation in two dose groups was observed in a dose-dependent manner with squamous cell carcinomas as the predominant tumor type at high exposure.

PM2 DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX is a potent direct-acting mutagen found in chlorinated drinking water. In the present study, the induction of DNA strand breaks and apurinic/apyrimidinic (AP) sites by MX in supercoiled PM2 DNA was examined using exonuclease III, which specifically cleaves the DNA at AP sites. The results showed that MX induced AP sites in great excess of direct strand breaks. In view of the known mutagenicity of AP sites, these results provide insight into the mechanism of MX-induced mutagenesis.

Identification of N-Acetylcysteine Conjugates of 1,2-Dibromo-3-chloropropane: Evidence for Cytochrome P450 and Glutathione Mediated Bioactivation Pathways

The haloalkane 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen, mutagen, nephrotoxin, and testicular toxin. The identification of N-acetylcysteine conjugates of DBCP provides information on GSH mediated and cytochrome P450 mediated bioactivation pathways in the expression of DBCP-induced toxicities. N-Acetylcysteine conjugates excreted in the urine of male Sprague-Dawley rats administered DBCP, C1D2-DBCP, C2D1-DBCP, C3D2-DBCP, or D5-DBCP (80 mg/kg) were purified by reverse-phase HPLC as their methyl ester derivatives and characterized by fast atom bombardment tandem mass spectrometry. These metabolites were also converted to tert-butyldimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) to facilitate the identification of N-acetyl-S-(2,3-dihydroxypropyl)cysteine (Ia), an apparent regioisomer of Ia, 2-(S-(N-acetylcysteinyl))-1,3-propanediol (Ib), N-acetyl-S-(3-hydroxypropyl)cysteine (IIa), and N-acetyl-S-(3-chloro-2-hydroxypropyl)-cysteine (III). Metabolites Ia, Ib, and III displayed quantitative retention of deuterium, an observation consistent with the formation of episulfonium ion intermediate(s) in their biogenesis. Mercapturate IIa retained three atoms of deuterium from D5-DBCP, and two atoms of deuterium from the dideuterio analogs (C1D2-DBCP and C3D2-DBCP), thus invoking P450 mediated formation of 2-bromoacrolein (2-BA) as an intermediate in the biogenesis of IIa. A mechanism is proposed in which conjugate addition of GSH to 2-BA, subsequent episulfonium ion formation, and addition of GSH afford 1,2-(diglutathion-S-yl)propanal. Glutathione mediated reduction is invoked to afford S-(3-hydroxypropyl)GSH which would be excreted in the urine as IIa. The quantitative retention of deuterium from C1D2-DBCP or C3D2-DBCP was indicative of isotopically sensitive branching of P450 metabolism at either C1 or C3 to afford 2-BA. C2D1-DBCP showed a 30% retention of 1 deuterium atom in IIa; the loss of the deuterium is consistent with 2-BA formation, whereas the retention of one deuterium atom is indicative of the formation of metabolite IIa through GSH conjugation of either 2,3-dibromopropanal or 2-bromo-3-chloropropanal. These data indicate that IIa is a marker metabolite for the potent direct-acting mutagen, 2-BA, or its metabolic precursors 2,3-dibromopropanal or 2-bromo-3-chloropropanal. Therefore, evidence has been presented for bioactivation of DBCP by glutathione and cytochrome P450 mediated mechanisms.

3‐Chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX) Induces gene mutations and inhibits metabolic cooperation in cultured chinese hamster cells

Investigations were carried out on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent direct-acting bacterial mutagen found in chlorinated tap water, for the potential to induce 6-thioguanine (6-TG) resistant mutations in cultured Chinese hamster V79 cells. The mutagenicity of MX was manifested when cells were treated with MX in Hanks balanced salt solution, but not in serum-free Eagle's minimal essential medium. A 12-fold higher mutation frequency over control was obtained at 12.5 micrograms/ml MX. In addition, the inhibitory effect of MX on metabolic cooperation between cocultivated 6-TG- sensitive (6-TGs) and -resistant (6-TGr) V79 cells was investigated. Recovery of 6-TGr cells significantly increased at 0.8-2.0 micrograms/ml MX due to impaired intercellular communication with 6-TGs cells. MX is believed to exert tumor-promoting and mutagenic activity in mammalian cells.

