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Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO2, Ag, CeO2, Co3O4, Fe3O4, NiO and SiO2) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8-oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO2, Co3O4 and SiO2. Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO2 and Co3O4, FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG. [ABSTRACT FROM AUTHOR]

The incidences of many cancers can be very different in men and women. Besides differences in exposures to putative causative agents, it is plausible that both genetic and epigenetic effects play roles in these differences. In addition, gender-specific lifestyle and behavioural factors may modulate the effects of exposure to genotoxins. This commentary focuses on several aspects of gender-related differences in responses to mutagens and carcinogens, including sensitivity to chromosome damage, the contribution of genotypic variation and the role of DNA methylation. It is concluded that the reasons for gender differences in cancer susceptibility remain largely unknown in many cases, and the subject deserves more attention and study. [ABSTRACT FROM PUBLISHER]

32P-HPLC is a recently published method to generate DNA adduct profiles after exposure to a complex of genotoxic substances. The low detection limit enables characterization of individual DNA adducts in the general population. The 32P-HPLC method was applied to human lymphocytes and granulocytes from Silesia, apolluted industrial region in the south of Poland. Human samples were collected at the end of winter and summer to investigate the seasonal influence on DNA adduct formation. In lymphocytes a strong seasonal variation was seen in total DNA adducts, with winter values exceeding the summer values by 7.33±3.56 times. Granulocytes did not show any seasonal variation. In winter-collected lymphocytes the DNA adduct levels were 21.4±16.6/108 normal nucleotides (NN) while the summer values were 2.96±2.46±108NN. Granulocytes had 8.06± 7.76 and 9.59±6.19 DNA adducts/108 NN during winter and summer respectively. The lymphocyte DNA adduct profile consisted of at least 16 individual or clusters of DNA adducts. All 16 had a clear winter influence, with a winter: summer ratio of 1.6–15.3, indicating exposure to a complex mixture of genotoxic substances. The DNA adducts analyzed in human lymphocytes had retention times similar to DNA adducts generated by poycyclic aromatic hydrocarbons. The suggested candidates for DNA adducts displayed a similar seasonal variation in airborne particles to that found in DNA adducts in lymphocytes of humans living in the area. This is the first application of the 32P-HPLC method to analysis of DNA adducts in human tissues. [ABSTRACT FROM PUBLISHER]

Analysis of DNA adducts demands both high sensitivity and good resolution. A high-performance liquid chromato-graphy method for P-postlabeled DNA adducts (P-HPLC) was used to investigate DNA adduct formation from 38 polycyclic hydrocarbons and biphenyls . The P-HPLC method proved to be useful for separation, detection and characterization of DNA adducts from most of the substances. The method used to form the DNA adducts, with calf thymus DNA, nucleotide 3'-phosphates and metabolic activation through S-9 liver homo-genate, gave poor quantitative reproducibility. However, the results showed that the P-HPLC method was suitable for characterizing DNA adducts from many substances. From 35 of the tested substances 365 DNA and nucleotide 3'-phospate adducts were detected and characterized concerning retention times. Of the adducts, 171 were detected in DNA and 39 of them from five substances were characterized concerning target nucleotides. The retention time library built can be used in future analyses of DNA with complex patterns of DNA adducts. [ABSTRACT FROM PUBLISHER]

2-Nitrofluorene (NF) is found in the environment mainly due to incomplete combustion, as in vehicles. The major class of metabolites in rats after oral administration is the reduced and acetylated metabolites, e.g. derivatives of 2-acetylaminofluorene (AAF). The intestinal microflora reduces the nitro function to an amine which is further metabolized via aeetylation and hydroxylations. In this study, NF and AAF were orally administered to germ-free and conventional rats with the aim of studying DNA adduct formation in different tissues with the P-post-labelling assay. Chromatographic detection was performed with TLC and on-line P detection after HPLC separation of DNA adducts. The major (95%) DNA adduct formed was dG-C8-AF for both NF and AAF. The potency to form DNA adducts successively declined with the combinations conventional/AAF, germ-free/AAF, conventional/NF and germ-free/NF. The DNA adduct dG-C8-AF was detected in all four tissues that were analysed, e.g. liver, kidney, lung and heart. The presence of intestinal microflora enhanced the formation of DNA adducts in the tissues studied. [ABSTRACT FROM PUBLISHER]

