Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells

1-Nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1-6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP) and 1,3,6-trinitropyrene (1,3,6-TNP) were tested for mutagenicity in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine-guanine phosphoribosyl transferase gene locus was quantified. While 1-NP and 1,3-DNP had only marginal direct-acting mutagenicity, 1,6-DNP, 1,8-DNP and 1,3,6-TNP showed definite mutagenicity, with specific mutagenic activities of 8.1, 21 and 54 mutants/10(6) survivors/micrograms . ml-1 respectively. The mutagenicity of 1-NP increased with increasing concentrations of Aroclor-1254 induced liver homogenate (S9) in the treatment medium. However, S9 at all concentrations tested decreased the mutagenicity of 1,6-DNP and 1,8-DNP. S9 at low concentrations enhanced the mutagenicity of 1,3-DNP and 1,3,6-TNP and that at high concentrations decreased their mutagenicity. The positive mutagenic response of the nitropyrenes suggests that they are potentially carcinogenic, and that further research into their possible human health risk should be performed.     

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.     

Mutagenic effects of nitropyrenes in a soybean test system

The mutagenic activity of 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) was assayed in heterozygous soybean plants (Y11y11), based on the appearance of mutational spots (yellow, dark green and twin) on the leaves. 1-NP, 1,3-DNP, 1,6-DNP and 1,8-DNP were direct-acting mutagens in a soybean test system, and mutagenicity was enhanced by addition of pyrene as a precursor. The mutagenicity of dinitropyrenes was enhanced by pretreatment with hepatic microsomal fractions of Aroclor 1254-treated rats. Binary and ternary isomeric mixtures of dinitropyrenes produced synergistic mutational response in the test system. The numbers of yellow and dark green spots per leaf increased by treatment with nitropyrenes. The frequency of twin spots did not change. Nitropyrenes stimulated the induction of forward and reverse mutations in soybeans. The number of light green spots (Y11y11) per leaf on homozygous soybeans (y11y11) increased markedly by treatment with 1-NP, 1,3-DNP, 1,6-DNP, and 1,8-DNP. These nitropyrenes would thus appear to cause point mutation and segmental loss as major effects.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro.

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02–0.8 μ g/plate (38–1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in “classical” nitroreductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 μg NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP₆, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

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-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.     

Airplane emissions: a source of mutagenic nitrated polycyclic aromatic hydrocarbons

Organic solvent extracts from airplane emission particulates are mutagenic for Salmonella typhimurium strain TA98. Using Salmonella tester strains deficient in enzymes required for the bioactivation of various nitroarenes, the mutagenicity present in these emissions was ascribed to the presence of nitrated polycyclic aromatic hydrocarbons. Based on the known aircraft particulate emission rates at U.S. airports, and using 1-nitropyrene (1-NP) and 1,8-dinitropyrene (1,8-DNP) as surrogates, it is calculated that at a minimum 7 kg 1-NP and 20 g, 1,8-DNP are emitted daily at a typical U.S. airport.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

Modulation of the mutagenicity of three dinitropyrene isomers in vitro by rat-liver S9, cytosolic, and microsomal fractions following chronic ethanol ingestion.

The effects of chronic ethanol feeding of rats on the ability of liver fractions to modulate the bacterial mutagenicity of three dinitropyrene isomers (1,3-, 1,6- and 1,8-DNP), which require bacterial enzymes but not an exogenous enzyme source for activation, were studied. The mutagenicity of the DNP isomers toward S. typhimurium TA98 and TA100 was attenuated in the presence of post-mitochondrial supernatants (S9) from both ethanol-fed and pair-fed rats albeit, that from the ethanol-fed group was more efficient in lowering the mutagenicity. The cytosolic fraction from ethanol-fed rats enhanced the mutagenicity of all of the DNP isomers in TA100. The most notable enhancement was with 1,3-DNP in which a more than 4-fold enhancement was obtained. Cytosol from pair-fed rats enhanced only the mutagenicity of 1,3-DNP, this by 2.9-fold. Cytosolic NADPH-nitroreductase activity from ethanol-treated rats toward 1,6-, 1,8- and 1,3-DNP was increased 2.8-, 1.7- and 1.3-fold, respectively over pair-fed controls. Cytosolic NADH-nitroreductase from ethanol-fed rats was increased with 1,3-DNP (1.7-fold) and 1,8-DNP (1.4-fold) as substrates, but not with 1,6-DNP. Microsomes decreased the mutagenicity of DNP similarly to S9, i.e., fractions from ethanol-fed rats were more efficient than those of pair-fed rats in deactivating all the DNP isomers. Per mg of protein, detoxification of DNP by S9 was more efficient than with microsomes, thus both cytosolic and microsomal enzymes are required for maximal detoxification. In summary, ethanol feeding modulates both the augmented cytosolic activation of DNP to mutagens and the deactivation of the direct-acting mutagenicity of DNP by microsomes. In combination, as is the case with S9, the microsomal detoxifying activity outcompetes the cytosolic activation.     