Expression of genes of nitroreductase and O-acetyltransferase of Salmonella typhimurium in Chinese hamster lung (CHL) cells

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver ( = 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.

Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo[4,5- f]quinoline (nitro-IQ) from IQ

The mutagenic effects of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA:arylamine N-acetyltransferase (NAT). The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC). One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts. The metabolite was identified as 2-nitro-3-methylimidazo[4,5- f]quinoline (nitro-IQ). Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Genotoxic activity of 1-chloromethylpyrene in stomach epithelium in vivo: insensitivity of the stomach scintillation UDS assay.

An acknowledged weakness of current testing programmes for genotoxic hazard has been the potential insensitivity of the established mouse bone marrow micronucleus test and rat liver unscheduled DNA synthesis (UDS) assays to direct-acting or short-lived mutagens, which may be consumed at the site of initial contact. In such cases, in vivo test systems sampling tissues such as the skin or the stomach would provide valuable data. To test these principles a stomach UDS assay was evaluated using the potent locally active mutagen 1-chloromethylpyrene (1-CMP). Contrary to expectations, no UDS response was observed 16 h following 1-CMP dosage by oral gavage. To confirm the integrity of the 1-CMP used for the stomach UDS assay, a sample of the stored chemical was re-evaluated in vitro and shown to be still strongly positive in the Ames assay and to have alkylating activity at least 15 min after incubation at stomach acid pH. No UDS response was observed when test dose levels were reduced or when earlier sampling times were used. Other genotoxic endpoints were examined in stomach. 32P-Postlabelling analysis revealed high levels of adduct formation in gastric DNA. An assay utilizing electrophoresis of DNA (the comet assay) showed the occurrence of DNA damage following dosing with 1-CMP in vivo. These positive results confirmed that 1-CMP should be regarded as a potential in vivo genotoxin. The failure to detect a UDS response to 1-CMP in stomach was investigated; a strong UDS response was observed in an in vitro hepatocyte UDS assay of 1-CMP indicating that the rat was capable of repairing 1-CMP-derived DNA adducts. Pretreatment of rats with hydroxyurea depressed the level of incorporation of thymidine into DNA both in negative and positive [methyl-N-nitrosoguanidine (MNNG)] controls. The results of these studies indicated that the protease digestion method employed did not selectively or efficiently sample those cells with any UDS response to 1-CMP or MNNG, and the activity seen for the latter was most likely due to the presence of S phase cells within the digests. As a result of the finding that UDS responses were not demonstrated for the potent direct-acting mutagens 1-CMP and MNNG, the protease digestion/scintillation method for stomach UDS does not appear to have general value in a screening programme for locally active genotoxic agents.

Mutagenic decomposition products of nitrosated 4-chloroindoles.

4-Chloro-6-methoxyindole, a constituent of fava beans, forms a potent direct-acting mutagen, 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime, when nitrosated. In order to better understand the properties of this mutagen, we have studied a readily-available analog, 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, prepared by nitrosation of 4-chloroindole. This analog is also mutagenic, and both mutagens decompose rapidly at neutral or higher pH to yield in each case a new, less potent mutagen which then reacts further to form a nonmutagenic final product. The two products arising from 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, on the basis of comparison of spectroscopic and chromatographic evidence with that from authentic standards, are 4-chloro-N-nitrosodioxindole and 4-chloroisatin; those arising from 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime appear to be the corresponding 6-methoxy analogs. The interplay of these pathways with respect to net biological activity, especially under gastric conditions, remains to be described.

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP 6 ( O-transacetylase-deficient) or TA98NR/1,8-DNP 6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [ 14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver ( = 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.

