DNA Adductome Analysis Identifies N-Nitrosopiperidine Involved in

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Article Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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DNA Adductome Analysis Identifies N‑Nitrosopiperidine Involved in the Etiology of Esophageal Cancer in Cixian, China Yukari Totsuka,*,† Yingsong Lin,‡ Yutong He,§ Kousuke Ishino,†,□ Haruna Sato,† Mamoru Kato,∥ Momoko Nagai,∥ Asmaa Elzawahry,∥ Yasushi Totoki,⊥ Hiromi Nakamura,⊥ Fumie Hosoda,⊥ Tatsuhiro Shibata,⊥ Tomonari Matsuda,∇ Yoshitaka Matsushima,# Guohui Song,@ Fanshu Meng,@ Dongfang Li,@ Junfeng Liu,§ Youlin Qiao,Δ Wenqiang Wei,Δ Manami Inoue,◆ Shogo Kikuchi,‡ Hitoshi Nakagama,¶ and Baoen Shan*,§ Downloaded via 178.159.100.28 on July 22, 2019 at 15:04:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Division of Carcinogenesis & Prevention, National Cancer Center Research Institute, Tokyo 104-0045, Japan Department of Public Health, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan § Cancer Institute, The Fourth Hospital of Hebei Medical University/The Tumor Hospital of Hebei Province, Shijiazhuang 050011, China ∥ Department of Bioinformatics, National Cancer Center Research Institute, Tokyo 104-0045, Japan ⊥ Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo 104-0045, Japan ∇ Research Center for Environmental Quality Management, Kyoto University, Shiga 520-0811, Japan # Department of Agricultural Chemistry, Tokyo University of Agriculture, Tokyo 156-8502, Japan @ Cixian Cancer Hospital, Cixian 056500, China Δ Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China ◆ Division of Prevention, Center for Public Health Sciences, National Cancer Center, Tokyo 104-0045, Japan ¶ National Cancer Center, Tokyo 104-0045, Japan ‡

S Supporting Information *

ABSTRACT: Esophageal cancer is prevalent in Cixian, China, but the etiology of this disease remains largely unknown. Therefore, we explored this by conducting a DNA adductome analysis. 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, which is in a low-incidence area. N2(3,4,5,6-Tetrahydro-2H-pyran-2-yl)deoxyguanosine (THP-dG) was the major adduct detected in samples from esophageal cancer patients in Cixian. The precursor of THP-dG, N-nitrosopiperidine (NPIP), exhibited a strong mutagenic activity under metabolic activation in the Ames test and a significant dose-dependent increase in mutation frequency during an in vivo mutagenicity test with guanine phosphoribosyltransferase (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 the liver and esophagus of animal samples. A similar mutational pattern was observed in the mutational signature of esophageal cancer patients that demonstrated weak correlation with THP-dG levels. These findings suggested that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents.

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INTRODUCTION

esophageal cancer in Cixian remain largely unknown partly because of a lack of well-designed case control or cohort

Esophageal cancer is the fourth leading cause of cancer deaths in China, which has a greater incidence in rural areas.1 Furthermore, the regions surrounding the Taihang Mountains in North China have among the highest rates of incidence of esophageal cancer in the world.2 One such high-incidence area is Cixian, which had an incidence of esophageal cancer 10-fold higher than those of low-incidence areas, such as Shanghai in the 1990s.3 However, the major contributing factors for © XXXX American Chemical Society

studies in this area. Notably, cigarette smoking and alcohol consumption, which are well-established risk factors for esophageal cancer in developed countries, were found to play Received: January 17, 2019 Published: June 21, 2019 A

