DNA adduct formation and oxidative stress from the carcinogenic urban air pollutant 3-nitrobenzanthrone and its isomer 2-nitrobenzanthrone, in vitro and in vivo.

The carcinogenic vehicle emission product 3-nitrobenzanthrone (3-NBA) is known to rearrange in the atmosphere to the isomer 2-nitrobenzanthrone (2-NBA), which exists in 70-fold higher concentration in ambient air. The genotoxicity of 2-NBA and 3-NBA was studied both in vitro (human cell lines A549 and HepG2) and in vivo (F344 female rats intra-tracheally administered 5 mg/kg body weight of 3-NBA) models, using the (32)P-HPLC and the single-cell gel electrophoresis (Comet assay) methods. In vitro, also the parent compound benzanthrone (BA) and the metabolite 3-aminobenzanthrone (3-ABA) were evaluated. 3-NBA gave highest levels of DNA adducts in the two cell lines, but significantly higher in HepG2 (relative adduct level approximately 500 adducts/10(8) normal nucleotides), whereas 2-NBA formed about one-third and one-twentieth of the DNA adduct amount in A549 and HepG2 cells, respectively. 3-ABA formed only minute amounts of DNA adducts and only in the A549 cells, whereas BA did not give rise to any detectable levels. The DNA adduct patterns from 3-NBA were similar between the two model systems, but differed somewhat for 2-NBA. The oxidative stress induced by BA was almost as high as what was observed for 3-NBA and 3-ABA in both cell lines, and 2-NBA induced lowest level of oxidative stress. The oxidative stress and DNA adduct level, in whole blood, was significantly increased by 3-NBA but not by 2-NBA. However, 2-NBA showed similar toxicity to 3-NBA, with respect to DNA adduct formation in vivo, hence it is important to further study 2-NBA as a potential contributor to health risk. While DNA adduct level in the 3-NBA-exposed animals reached a peak around 1 and 2 days after instillation, 2-NBA-treated animals showed a tendency towards a continuing increase at the end of the study.

DNA damage and acute toxicity caused by the urban air pollutant 3-nitrobenzanthrone in rats: characterization of DNA adducts in eight different tissues and organs with synthesized standards.

3-Nitrobenzanthrone (3-NBA) is an urban air pollutant and rat lung carcinogen that is among the most potent mutagens yet tested in the Salmonella reversion assay. In the present study, 1 mg 3-NBA was administered orally to female F344 rats and DNA adduct formation was examined in liver, lung, kidney and five sections of the gastrointestinal (GI) tract at 6 hr, and 1, 2, 3, 5, and 10 days after administration. The DNA adduct patterns, analyzed by (32)P-postlabelling followed by HPLC separation, were similar in all tissues and organs. Five of the adduct peaks cochromatographed with synthesized DNA adduct standards. Three of these unequivocally determined standards, dGp-C8-N-ABA, dGp-N2-C2-ABA, and dAp-N6-C2-ABA, were of the nonacetylated type, suggesting that at least part of the pathway for activation of 3-NBA proceeds through O-acetylation of the hydroxylamine intermediate. The two other DNA adduct standards, dGp-C8-C2-N-Ac-ABA, and dGp-N2-C2-N-Ac-ABA, were of the acetylated type, but there was some ambiguity in the characterization of these DNA adducts, since they varied inconsistently between samples and they also aligned with peaks found in controls. At 6 hr after treatment, the level of DNA adducts was highest in glandular stomach (relative adduct labeling (RAL), approximately 70 adducts/10(8) normal nucleotides (NN)); adduct levels in this organ decreased at 24 hr, but increased afterwards. DNA adduct levels in the majority of organs were characterized by an early increase (from 6 hr to 3 days), which was followed by a decrease at 5 days and a maximum level 10 days after administration (RAL approximately 120 adducts/10(8) NN for the lung, kidney and glandular stomach, approximately 80 adducts/10(8) NN for the forestomach and ceacum, and approximately 40 adducts/10(8) NN for the liver, small intestine, and colon). This pattern was consistent with pathological observations during autopsy showing high levels of tissue damage in the GI tract; the tissue damage included hemorrhages, loss of villous surface structure in the small intestine, as well as intestine fragility and oedema of the adipose tissue around the GI-tract. Tissue damage decreased and DNA adduct levels increased at 10 days after administration. These observations suggest that 3-NBA not only exerts acute toxic effects, but that the bioavailability is affected by storage in tissues and later becomes available, resulting in the increased DNA adduct levels at the later time points of collection.