Mutagenesis and DNA adduct formation by 1-nitropyrene in Chinese hamster ovary cells without exogenous metabolic activation

The direct-acting mutagenicity of 1-nitropyrene (1-NP), a tumorigenic environmental pollutant and model nitropolycyclic aromatic hydrocarbon, was studied in Chinese hamster ovary (CHO) cells. Previous reports indicated that 1-NP, a potent direct-acting mutagen in Salmonella typhimurium, was mutagenic in CHO cells only in the presence of an exogenous activation system. In this study, a DNA-repair-deficient CHO cell line, CHO-UV5, and the repair-proficient CHO-K1-BH 4 cell line were used to measure the direct-acting mutagenicity of highly purified samples of 1-NP. Exposure of CHO-K1-BH 4 cells for 5, 12, and 24 hr did not increase mutations at the hypoxanthineguanine phosphoribosyl transferase ( hprt) locus ( p > 0.1; paired t test; n = 5–6), while treatment of CHO-UV5 cells for 24 hr produced a statistically significant induction of mutants (22 ± 6 mutants per 10 6 clonable cells vs a solvent control value of 9 ± 3 per 10 6 cells; p < 0.01; n = 6). 32P-Postlabeling analysis of the DNA adducts formed in CHO-UV5 cells following 1-NP exposure revealed the presence of a single major adduct. Based on its chromatographic properties, its sensitivity to nuclease P1 digestion, and its resistance to hydrazine treatment, the adduct appeared to be N-(deoxyguanosin-8-yl)-1-aminopyrene, which was presumably produced by nitroreduction of 1-NP to N-hydroxy-1-aminopyrene. These results suggest that the use of extended treatment times and DNA-repair-deficient cells may be required to assess adequately the mutagenicity of nitropolycyclic aromatic hydrocarbons in CHO cells.

Formation of C8-modified deoxyguanosine and C8-modified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism mediated by rat and mouse liver microsomes and cytosols

2-Nitropyrene, the geometric isomer of the most studied nitropolycyclic aromatic hydrocarbon (nitro-PAH), 1-nitropyrene, is an environmental contaminant detected in ambient air and a potent direct-acting mutagen. Its metabolic activation leading to the formation of DNA adducts was studied. The activated metabolite, N-hydroxy-2-aminopyrene, was prepared and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA, the resulting nucleosides were separated by HPLC, and the adducts were characterized by mass and proton NMR spectral analysis. Both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene, in a 5:2 ratio, were identified. These adducts were then utilized as standards to identify the DNA adducts formed from reaction of [3H]2-nitropyrene with DNA mediated by liver microsomes and cytosols of mouse and rat. In all cases, both adducts were formed. The quantities of the two adducts formed in each system were: mouse liver microsomes (11.3 pmol [3H]2-nitropyrene/mg DNA), rat liver microsomes (23), mouse liver cytosol (11.4) and rat liver cytosol (5.1). Thus, these adducts were formed in highest yield from rat liver microsomes and the lowest from rat liver cytosol. The deoxyguanosine/deoxyadenosine adduct ratio was higher from rat and mouse liver microsomes (7.8:9.2) than from rat and mouse liver cytosols (2.5:3.1). Our results represent the first direct demonstration of a C8-deoxyadenosine adduct being formed as a major product from the reaction of a nitro-PAH metabolite with DNA.

MNDO calculations of highly mutagenic (chloromethyl)benzo[a]pyrenes

(Chloromethyl)benzo[a]pyrenes (ClMeB[a]P) are among the most potent direct-acting mutagens (i.e., not requiring metabolic activation) detected by the Ames assay using strain TA98 and TA100. We wanted to determine if molecular quantum mechanics would yield insights into the reactivity and mutagenicity of these compounds. Calculations using the MNDO semiempirical method (MOPAC program) were carried out on a series of ClMeB[a]P. The experimental rates of solvolysis of ClMeB[a]P in 50% aqueous acetone correlated well (r = -0.95) with the differences between the calculated heats of formation of ClMeB[a]P and those of their respective carbocations, supporting the hypothesis that ClMeB[a]P react via carbocations. None of the calculated parameters correlated with mutagenicity. Thus, the prediction of mutagenicity of ClMeB[a]P in the Ames assay is more complex than the simple formation of carbocation intermediates.