Identification of dinitropyrenes in diesel-exhaust particles. Their probable presence as the major mutagens

The direct-acting mutagens in diesel particulate extracts were identified. It is concluded that the major mutagens are in all probability 1,6- and 1,8-dinitropyrene (DNP). 1-Nitropyrene (NP) and 3-nitrofluoranthene (NF) were also present. The DNP isomers contributed 43% of the total mutagenic activity of the crude extracts, whereas 1-NP (or 3-NF) was responsible for less than 10% of the activity. The quantities of 1,6- and 1,8-DNP were 1.2 and 3.4 ppm of the crude extracts, respectively, and the induction of both DNPs in the diesel particulate matter corresponded to about 1.7-4.8% by weight of the 1-NP content (70.5 ppm in the crude extracts).     

Nitropyrene-induced DNA repair in Clara cells and alveolar type-II cells isolated from rabbit lung

DNA excision repair, as measured by unscheduled DNA synthesis (UDS), was examined in different cell types of rabbit lung exposed to nitropolycyclic aromatic hydrocarbons (NO-PAH) in vitro. Dose-related increases in UDS were observed. 1,6-Dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) induced UDS more effectively in alveolar type-II cells compared with Clara cells. On the other hand, 1-nitropyrene (1-NP) caused a weak UDS response in Clara cells but no DNA repair in alveolar type-II cells.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Metabolic activation of 2,4-dinitrobiphenyl derivatives for their mutagenicity in Salmonella typhimurium TA98

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we tested the mutagenic potency of 18 kinds of nitroarenes including nitrated biphenyl, fluorene, phenanthrene and pyrene on Salmonella typhimurium TA98 in the absence and presence of S9 mix. The mutagenicities of 2,4-dinitrobiphenyl derivatives and 4-nitrobiphenyl were enhanced by the addition of S9. 2,4,6-Trinitrobiphenyl (3 net rev./10 micrograms without S9) was activated 60-fold by the mammalian metabolic system (181 net rev./10 micrograms with 10% S9). The mutagenic potency of 2,4,2',4'-tetranitrobiphenyl in TA98, TA98NR and TA98/1,8-DNP6 was also enhanced by the addition of 10% S9. But 1-nitropyrene and 1,3-dinitropyrene, which are well-known mutagens and carcinogens, were deactivated to 3% and 0.4%, respectively, by the addition of 10% S9. Separate addition of microsomal and cytosolic fractions slightly activated the mutagenicity of 2,4,6-trinitrobiphenyl, and 2,4,2',4'-tetranitrobiphenyl was activated not only by S9 but also by the cytosolic fraction.     

Identification and quantification of highly mutagenic nitroacetoxypyrenes and nitrohydroxypyrenes in diesel-exhaust particles

Heavy-duty diesel-exhaust particles were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor-induced rat liver (S9 mix). The neutral and acidic fractions showed high mutagenicity with TA98 in the absence of S9 mix, the acidic fraction having the highest specific activity. In the absence of S9 mix, the mutagenicity of crude, neutral and acidic fractions was greater in TA98 than in TA98NR and TA98/1,8-DNP6. Chemically-synthesized nitroacetoxypyrenes and nitrohydroxypyrenes were fractionated into the neutral and acidic fractions, respectively. These nitroarenes were purified by high-performance liquid chromatography and their mutagenicity was measured with the 4 strains. With TA98 in the absence of S9 mix, 1-nitro-3-acetoxypyrene, 1-nitro-6/8-acetoxypyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6/8-hydroxypyrene induced 16 700, 336, 992, 94 His+ revertants per plate per nmole, respectively. In the absence of S9 mix, the level of mutagenicity of these nitroarenes was highest in TA98, lowest in TA98/1,8-DNP6 and intermediate in TA98NR. The neutral and acidic fractions of diesel-exhaust particles were analyzed by gas chromatography-mass spectrometry and gas chromatography-mass fragmentography. The neutral fraction was found to contain nitroacetoxypyrenes, 1-nitropyrene, 1,6-dinitropyrene, while nitrohydroxypyrenes were detected in the acidic fraction. The amounts of 1-nitro-3-acetoxypyrene, 1-nitropyrene, 1,6-dinitropyrene and 1-nitro-3-hydroxypyrene were 6.3, 62, 0.81, and 70 ng per mg of crude extract, and accounted for 12, 3.6, 8.0, and 9.0%, respectively, of mutagenicity of the crude extract in TA98 in the absence of S9 mix.     