The urinary mutagenicity (unconjugated forms) after administration of 2, 7-dinitrofluorene (2, 7-dNF) orally or i.p. was lower compared to 2-nitrofluorene (NF) administration. When partial hepatectomy (PH) was performed before i.p. administration, both substances had higher excretion of mutagens in which 2, 7-dNF increased dramatically and showed a higher level of mutagenicity compared to NF. NF and 2, 7-dNF formed DNA adducts in liver tissue. By different routes of administration (oral or i.p.) of the same substance at the same dose (200 mg/kg body wt), the patterns of DNA adducts were different. It seemed to be that PH, which was performed 18 h before i.p. administration, had no significant effects on the amount of DNA adducts. Generally, the total amount of DNA adducts after oral administration was higher compared to i.p. administration. Dramatic increases of the nitroreduced DNA adducts were noticed after oral compared to i.p. administration. When given i.p., both substances showed initiating capacity in foci formation both at 50 mg/kg body wt and 200 mg/kg body wt. When NF and 2, 7-dNF were administered orally by single gavage, 2, 7-dNF was more potent as an initiator in foci formation compared to NF and the initiating capacity of the two substances was higher compared to i.p.administration. The great difference between these two nitro-PAHs seen in the bacterial tests for mutagenicity was not seen in the genotoxic experiments. The results indicate that both NF and 2, 7-dNF formed DNA adducts and preneoplastic lesions after both i.p. and oral administration. After oral administration, both substances were more potent in causing DNA adduct and foci formation compared to i.p. administration. 2, 7-dNF was more potent as an initiator than NF especially after oral administration. The urinary excretion of unconjugated mutagens did not indicate the genotoxic effects of the parent substance. [ABSTRACT FROM PUBLISHER]

A further development of an HPLC method to analyze P-postlabeled DNA adducts is presented. The method is based on on-line detection of P radioactivity after separation by reversed-phase chromatography. The method has an advantage in that the postlabeling mixture can be injected directly into the HPLC system without any prior purification, with the background radioactivity on a low level. The analysis includes the whole range of substances from orthophosphate to non-polar DNA adducts, which makes it possible to analyze normal nucleotides and ATP together with DNA adducts. The analytical system has a high reproducibility and separates complex mixtures of DNA adducts. The slightly lower sensitivity compared to the TLC method is compensated for by the possibility of injecting large amounts of DNA into the system without affecting the analytical properties. The system can be applied to different DNA adducts as well as complex mixtures of DNA adducts. [ABSTRACT FROM PUBLISHER]

DNA adducts have been detected in laboratory animals after exposure to carcinogens as well as in human populations with known or suspected risk of developing cancer. Examples are smokers, coke and aluminium workers, urban citizens and roofers. The formation of DNA adducts is an early event in carcinogenesis which can be used for measuring target dose and as a biomarker for genotoxk risk. A method of analyzing P-postlabeled DNA adducts on reverse HPLC with on-line detection of P has been developed. The method permits direct injection of the P-postlabeling mixture into the analytical system without prior purification with background radioactivity on a low level. The method can be used in parallel with TLC analyses of P-postlabeled DNA adducts to improve the analytical capacity. The time for analysis of a typical single sample by HPLC and TLC is 30–60 min and 6–24 h respectively. A high (2 M) salt concentration in the HPLC eluent reduces the P background considerably. Also the peak tailing was substantially diminished, giving an ability to separate DNA adducts equal to or better than the TLC method. The method has been applied to 2-nitrofluorene (NF), a carcinogenic air pollutant, and -acetyl-2-aminofluorene (AAF), a model carcinogen which is also a metabolite of NF. A number of DNA adducts are formed in the livers of rats. After oral administration of AAF and NF, DNA adducts in the liver have been characterized as dG-C-AF and dG-C-AAF. The major DNA adduct found in both NF- and AAF-administered animals was dG-C-AF. The described HPLC method can, with minor adjustments, generally be used to analyze P-postlabeled DNA adducts. [ABSTRACT FROM PUBLISHER]

Absorption, metabolism and DNA binding of 2-nitrofluorene (NF) was studied in isolated, perfused and ventilated rat lungs and in lung microsomal incubations. Comparisons were made between control animals and animals treated with β-naphtho-flavone (BNF), a 2,3,7,8-tetrachlorodibenzo()dixon (TCDD) receptor ligand and inducer of cytochrome P450IA1. Clearance of NF increased significantly in the isolated, perfused and ventilated lungs after BNF dosage, from 0.55 ± 0.06 ml/min to 2.37 ± 0.62 ml/min ( < 0.05, =5–6). As a consequence of this, the mean residence time (MRT) for NF decreased when NF was dosed directly to the perfusion buffer, from 213 ± 23 min (=6) to 48 ± 9 min (=6), and after intratracheal dosage from 289 ± 101 min (=5) to 135 ± 72 min (=5). Irreversible binding of NF metabolites to DNA increased 2-fold after treatment with BNF when NF was dosed to the lung perfusion buffer. Treatment with BNF increased the rate of lung microsomal NF metabolism significantly, from 54 ± 5 to 106 ± 11 pmol/min/mg protein (P < 0.05, =6–12). Formation of the monohydroxylated metabolite X-OHNF was inhibited by addition of α-naphthoflavone (50 µM), by 89 and 98% with lung microsomal fractions from control and BNF-treated rats respectively. In contrast, proadifen (50 µM) preferentially inhibited formation of 9-OHNF, by 42 and 33% in incuba tions with lung microsomal fractions from control and BNF-treated animals. Anti-P450IIB1-IgG inhibited fonnatlon of 9-OHNF by 96 and 45% with lung microsomes from control and BNF-treated rats respectively. Formation of X-OHNF was unaffected by addition of anti-P-450IIB1-IgG in both cases. These results show that both constitutive and inducible microsomal rat lung enzymes metabolize NF. A constitutive enzyme, most likely cytochrome P450IIB1, catalyzes metabolic attack on NF with high preference for the 9-position. A BNF-inducible microsomal enzyme, most likely cytochrome P450IA1, catalyzes hydroxylation of NF both in the 9-position and in other positions. Increased metabolic clearance, metabolism and DNA binding of NF after BNF treatment suggest that the level and speficity of cytochrome P450 isozymes may be important determinants for toxicity and availability of NF in the rat lung. [ABSTRACT FROM PUBLISHER]