Detection and mechanism of formation of the potent direct-acting mutagen 2-bromoacrolein from 1,2-dibromo-3-chloropropane

The nematocide 1,2-dibromo-3-chloropropane (DBCP) was converted to products which are mutagenic for Salmonella typhimurium TA 100 in the presence of rat liver microsomes, NADPH, and oxygen. Typical in vivo and in vitro inhibitors of cytochrome P-450 decreased DBCP mutagenicity in the presence of microsomes. Addition of glutathione to cytosolic preparations failed to bioactivate DBCP to mutagenic metabolites. Mutagenicity studies with selectively deuterated analogs showed that substitution of deuterium for hydrogen at C-1 or C-3 of DBCP modestly decreased mutagenicity, but that deuteration at both C-1 and C-3 markedly decreased mutagenicity. The formation rates of the potent direct-acting mutagen, 2-bromoacrolein (2-BA), in incubations of DBCP and its deuterated analogs with rat liver microsomes, correlated with the isotope effects on mutagenicity. Characterization of 2-BA was accomplished by gas chromatography-mass spectrometry using positive-ion chemical ionization. Mass spectral analysis of 2-BA formed from specifically deuterated analogs of DBCP indicated that initial oxidative dehalogenation at C-1 followed by a spontaneous β-elimination reaction was the preferred pathway in the formation of 2-BA from DBCP. These results demonstrate that mutagenic metabolites of DBCP are formed by cytochrome P-450-mediated oxidative metabolism, and that 2-BA is a major mutagen formed.

Dose-response relationship for chromosome aberrations induced by fecapentaene-12 in human lymphocytes

Chromosome analyses were carried out in human lymphocytes exposed to a synthetic racemic all- trans fecapentaene-12 at 2–24 μM. A dose-dependent increase of the incidences of chromatid-type changes with distinct saturation at higher doses could be observed. The results reveal for the first time that fec-12 is a potent direct-acting mutagen in human lymphocytes.

Metabolism of 1-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites.

1-,3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypes of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derived from a carcinogenic parent PAH, benzo[a]pyrene, are environmental contaminants and potent bacterial mutagens. In this study, the aerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomes was studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol, while metabolism under hypoxic conditions yielded 1-amino-BaP. The metabolites formed from aerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9,10-dihydrodiol by liver microsomes of untreated rats and rats pretreated with 3-methylcholanthrene and phenobarbital were quantified. Comparison of these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-position of BaP markedly affects the regioselectivity of the P-450-containing enzymes. 1-Nitro-BaP and the three metabolites were potent mutagens in Salmonella typhimurium TA98, both in the absence and in the presence of an exogenous metabolic activation system (S9). The direct and S9-mediated mutagenicities of 1-nitro-BaP and the two dihydrodiols were decreased in the nitroreductase-deficient strain TA98NR, while TA98/1,8-DNP6, an O-acetylase-deficient strain, was less sensitive to the two dihydrodiols, both with and without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equally mutagenic in all three tester strains. These observations indicate that: the metabolism of 1-nitro-BaP involves several pathways leading to mutagenic activation; the major activation pathways of 1-nitro-BaP involve nitroreduction; nitroreduction followed by O-acetylation is the major activation pathway of 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol; and 1-amino-BaP is a potent direct-acting mutagen.