Oxidative stress and DNA damage caused by the urban air pollutant 3-NBA and its isomer 2-NBA in human lung cells analyzed with three independent methods.

The air pollutant 3-nitrobenzanthrone (3-NBA), emitted in diesel exhaust, is a potent mutagen and genotoxin. 3-NBA can isomerise to 2-nitrobenzanthrone (2-NBA), which can become more than 70-fold higher in concentration in ambient air. In this study, three independent methods have been employed to evaluate the oxidative stress and genotoxicity of 2-NBA compared to 3-NBA in the human A549 lung cell line. HPLC-EC/UV was applied for measurements of oxidative damage in the form of 8-oxo-2'-deoxyguanosine (8-oxodG), (32)P-HPLC for measurements of lipophilic DNA-adducts, and the Comet assay to measure a variety of DNA lesions, including oxidative stress. No significant oxidative damage from either isomer was found regarding formation of 8-oxodG analysed using HPLC-EC/UV. However, the Comet assay (with FPG-treatment), which is more sensitive and detects more types of damages compared to HPLC-EC/UV, showed a significant effect from both 3-NBA and 2-NBA. (32)P-HPLC revealed a strong DNA-adduct formation from both 3-NBA and 2-NBA, and also a significant difference between both isomers compared to negative control. These results clearly show that 2-NBA has a genotoxic potential. Even if the DNA-adduct forming capacity and the amount of DNA lesions measured with the (32)P-HPLC and Comet assay is about one third of 3-NBA, the high abundance of 2-NBA in ambient air calls for further investigation and evaluation of its health hazard.

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

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

32P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products.

The carcinogenicity of petroleum products is mainly due to their content of polycyclic aromatic compounds (PACs). These compounds may be activated metabolically and react with DNA to form DNA adducts, which is a critical event in the initiation of cancer. One of the most common techniques for analyzing DNA adducts is (32)P-postlabeling. The chromatographic method often used has been (32)P-TLC (thin-layer chromatography), but the more recently developed (32)P-HPLC (high-performance liquid chromatography) method has shown advantages. The aim of this study was to test the hypothesis that the (32)P-HPLC method has a better ability of detecting DNA adducts derived from petroleum products than (32)P-TLC. It was found that some DNA adducts migrated from the application point in (32)P-TLC in such a way that it is doubtful if they could be detected and quantified properly. It was also found that, when using (32)P-HPLC, it is possible to use the same protocol for substances with a wide variety of DNA adduct forming potential, whereas (32)P-TLC needs to be optimized regarding time of exposure and/or the amount of DNA applied. Further, a pattern of recognition in (32)P-HPLC enables a selective assessment of DNA adducts derived from complex mixtures whereas (32)P-TLC is very limited when analyzing complex mixtures due to poor resolution. With more knowledge about the properties of the most mutagenic DNA adducts in HPLC, it could be possible to know also which pattern corresponds to a mutagenic or carcinogenic oil. Consequently, (32)P-HPLC is a good alternative when assessing the genotoxicity of petroleum products.

DNA adduct formation and physiological effects from crude oil distillate and its derived base oil in isolated, perfused rat liver.

A distillate, D431, originating from Venezuelan heavy crude oil and a severely hydro-treated base oil, BO100, derived from this distillate, were tested for DNA adduct formation capacities and overall impact on liver functions. D431 had earlier showed DNA adduct formation in vitro but not in vivo in the rat. In this study, isolated rat liver perfusions were performed to elucidate whether the lack of DNA adducts in vivo was because of lack of uptake or metabolism. The oils were extracted with dimethyl sulfoxide and the extracts added to the perfusion system. Bile production, lactate metabolism and perfusate flow through the organ, which are parameters that reveal the condition of the liver, were continuously monitored. Samples of liver were collected once every hour during perfusion for DNA adduct analysis with (32)P-HPLC. The results for the distillate D431 showed that the production of bile and metabolism of lactate decreased while DNA adduct formation increased with time. The DNA adduct pattern formed in the D431-treated livers was similar to that found earlier in in vitro studies performed on calf thymus DNA (CT-DNA). In the case of DNA adduct formation, virtually no difference with dose was seen, suggesting that perhaps a point of saturation of, for instance, enzymatic systems was reached. The results for base oil BO100 showed no significant difference regarding bile production, lactate metabolism and DNA adduct formation when compared with the control, indicating no toxic or genotoxic activity.