2-Nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, is present in exhaust from diesel- and petrol-driven vehicles, in exhaust from kerosene heaters, and in urban air and river sediments. Therefore the possible consequences of human exposure are important to elucidate. In the present study the initiating and promoting activity of NF was studied in a liver model for chemical carcinogenesis in the rat. Furthermore, the metabolism and formation of genotoxic metabolites of NF were investigated. NF was found to be a moderate initiator and a weak promoter when compared to diethylnitrosamine (DEN) and 2-acetylamino-fluorene (AAF) respectively, both of which are potent carcinogens. Animals given NF excreted more urinary mutagenicity when compared to animals given AAF or DEN. [ABSTRACT FROM PUBLISHER]

After a single oral dose of 2-nitro[9-C]fluorene (NF) to germfree and conventional rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and feces. The mutagenicity excreted from germfree animals exceeded the mutagenicity excreted from conventional animals. Absence of the microflora resulted in lack of nitroreduction and excreted metabolites from germfree rats were mono- or dihydroxylated nitrofluorenes, as assessed by liquid chromatography/mass spectrometry (LC/MS). These hydroxy-NFs were associated with the high direct-acting mutagenicity that was excreted in germfree animals. Only traces of NF were found in urine from germfree rats and no NF was detected in urine from conventional animals. Thus, in contrast to a number of reports on other mutagenic compounds, these results would tend to indicate that the intestinal microflora causes a reduced excretion of mutagenic metabolites of NF. [ABSTRACT FROM PUBLISHER]

The metabolism of 2-nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, was studied in the isolated perfused rat lung and liver. NF has been identified in urban air and diesel exhaust and occurs in the gas, as well as in the particulate phase. Therefore, it is conceivable that the lung represents one point of entry of this compound into the body. The lung metabolizes NF to hydroxylated NFs, mainly 9-hydroxy-NF, independently of the route of administration (intravascular or intratracheal). After intratracheal administration, NF is rapidly excreted into the perfusate, indicating that other organs might be exposed to unmetabolized NF. The liver excretes NF metabolites as biliary glucuronides. Untreated bile is not mutagenic. However, after β-glucuronidase treatment of bile, direct-acting mutagens were detected. The mutagenic metabolites in β-glucuronidase-treated bile were the same as identified in the perfusate of the isolated lung. Since β-glucuronidase is an enzyme found in the human intestinal microflora, inhalation of NF could result in the liberation of genotoxic metabolites in the colon. [ABSTRACT FROM PUBLISHER]

After a single oral dose of 2-nitro [9-C]fluorene to rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and faeces, urine being the major excretory route. The major part of the urinary mutagenicity was associated with the unconjugated fraction of metabolites which consisted of a range of hydroxylated acetylaminofluorenes in addition to a number of hydroxylated nitrofluorenes. The formation of hydroxylated acetylaminofluorenes supports the contention that 2-nitrofluorene, , enters the metabolic pathway of the well-known carcinogen 2-acetylaminofluorene. The single most mutagenic metabolite of 2-nitrofluorene was a hydroxylated nitrofluorene; the formation of this metabolite was markedly increased when the rats had been treated with β-naphthoflavone. Finally, an alternative pathway is discussed for the formation of 1- and 3-hydroxyacetylaminofluorenes. [ABSTRACT FROM PUBLISHER]

2-Nitrofluorene (NF) is a potent genotoxic substance found in environments where incomplete combustion takes place. NF is a mutagen and a carcinogen in animal models. NF or 7, 12-dimethylbenz[]anthracene (DMBA) was administered once topically to female SENCAR mice followed by promotion by 12--tetradecanoylphorbol-13-acetate (TPA) in two different studies. TPA (2–5 μg) was administered twice a week for 13 or 19 weeks after initiation of DMBA (5–10 μg) or NF (50–1500 μg). DMBA administration (positive control) resulted in the formation of many papillomas, which were seen from week 8–9 after initiation. The negative controls administered acetone only were free of tumours. NF administration did not result in papilloma formation, even at high initiating doses of NF combined with a dose of TPA causing a systemic toxic effect in terms of a significant reduced body weight. The mechanism behind the absence of papilloma formation after administration of genotoxic and carcinogenic NF remains to be investigated. [ABSTRACT FROM PUBLISHER]