Mutagenic activity and quantification of nitroarenes in surface soil in the Kinki region of Japan

To clarify the mutagenic potential of surface soil in the Kinki region of Japan, particularly in Osaka and neighboring cities, 62 surface soil samples were collected and their organic extracts were examined by the Ames/Salmonella assay. All of the samples were mutagenic toward TA98 in both the presence and absence of a mammalian metabolic activation system (S9 mix). While all of the samples showed mutagenicity toward TA100 with S9 mix, only 45/62 (73%) were mutagenic without S9 mix. Fifty (81%) of the samples showed higher activity toward TA98 than TA100. The mean values of the mutagenicities of soil samples collected in Osaka prefecture (n=35) toward TA98 with and without S9 mix were 2315 and 1630 revertants per gram of soil, respectively, and these were 2.9 and 2.6 times as high as the values for samples from other prefectures (n=27), respectively. Three dinitropyrene (DNP) isomers, i.e. 1,3-, 1,6- and 1,8-DNP, and 3-nitrobenzanthrone (NBA) in the surface soil samples were quantified by fluorometric detection of the corresponding amino compounds, i.e. diaminopyrene isomers and 3-aminobenzanthrone, using high-performance liquid chromatography (HPLC). The three DNP isomers were detected in all of the soil samples (n=26) that were mainly collected in Osaka prefecture, and the amounts of 1,3-, 1,6- and 1,8-DNP were 6-1526, 11-1772 and 10-2092pg/g of soil, respectively. The contribution ratios of 1,3-, 1,6- and 1,8-DNP to the mutagenicity of soil extracts toward TA98 without S9 mix were 0.2-12, 0.3-12 and 0.5-27%, respectively. The amount of 3-NBA in soil samples (n=8) was 144-1158pg/g of soil, and the contribution ratio of 3-NBA to the mutagenicity of soil extracts was 2-38%. These results suggest that the surface soils in the Kinki region were highly polluted with mutagens and the pollution levels in Osaka prefecture were higher than those in other areas. DNP isomers and 3-NBA may be major mutagens that contaminate surface soil in this region.     

Aerobic and anaerobic reduction of nitrated pyrenes in vitro

Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1-nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds.     

Genotoxicities of nitropyrenes and their modulation by apigenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems

Four naturally occurring compounds, indole-3-carbinol (I3C), apigenin (Api), ellagic acid (EA) and tannic acid (TA), were tested for their inhibitory effects against 1-nitropyrene- (1-NP) or 1,6-dinitropyrene (1,6-DNP)-induced genotoxicity in Salmonella tester strains and Chinese hamster ovary (CHO) cells. Api and TA strongly inhibited the bacterial mutagenesis induced by nitropyrenes, while 13C and EA had little or no effect. For example, in TA98, 0.2 μmole Api resulted in 48% and 56% inhibition of the mutagenicity induced by 4 nmole 1-NP and 0.035 nmole 1,6-DNP, respectively. With an equal dose, expected, a good correlation was observed between the antimutagenicity of nitropyrenes and their inhibitory effect on nitroreductase activity. This indicated that one of the possible antimutagenic mechanisms of Api or TA was to inactivate the metabolism of nitropyrenes. Two biological end-points, cytotoxicity and sister-chromatid exchange (SCEs), were used to screen the antigenotoxic effects of these compounds in CHO cells. At the sub-cytotoxic dose, 13C, Api and TA all protected against the cytotoxicity induced by 1-NP and 1,6-DNP, but only TA and Api gave a significant reduction of the frequency of SCEs. Moreover, this reduction was found to be highly dose-dependent.     

Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines

1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G⁻, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G⁻ and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required.The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.     

Mutagenicity of the reaction products of dibenzo-p-dioxin with nitrogen oxides.

Dibenzo-p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1:3, 1:6 and 1:18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP6 without S9 mix. In a gas chromatography/mass spectrometry study, 2-nitrodibenzo-p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1:18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo-p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs.     

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.