Abstract A24: Exploration of esophageal cancer etiology using comprehensive DNA adduct analysis (DNA adductome analysis)
Authors: Nakagama Hitoshi , Totsuka Yukari , Lin Yingsong , He Yutong , Sato Haruna , Matsuda Tomonari , Matsushima Yoshitaka , Kato Mamoru , Elzawahry Asmaa , Totoki Yasushi , Shibata Tatsuhiro , Shan Baoen 
Published: June 2022

Abstract
Objective: China has among the highest incidence and mortality rates of esophageal cancer in the world. Cixian, one of the high-incidence areas, demonstrates a much higher incidence when compared with other rural or urban areas in China. To address the etiology of esophageal cancer in Cixian, we carried out a comprehensive DNA adduct analysis (DNA adductome) using surgical specimens that were collected from esophageal cancer patients living in Cixian and Shijiazhuang (low-incidence area for esophageal cancer).
Methods: Both tumorous and nontumorous tissues were collected from patients who underwent surgical procedures at Cixian Cancer Hospital and the Fourth Hospital of Hebei Medical University. A peripheral blood sample was also collected in each hospital. DNA adductome analysis was performed with LC-QTOF mass spectrometer by using DNA derived from surgical specimen. Whole-exome sequencing analysis was also conducted using esophageal tumor/peripheral blood paired samples. Mutation analyses of candidate chemical substance, which was predicted by DNA adductome analysis, were performed with Ames assay and gpt delta transgenic rats.
Results: Multiple DNA adducts were detected in samples from both areas in the DNA adductome analysis. The 2D-PCA scores plot of the DNA adducts showed a clear clustering of the high- and low-incidence areas, and the associated loadings plot demonstrated that several DNA adducts made a greater contribution to the high-incidence area. By referring to an in-house DNA adduct database, N2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)deoxyguanosine (THP-dG), which was derived from N-nitrosopiperidine (NPIP), emerged as a major DNA adduct. In order to confirm the DNA adduct that was highly correlated to the high-incidence area, we synthesized authentic 15N5-THP-dG and analyzed it by quantitative LC-MS/MS apparatus. An elution peak, which appeared at the same position of authentic 15N5-THP-dG, was observed in the surgical specimens collected from the high-incidence area. Examination of THP-dG adduct in peripheral blood samples demonstrated that the adduct levels were significantly higher in the high-incidence area than in the low-incidence area. Moreover, NPIP exhibited a strong mutagenic activity under metabolic activation in the Ames test and a significant dose-dependent increase in mutation frequency during in vivo mutagenicity tests with gpt delta rats. The NPIP-induced mutation was dominated by A:T to C:G transversions, followed by G:C to A:T and A:T to G:C transitions, in both liver and esophagus of animal samples. Similar mutational patterns were observed in the mutational signatures of esophageal cancer patients, which demonstrated weak correlation with THP-dG levels.
Conclusion: This is the first study that used DNA adductome analysis to reveal the etiology of esophageal cancer in Cixian. These findings suggested that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents.
Citation Format: Hitoshi Nakagama, Yukari Totsuka, Yingsong Lin, Yutong He, Haruna Sato, Tomonari Matsuda, Yoshitaka Matsushima, Mamoru Kato, Asmaa Elzawahry, Yasushi Totoki, Tatsuhiro Shibata, Baoen Shan. Exploration of esophageal cancer etiology using comprehensive DNA adduct analysis (DNA adductome analysis) [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A24.