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.     

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.     

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.     

SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals

The umu test system is a newly developed method to evaluate genotoxic activities of a wide variety of environmental carcinogens and mutagens (Oda et al., 1985). In the present study, we further examined the abilities of 151 chemicals to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002. Among the chemicals examined, 72 compounds induced umu gene expression, which could be defined on a basis of increased beta-galactosidase activity by 2-fold over the background level. The potent genotoxic compounds without metabolic activation were adriamycin, bleomycin, daunorubicin, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, N-ethyl-N'-nitro-N-nitrosoguanidine, furylfuramide, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, 1-nitropyrene and 4-nitroquino-line-1-oxide. In the presence of S9, aflatoxin B1, 2-aminoanthracene, Glu-P-1, IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2 also induced umu gene expression markedly. Several chemicals such as 2-acetylaminofluorene, 9-aminoacridine, azobenzene, benzanthracene, benzidine, diethyl nitrosamine, 1-nitronaphthalene, paraquat, potassium dichromate and sodium nitrite were weakly genotoxic and the induction by these compounds could be detected only when the incubation time was prolonged from 2 h to 5 h. Data are also presented that some of the chemicals such as dimethyl sulfoxide, m-dioxan, 5-fluorouracil and paraquat, which have been reported to be non-mutagenic in Ames/Salmonella assay, were found to be active in inducing umu gene expression, while the known mutagenic compounds including acrylonitrile, 4,4'-dinitrobiphenyl, furfural, methylene chloride, 1-naphthylamine, sodium azide, o-tolidine and o-toluidine were non-genotoxic in the present assay system.     

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.     

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/10⁶ survivors/μg·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.     

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.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.     

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.     

Cytotoxicity and genotoxicity of ingenamine G isolated from the Brazilian marine sponge Pachychalina alcaloidifera

Marine sponges belonging to the order Haplosclerida are one of the more prolific sources of new natural products possessing various biological activities. The present study examined the cytotoxic and genotoxic potential of ingenamine G, an alkaloid isolated from the Brazilian marine sponge Pachychalina alcaloidifera. Ingenamine G displayed a moderate cytotoxic activity against human proliferating lymphocytes evaluated by the MTT assay (IC(50) 15 microg/mL). The hemolytic assay showed that ingenamine G cytotoxic activity was not related to membrane disruption. The comet assay and chromosome aberration analysis were applied to determine the genotoxic and clastogenic potential of ingenamine G, respectively. Cultured human lymphocytes were treated with 5, 10, 15 and 20 microg/mL of ingenamine G during the G(1), G(1)/S, S (pulses of 1 and 6 h), and G(2) phases of the cell cycle. All tested concentrations were cytotoxic, reduced significantly the mitotic index, and were clastogenic in all phases of the cell cycle, especially in S phase. While an increase in DNA-strand breaks was observed starting with the concentration corresponding to the IC(50). The presence of genotoxicity and polyploidy during interphase and mitosis, respectively, suggests that ingenamine G at high concentrations is clastogenic and indirectly affects the construction of mitotic fuse.     

Genotoxicity of a variety of nitroarenes and other nitro compounds in DNA-repair tests with rat and mouse hepatocytes

Genotoxicity of a variety of nitroarenes and other compounds was examined in DNA-repair tests with rat or mouse hepatocytes. Out of 15 nitroarenes tested, 9 compounds, i.e., 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, 1-nitro-3-acetoxypyrene, 3-nitrofluoranthene, 2-nitrofluorene, 2,7-di-nitrofluorene and 5-nitroacenaphthene elicited positive response of DNA repair in the tests with rat and mouse hepatocytes. Among the positive chemicals, the DNA-repair level of the 3 dinitropyrene isomers was much higher than other nitroarenes. The results indicate that a number of nitroarenes are metabolically activated in the primary culture of rodent hepatocytes, and suggest potential carcinogenicity of 1-nitropyrene and 1-nitro-3-acetoxypyrene the carcinogenicity of which is either not clear or unknown. Of the other nitro compounds, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide as well as 4-nitroquinoline 1-oxide were clearly genotoxic in the assays with hepatocytes of both species. However, 5-nitro-2-furaldehyde semicarbazone was negative in both assays with hepatocytes of 2 species.     

The effects of 1,6-dinitropyrene on spindle morphology in transformed human cells

The effects of 1,6-dinitropyrene (1,6-DNP) on the fidelity of cell division were studied in the transformed human fibroblast cell line MRC5VA. Over a dose range of 0.1-10 micrograms/ml of 1.6-DNP, we observed significant increases in the levels of abnormal division stages, associated with damage to the spindle apparatus of the cell. Qualitative changes in spindle morphology and a quantitative decrease in pole-to-pole spindle length were also observed with increasing doses of 1.6-DNP. Such changes in the size and morphology of the spindle corresponded with an accumulation of cells blocked at metaphase. The presence of catalase did not modify the response, suggesting that the effects on the spindle apparatus and cell division were not caused by the generation of radicals but by the direct action of 1.6-DNP.     

