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

      Nagy, Eszter; Adachi, Shuichi; Takamura-Enya, Takeji; Zeisig, Magnus; Moller, Lennart (2007-03)
      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.

      Nagy, Eszter; Adachi, Shuichi; Takamura-Enya, Takeji; Zeisig, Magnus; Moller, Lennart (2006-08)
      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.