title: Adduct formation and repair, and translesion DNA synthesis across the adducts in human cells exposed to 3-nitrobenzanthrone.
abstract: 3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a potent environmental mutagen that is found in diesel exhaust fumes and airborne particulates. It is known to produce several DNA adducts, including three major adducts N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-ABA), 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone (dA-N(6)-C2-ABA), and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-C2-ABA) in mammalian cells. In the present study, we measured the quantity of the formation and subsequent reduction of these adducts in human hepatoma HepG2 cells that had been treated with 3-NBA using LC-MS/MS analysis. As a result, dG-C8-N-ABA and dG-N(2)-C2-ABA were identified as major adducts in the HepG2 cells, and dA-N(6)-C2-ABA was found to be a minor adduct. Treatment with 1μg/mL 3-NBA for 24h induced the formation of 2835±1509 dG-C8-N-ABA and 3373±1173 dG-N(2)-C2-ABA per 10(7) dG and 877±330 dA-N(6)-C2-ABA per 10(7) dA in the cells. The cellular DNA repair system removed the dG-C8-N-ABA and dA-N(6)-C2-ABA adducts more efficiently than the dG-N(2)-C2-ABA adducts. After a 24-h repair period, 86.4±11.1% of the dG-N(2)-C2-ABA adducts remained, whereas only 51.7±2.7% of the dG-C8-N-ABA adducts and 37.8±1.7% of the dA-N(6)-C2-ABA adducts were present in the cells. We also evaluated the efficiency of bypasses across these three adducts and their mutagenic potency by introducing site-specific mono-modified plasmids into human cells. This translesion DNA synthesis (TLS) assay showed that dG-C8-N-ABA blocked DNA replication markedly (its replication frequency was 16.9±2.7%), while the replication arrests induced by dG-N(2)-C2-ABA and dA-N(6)-C2-ABA were more moderate (their replication frequencies were 33.3±6.2% and 43.1±7.5%, respectively). Mutagenic TLS was observed more frequently in replication across dG-C8-N-ABA (30.6%) than in replication across dG-N(2)-C2-ABA (12.1%) or dA-N(6)-C2-ABA (12.1%). These findings provide important insights into the molecular mechanism of 3-NBA-mutagenesis.
authors.affiliation: Osaka Prefecture University, Osaka, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kawanishi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fujikawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishii
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishida
authors.forename: Yuka
authors.initials: Y
authors.lastname: Higashigaki
authors.forename: Takaharu
authors.initials: T
authors.lastname: Kanno
authors.forename: Tomonari
authors.initials: T
authors.lastname: Matsuda
authors.forename: Takeji
authors.initials: T
authors.lastname: Takamura-Enya
authors.forename: Takashi
authors.initials: T
authors.lastname: Yagi
mesh_terms: D001551:Benz(a)Anthracenes / Q000633:toxicity*; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D045744:Cell Line, Tumor; D004247:DNA / Q000096:biosynthesis*; D018736:DNA Adducts; D004260:DNA Repair / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics; D009153:Mutagens / Q000633:toxicity; D053719:Tandem Mass Spectrometry
chemical_list: C475568:3-aminobenzanthrone; D001551:Benz(a)Anthracenes; D018736:DNA Adducts; D009153:Mutagens; D004247:DNA