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.     

The present lack of evidence for unique rodent germ-cell mutagens

Six chemicals, diethylhexyl phthalate (DEHP), ethanol, cyclohexylamine (CHA), sodium saccharin (NaS), cadmium chloride (CdCl2) and triflupromazine (TFP), were suggested to be unique germ-cell mutagens (Auletta and Ashby, 1988) by the GeneTox Workgroups of the U.S. Environmental Protection Agency (EPA). If this is a correct classification it would have major consequences when screening for mutagenicity and when labelling genotoxic substances. However, our re-evaluation of the GeneTox literature, including some more recent publications, has failed to find substantive evidence that any of these chemicals have been unequivocally established as having unique mutagenic activity in germ cells. For DEHP, NaS and TFP the evidence for genotoxic/mutagenic effects is questionable, in both germinal and somatic cells. Ethanol and CdCl2 showed clastogenic activity, but it was not restricted to germ cells. Both, ethanol and cadmium salts, appear to induce aneuploidy. The unconfirmed clastogenic effect of CHA was restricted to rats, but it occurred in both bone marrow and spermatogonia. Therefore, the general observation that rodent germ-cell mutagens are also genotoxic in somatic cells in vivo (Brusick, 1980; Holden, 1982) remains valid.     

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.     

Chromosome-damaging activity of a ruthenium radiosensitizer, RuCl2 (DMSO)2 (4-nitroimidazole)2, in Chinese hamster ovary cells in vitro

The metal complex, RuCl2 (DMSO)2 (4-nitroimidazole)2, 1, which has hypoxic radiosensitizing properties, was examined for genotoxic activity, as measured by the in vitro induction of chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells. A dose-dependent increase in the frequencies of metaphases with chromatid aberrations was observed for 1. Addition of S9 liver microsomal mixture and 1 to the cultured CHO cells did not alter the clastogenic activity noted for the complex itself. The clastogenic (chromosome damaging) activity of a precursor complex, cis-RuCl2(DMSO)4 and the ligand, 4-nitroimidazole (4-NO2-Im) were found to be less than that of 1 at corresponding concentrations. A comparison with two drugs used clinically with radiation, cis-dichlorodiammineplatinum(II) (cis-DDP) and misonidazole (miso), indicated that the clastogenic activity of 1 was similar to miso and much less than that of cis-DDP.     

Comparison of two extraction procedures for the assessment of sediment genotoxicity: implication of polar organic compounds

Four sediment samples (Va?ne Airport VA, Va?ne Center VC, Va?ne North VN and Reference North RN) were collected in the Berre lagoon (France). Sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs) by use of pressurized fluid extraction with a mixture of hexane/dichloromethane followed by HPLC with fluorescence detection analysis. Organic pollutants were also extracted with two solvents for subsequent evaluation of their genotoxicity: a hexane/dichloromethane mixture intended to select non-polar compounds such as PAHs, and 2-propanol intended to select polar contaminants. Sediment extracts were assessed by the Salmonella/microsome mutagenicity test with Salmonella typhimurium TA98+S9 mix and YG1041±S9 mix. Extracts were also assessed for their DNA-damaging activity and their clastogenic/aneugenic properties by the comet assay and the micronucleus test with Chinese Hamster ovary (CHO) cells. The PAH concentrations were 611ngg(-1)dw, 1341ngg(-1) dw, 613ngg(-1)dw and 482ngg(-1)dw for VA, VC, VN and RN, respectively. Two genotoxic profiles were observed, depending on the extraction procedure. All the non-polar extracts were mutagenic for TA98+S9 mix, and VA, VC, VN sediment samples exerted a significant DNA-damaging and clastogenic activity in the presence of S9 mix. All the polar extracts appeared mutagenic for TA98+S9 mix and YG104±S9 mix, and VA, VC, VN were genotoxic and clastogenic both with and without S9 mix. These results indicate that the genotoxic and mutagenic activities mainly originated from PAHs in the non-polar extracts, while these activities came from other genotoxic contaminants, such as aromatic amines and nitroarenes, in the polar extracts. This study focused on the important role of uncharacterized polar contaminants such as nitro-PAHs or aromatic amines in the global mutagenicity of sediments. The necessity to use appropriate extraction solvents to accurately evaluate the genotoxic hazard of aquatic sediments is also highlighted.     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

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.