DOI: 10.1021/acs.chemrestox.9b00017 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Ltd. (Osaka, Japan). DNase II from porcine spleen and acid phosphatase from potato were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). Difco Nutrient Broth and Cofactor-I were purchased from Becton, Dickinson and Company (Sparks, MD) and Oriental Yeast Co., Ltd. (Tokyo, Japan), respectively. The S9 mix was purchased from IEDA Trading Corp. (Tokyo, Japan). All other chemicals were of analytical grade and were purchased from Wako. Sample Collection. Esophageal cancer patients residing in the high-incidence area of Cixian, Hebei Province, were recruited from Cixian Cancer Hospital, and those residing in the low-incidence area of Shijiazhuang, which is the capital city of Hebei Province and had an incidence rate of 15 per 100000 people in 2015, were recruited from the Fourth Hospital of Hebei Medical University. In 2011, the agestandardized incidence rate of esophageal cancer in Cixian was 106.74 per 100000 men and 75.41 per 100000 women.3 The incidence rate in Shijiazhuang approximated the average rate in China, which was 14.1 per 100000 people in 2013.13 A written informed consent was obtained from each participant, and the research plan was approved by the Institutional Review Board of the Hebei Cancer Institute and the National Cancer Center in Tokyo, Japan. Resection tissues, including both tumorous and nontumorous tissues, were collected from esophageal cancer patients who underwent surgical procedures in each hospital. The same protocol was used to collect tissue biospecimens in each hospital. First, 0.5 g of tissue was collected from the central portion of the tumor, and 2 g of nontumorous tissue was collected from two nontumorous sites, namely, the proximate and distal sites approximately 5 cm from the central tumor area (1 g per site). The tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C in a freezer. A peripheral blood sample of 2 mL was also collected in each hospital and stored at −80 °C until the samples were analyzed. For this study, DNA samples were used for 32 and 31 subjects from highand low-incidence areas, respectively. After obtaining export approval from the Chinese National Office for the Management of Human Genetic Resources, we transported the tissue biospecimens to the laboratory at the National Cancer Center in Tokyo, Japan, to perform the DNA adductome analysis. Each of the study subjects was also asked to fill out a questionnaire that solicited information about demographic characteristics and lifestyle factors, including cigarette smoking and alcohol consumption. All of the tumor samples were squamous cell carcinoma. Table S2 shows the characteristics of the patients, including gender, age, smoking, and drinking. The age of the patients ranged from 43 to 78 years old, and there were no significant differences in gender, sex, or lifestyle habits between patients from the two areas. DNA Adductome Analysis. DNA was extracted and purified using a Gentra Puregene tissue kit (Qiagen, Valencia, CA) from the nontumorous tissues of esophageal cancer patients in Cixian (n = 7) and Shijiazhuang (n = 8). Tissue specimens were minced with scissors into tiny pieces, and then DNA extraction was performed according to the manufacturer’s instructions except that desferroxamine (final concentration of 0.1 mM) was added to all solutions to prevent the formation of oxidative adducts during the purification step. The extracted DNA was stored at −80 °C until DNA adductome analysis was performed. DNA samples were enzymatically digested using a modified method proposed by Ishino et al.12 First, 50 μg of DNA was placed in sodium acetate buffer (pH 5.3, final concentration of 60 mM) containing MgSO4 (final concentration of 40 mM) and NaCl (final concentration of 100 mM) in DNase II (from porcine spleen) for 30 min. Then, phosphodiesterase II (from bovine spleen) was added, and the sample was incubated for a further 30 min at 37 °C, following which acid phosphatase (from potato) was added and the sample was incubated for an additional 2 h at 37 °C. The sample was then purified using a Vivacon500 instrument (10 kDa molecular weight cutoff filters; Sartorius AG, Goettingen, Germany), and the reaction mixture was centrifuged at 4 °C and 10000g for 15 min using Ultrafree (0.2 μm pore; Millipore Co., Billerica, MA). The filtrate was then used for DNA adductome analysis. Details concerning the DNA adductome analysis were published previously.12 Briefly, LC−MS analysis was performed using a

an only minor role in high-incidence areas surrounding the Taihang Mountains, including Cixian.4 One likely explanation for the exceptionally high incidence of esophageal cancer in Cixian residents is the persistent exposure to certain chemical carcinogens. Nitrosamine has long been suspected as an etiological factor for esophageal cancer, and ecological studies conducted in Cixian have suggested the possible involvement of nitrate and nitrite, which are precursors of nitrosamine.5 However, definitive evidence of a causal role is lacking. Furthermore, a number of Nnitrosamines have been shown to be carcinogenic in experimental animals,6,7 making it unclear which of the hundreds of types of N-nitrosamines may be causally related to esophageal cancer. Carcinogen exposure can induce DNA damage and mutations in genes that are involved in the regulation of cellular growth, proliferation, and death, leading to tumorigenesis.8 The formation of DNA adducts, which results from interactions between the carcinogen and DNA, is a necessary event in the early stages of carcinogenesis,9 making the accurate measurement of DNA adducts critical for assessing the potential carcinogenic effects of specific exposures. Technical advances have seen DNA adduct measurement evolving from targeting a single DNA adduct into detecting and quantifying multiple DNA adducts.10 On the basis of the detection of the neutral loss of a 2′-deoxyribose moiety ([M + H]+, −116.04736), Kanaly et al. established a method that could detect hundreds of DNA adducts using liquid chromatography coupled with tandem mass spectrometry (LC−MS/MS).11 Building on their methods, we developed our own comprehensive DNA adductome analysis using an ultraperformance liquid chromatography-quadrupole time-offlight (UPLC-QTOF) mass spectrometer (MS).12 One advantage of our approach is that the high-resolution and accurate MS analysis provides us with information about the chemical structures of the detected DNA adducts, which can then be identified by referring to a list of 130 DNA adducts with known m/z [M + H]+ values that we compiled (Table S1). In a previous DNA adductome analysis, we detected 42 types of DNA adducts in the lungs of mice that had been exposed to magnetite nanoparticles (MGTs), which are genotoxic substances that are widely used in medicinal and industrial fields, among which 3,N4-ethenodeoxycytidine (εdC) was identified as the major contributor to the genotoxicity caused by MGT exposure.12 Therefore, in this study, we used DNA adductome analysis to explore the etiology of esophageal cancer in Cixian. Given the exceptionally high incidence of esophageal cancer in Cixian and the indication that precursors of nitrosamine may play a role in the etiology of this disease,5 our major hypothesis was that the detection of possible nitrosamine− DNA or other unknown adducts in surgical specimens originating from Cixian esophageal cancer patients that were absent from or present at a low level in tissues from a lowincidence area, coupled with confirmation of the mutagenicity of any detected chemical carcinogens, would provide evidence supporting the role of carcinogen exposure in the development of esophageal cancer in Cixian.