11- and 12-Chloromethylbenzo[ a]pyrene are potent direct-acting mutagens in the Ames assay

11- and 12-Chloromethylbenzo[ a]pyrene are potent direct-acting mutagens in the Ames assay

1-Nitropyrene reduction by Salmonella typhimurium, V79 Chinese hamster and primary rat liver cells

1-Nitropyrene is an unusually potent direct-acting (in the sense that tissue homogenate is not required for activation) mutagen in the Ames assay (Mermelstein et al., 1981). Recently, there has been considerable interest in 1-nitropyrene as an environmental mutagen because the compound has been found at low concentrations (1-100 ppm) in particles collected from ambient urban air (Gibson, 1982); from fireplace emissions (Gibson, 1982); and from diesel engine exhaust (Pederson and Siak, 1981; Salmeen et al., 1982). In some cases 1-nitropyrene accounted for a few to 10°70 of the direct-acting mutagenicities observed for organic solvent extracts of the particles (Gibson, 1982; Salmeen et al., 1982). Despite its potency in the Ames assay, 1-nitropyrene is not a direct-acting mutagen in mammalian cell culture assays so far tested (I(arpinski et al., 1982; Li and Dutcher, 1983; our data, unpublished). (It may be a weak mutagen for some cells with $9 activation (Li and Dutcher, 1983).) One conjecture to explain the difference between the mutagenicity of 1-nitropyrene in bacteria and in mammalian cells is that the bacteria have a unique nitroreductase activity (Karpinski et al., 1982; Mermelstein et ah, 1981). This conjecture was based on reasonably strong evidence that 1-nitropyrene reduction is necessary for the bacterial mutagenicity of the compound. Herein we describe experiments in which we tested this conjecture with measurements of nitropyrene reduction by intact Salmonella bacteria which are sensitive to mutation by 1-nitropyrene; by Salmonella bacteria which are resistant to mutation by 1-nitropyrene; and by two types of mammalian cells V79 Chinese hamster cells (not sensitive to mutation by 1-nitropyrene), and primary rat liver cells (non-dividing cells). We found that all of the bacterial strains and both types of mammalian cells reduced 1-nitropyrene to 1-aminopyrene. We conclude that reduction of nitropyrene is not unique to bacteria which are sensitive to mutation by 1-nitropyrene and that, although 1-nitropyrene reduction may be necessary, it is not sufficient, for the mutagenicity of 1-nitropyrene.

N-Mononitrosopyridinol carbamate: The principal mutagen formed by nitrosation of pyridinol carbamate

The principal mutagen formed by the reaction of equimolar amounts of pyridinol carbamate and nitrite was isolated, and identified as N-mononitrosopyridinol carbamate on the basis of its spectroscopic data. By microbial mutagenesis assay, the nitroso compound was proved to be a potent directacting mutagen towards base-pair substitution strains.

Contribution of 1-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts

Projections of future increases in the number of diesel powered U.S. passenger cars have raised concerns about potential adverse human health effects associated with diesel engine particulate exhaust (Pepelko et al., 1980). One concern has been carcinogenic polycyclic aromatic hydrocarbons (PAH), known for many years to be associated with diesel exhaust particles. A more recent concern, raised by experiments with the Ames Salmonella mutagenesis assay, has been the discovery that PAH may not be the only potential carcinogens in the diesel exhaust particles (Huisingh et al., 1978; Wang et al., 1978; Pepelko et al., 1980). These experiments have shown that organic solvent extracts of diesel exhaust particles are directly mutagenic (i.e., mutagenic without activation by mammalian tissue homogenate), whereas the predominant PAH identified in the extracts are mutagenic in the Ames assay only with activation (Searle, 1976; McCann and Ames, 1976; Gibson et al., 1978). We (Schuetzle et al., 1981) and others (Erickson et al., 1979) have identified many oxygenated derivatives of PAH in these extracts. Many of these derivatives could be direct-acting mutagens, but little is known yet about their mutagenicities or their concentrations in the extracts. One of the PAH derivatives identified was l-nitropyrene (I-NP) (Schuetzle et al., 1982), recently shown to be an extremely potent direct-acting mutagen in the Ames assay (Wang et al., 1980; LOfroth et al., 1980, Rosenkranz et al., 1980). Herein we report a rigorous determination of the percentage contribution of 1-NP to the direct-acting Ames assay mutagenicities of extracts of exhaust particles collected from 3 different diesel powered passenger vehicles. The percentage contribution, P, of 1-NP is given by: