Adduct formation and repair, and translesion DNA synthesis across the adducts in human cells exposed to 3-nitrobenzanthrone

• Published in: Mutation research. Genetic toxicology and environmental mutagenesis 2013-05, Vol.753 (2), p.93-100
• Main Authors: Kawanishi, Masanobu, Fujikawa, Yoshihiro, Ishii, Hiroshi, Nishida, Hiroshi, Higashigaki, Yuka, Kanno, Takaharu, Matsuda, Tomonari, Takamura-Enya, Takeji, Yagi, Takashi
• Format: Article
• Language: English
• Subjects: 3-Nitrobenzanthrone; Airborne particulates; Cells; DNA adduct; DNA repair; Mutagenesis; Mutation; Protein synthesis; Translesion DNA synthesis
• ISSN: 1383-5718; 1879-3592
• DOI: 10.1016/j.mrgentox.2013.03.005

•3-Nitrobenzanthrone formed dG-C8-N-ABA and dG-N2-C2-ABA as major adducts.•The DNA repair system was less efficient at removing dG-N2-C2-ABA.•dG-C8-N-ABA blocked DNA replication markedly.•TLS across dG-C8-N-ABA resulted in a significantly increased mutation frequency. 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-N6-yl)-3-aminobenzanthrone (dA-N6-C2-ABA), and 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-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-N2-C2-ABA were identified as major adducts in the HepG2 cells, and dA-N6-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-N2-C2-ABA per 107 dG and 877±330 dA-N6-C2-ABA per 107 dA in the cells. The cellular DNA repair system removed the dG-C8-N-ABA and dA-N6-C2-ABA adducts more efficiently than the dG-N2-C2-ABA adducts. After a 24-h repair period, 86.4±11.1% of the dG-N2-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-N6-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-N2-C2-ABA and dA-N6-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-N2-C2-ABA (12.1%) or dA-N6-C2-ABA (12.1%). These findings provide important insights into the molecular mechanism of 3-NBA-mutagenesis.