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EXPERIMENTAL PROCEDURES

Chemicals. 2′-Deoxyguanosine, bovine spleen phosphodiesterase II, and high-performance liquid chromatography (HPLC)-grade acetonitrile were purchased from Wako Pure Chemical Industries, B


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mass spectrometer (SCIEX, Framingham, MA) to obtain the fragmentation data for the pooled human DNA sample with highresolution accurate mass (HRAM)−MS/MS analysis. The HPLC conditions were as follows: column, Synergi Fusion-RP (2.5 μm particle size, 2.0 mm × 100 mm; Phenomenex, Torrance, CA); flow rate, 0.4 mL/min; gradient system of acetonitrile in 10 mM ammonium acetate (pH 5.3) from 0 to 20 min and a linear gradient from 1% to 3% from 20 to 25 min and from 3% to 85%. THP-dG was also analyzed using a human blood sample collected from the highincidence area in PI mode with the major fragment ions at m/z 352.1 → 236.1, which corresponds to the loss of the deoxyribose moiety (THP-G), and m/z 352.1 → 152.05, corresponding to the guanine base (G) with a cone voltage of 40 V and a collision energy of 20 eV, and the mass tolerance was set to ±0.02 Da. Mutagenicity Assay. Mutagenicity was examined using the preincubation method with Salmonella typhimurium TA100 and TA1535 in the presence of S9 mix (0.05 mL of S9 in a total volume of 0.5 mL). NPIP was dissolved in dimethyl sulfoxide (DMSO) and added to the test strain at doses of 1, 2, and 5 μg per plate, and the number of hisG+ revertant colonies was counted. DMSO was used as a solvent control. The mutagenic activities of the samples were calculated from the linear portions of the dose−response curves that were obtained from three doses on duplicate plates in at least two independent experiments. Analysis of the Global Mutational Profiles of NPIP in a Salmonella Strain. The hisG+ colonies were randomly isolated, and genomic DNA was extracted from 77 clones from the control group and 50 clones from the NPIP-treated groups using a Puregene Cell and Tissue Kit (Qiagen). The mutational profiles that were induced by NPIP were then examined using whole-genome sequencing, as described previously.16,17 The sequence reads were aligned to reference sequence NC_003197 (Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 chromosome, complete genome, 4857432 bp) using CLC Genomics Workbench version 5. In Vivo Mutation Analysis of NPIP Using Experimental Animals. Six-week-old male F344 guanine phosphoribosyltransferase (gpt) delta rats (n = 15), which carry 5−10 copies of λ EG10 DNA on chromosome 4,18,19 were purchased from Japan SLC (Shizuoka, Japan). The animals were provided with food (CE-2 pellet diet; CLEA Japan, Inc., Tokyo, Japan) and tap water ad libitum and were maintained under a 12 h light/dark cycle at 22 ± 2 °C and a relative humidity of 55 ± 10%. After the animals had been quarantined for 1 week, the experiments were conducted according to the Guidelines for Animal Experiments in the National Cancer Center, following approval by its Experimental Animal Research Committee. Two groups of male gpt delta rats (n = 5 per group) were orally administered five consecutive doses of 33 or 66 mg of NPIP/kg per week for 4 weeks, and rats in a third control group (n = 5) were treated with distilled water. The rats were sacrificed at 14 weeks of age (6 weeks after NPIP administration). The liver and esophagus were removed from each rat and stored at −80 °C until the DNA was extracted. High-molecular weight genomic DNA was extracted using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions, and λ EG10 phages were rescued using Transpack Packaging Extract (Stratagene, La Jolla, CA). The gpt mutagenesis assay was performed following a previously described procedure.20 The 6-thioguanine (6-TG)-resistant colonies were cultured overnight at 37 °C in lysogeny broth containing 25 μg/ mL chloramphenicol, harvested by centrifugation (7000 rpm for 10 min), and stored at −80 °C. The mutational spectra of the 6-TG coding sequences were determined using polymerase chain reaction (PCR) and direct sequencing, as described previously.20 Sequence analysis was performed at Takara Bio Inc. (Mie, Japan). Whole-Exome Sequencing of Human Esophageal Tumors. Twenty-five esophageal tumor/peripheral blood paired samples were used for whole-exome sequencing library preparation using the SureSelect Human All Exon V5 + linc Kit (Agilent Technologies) according to the manufacturer’s instructions. Briefly, 1 μg of doublestranded DNA was fragmented by a Covaris S2 Focused ultra-

