Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.     

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.     

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.     

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.     

Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter

Of the many nitroarenes, dinitropyrenes (DNPs) have the potential to revert Salmonella typhimurium his- mutants. This study was conducted to investigate the potential mutagens present in airborne particulate matter collected in Santiago, Chile. 5 organic substances extracted with dichloromethane showed mutagenic rates of from 38.9 to 287 revertants per m3 of air for S. typhimurium his- strain TA98 without S9 mix. 4 of the samples had greatly reduced mutagenicity for strain TA98/1,8DNP6 but not for strain TA98NR. The 1-nitropyrene (1-NP) content accounted for 0.06-0.15 microgram per g of particulate, as determined by high-performance liquid chromatography (HPLC), but the contribution of the compound to mutagenicity was less than 1% of the total activity. On the other hand, by using two columns in the HPLC, DNPs of 1,6- and 1,8-isomers were detected in the samples pooled after the determination of 1-NP, and the amount of the derivatives was about 0.2 microgram per g of particulate matter.     

Nitropyrenes are inducers of polyoma viral DNA synthesis

The biological activity of a series of nitropyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a ts-a mutant of polyoma virus. Concentrations of 10-30 micrograms/ml of 1-nitropyrene (1-NP) induced viral replication, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 micrograms/ml) to the culture medium. The response was less than that obtained with 0.1 micrograms/ml of the activated metabolite of benzo[a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replication, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5-2.0 microgram/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 microgram/ml indicated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modification seen with 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.     

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).     

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.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

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.     

Effect of pH on the activity and stability of clastogens in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells

The effect of the pH of the medium on the clastogenic activity of several direct-acting and indirect clastogens was evaluated in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells. Furthermore, the stability of the chemicals in the cell culture medium was measured by HPLC over the pH range of 5.0-11.0. The activity of the direct-acting clastogens mitomycin C (MMC), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and 4-nitroquinoline-1-oxide (4NQO) at various pH values depended on their stability. In the case of ENNG, its clastogenic activity decreased to about one-fifth at pH 9 but was about twice as high at acidic pH compared with that at pH 7.4. This is consistent with the observation that ENNG is unstable at basic pH; the residual content of ENNG was 0.5% of the initial amount in cell culture medium at pH 9.0 after a 2-h incubation. 4NQO was unstable at strongly basic pH (pH 10-11), and MMC was unstable at pH 5.0 and pH 11.0. The frequencies of chromosomal aberrations induced by MMC and ENNG were correspondingly decreased at these pH values. On the other hand, the clastogenicities of the indirect clastogens 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN), which require metabolic activation, were reduced at pH 10-11 and pH 5.8. The frequencies of chromosomal aberrations at these pHs were almost equal to negative control values. These chemicals were stable in the medium in the absence of S9 mix over the pH range of 5.0-11.0. Thus clastogenicity of indirect-acting clastogens is reduced under extreme pH conditions, probably because of the instability or nonformation of the active form. The present results indicate that the clastogenic activity of any compound will depend on its stability in the medium irrespective of its direct- or indirect-acting nature. In addition, some of the chemicals that are recognized as clastogens presumably might induce chromosomal aberrations by means of acidic pH itself. It is, therefore, important to take account of the pH of the treatment medium in evaluating the clastogenicity of chemicals.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

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.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1,3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.     

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.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1, 3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.     

1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells

We have investigated the cytogenetic effect of 1,6-dinitropyrene (1,6-DNP) in Chinese hamster V79 cells. The chemical caused a dose-dependent increase in the incidence of initial and full C-mitoses, polyploid mitoses, ana-telophases with lagging chromosomes, non-disjunction and multipolar configurations, in a range of 0.05-5 microM. These findings indicate that 1,6-DNP interferes with the functioning of the spindle apparatus in V79 cells. Early signs of spindle disturbances were seen at 1,6-DNP concentrations which only moderately reduced cell growth and division. Analysis of structural chromosomal aberrations revealed the appearance of chromatid-type aberrations with open breaks and exchanges accompanied by gaps. The results indicate that 1,6-DNP is both a spindle-disturbing and a clastogenic agent in V79 cells.     

Mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro

The mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line, CHL. BHA, tert-butylhydroquinone (BHQ), tert-butylquinone (BQ) and BHA dimer (diBHA) did not show any mutagenic potential with and without S9 mix in the reverse mutation assay. In addition to the above 4 chemicals, 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), 3-tert-butylanisole-4,5-quinone (BHA-o-Q), and tert-butylquinone oxide (BQO) were tested in the chromosomal aberration test. BHA, BHQ and BQ induced chromosomal aberrations only in the presence of S9 mix, while BHA-OH, BHA-o-Q and BQO induced chromosomal aberrations only without S9 mix. DiBHA, however, showed no clastogenic potential with and without S9 mix. The present findings suggest that BHA-OH, BHA-o-Q or BQO may contribute to the clastogenicity of BHA in the presence of S9 mix.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

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.