nanoACQUITY UPLC system (Waters, Milford, MA) equipped with a Xevo QTOF mass spectrometer (Waters, Manchester, U.K.), which was equipped with an electrospray ionization (ESI) source and controlled by MassLynx version 4.1. The digested DNA samples were separated on an ACQUITY UPLC BEH130 C18 column [1.7 μm, 1.0 mm (inside diameter) × 150 mm] at a flow rate of 25 μL/min. The column temperature was set to 40 °C, and water and methanol were used as mobile phases A and B, respectively. Chromatographic separation was performed by gradient elution: 1% B from 0 to 5 min, linear gradient to 10% B from 5 to 10 min, linear gradient to 80% B from 10 to 35 min, and 80% B from 35 to 45 min. The MS parameters were set as follows: mass range scanned from 50 to 1000 with a scan duration of 0.5 s (1.0 s total duty cycle), capillary voltage of 3.7 kV, sampling cone voltage of 40 V, extraction cone voltage of 4 V, source temperature of 125 °C, and desolvation temperature of 250 °C. Nitrogen gas was used as the desolvation gas (flow rate of 800 L/h) and cone gas (flow rate of 30 L/h). All data were collected in positive ion mode. Multiscale entropy (MSE) analysis was performed on the mass spectrometer set at 3 V for a low collision energy and a ramp of 10−25 V for a high collision energy during the acquisition cycle. A cone voltage of 20 V was used. LC−MS Data Processing. The raw data files obtained from the LC−MS runs were analyzed using MassLynx version 4.1 and MarkerLynx version 4.1 (Waters). These applications detect, integrate, and normalize the intensities of the peaks to the sum of peaks within a particular sample. The resulting multivariate data set, which consisted of the peak number (based on the retention time and m/z), sample name, and normalized peak intensity, was analyzed with S-plot using SIMCA-P+11.5 (Umetrics AB). The LC−MS parameters were set as follows: mass tolerance of 0.05 Da, apex track parameters being a peak width at 5% height (seconds) of 15, a peak-to-peak baseline noise of 50, and apply smoothing = Yes, and collection parameters being an intensity threshold (counts) of 100, a mass window of 0.05, a retention time window of 0.10, a noise elimination level of 6, and deisotope data = Yes. Confirmation and Quantification of THP-dG. DNA was extracted from nontumorous lesions of the esophagus or the peripheral blood using a Gentra Puregene tissue kit. Authentic [15N5]THP-dG and THP-dG were synthesized following a previously described procedure14,15 and analyzed using the Waters 2795 LC system (Waters, Manchester, U.K.) interfaced with a Quattro Ultima triple-stage quadrupole mass spectrometer (Waters). The HPLC conditions were as follows: column, Shim-Pack (5 μm particle size, 2.0 mm × 150 mm; Shimadzu, Kyoto, Japan); flow rate, 0.2 mL/min; solvent system, a linear gradient from 2% to 95% acetonitrile in 5 mM ammonium formate (pH 6.5) over 30 min. Quantitative measurements of THP-dG and [15N5]THP-dG were taken by monitoring the multiple-reaction monitoring (MRM) transitions of the major fragment ions corresponding to the loss of a deoxyribose moiety and guanine base, m/z 352.1 → 236.1 (THP-G) and m/z 352.1 → 152.05 (G) for THP-dG and m/z 357.1 → 241.1 (THP-[15N5]G) and m/z 357.1 → 157.05 ([15N5]G) for [15N5]THP-dG, respectively, with a cone voltage of 35 V and a collision energy of 10 eV. When authentic [15N5]THP-dG was analyzed under these conditions, double peaks were eluted at retention times (tR) of 23.36 and 23.90 min, respectively, indicating the presence of an isomer.14 For quantitative analysis of THP-dG in human samples, DNA (∼40 μg) was enzymatically digested using the same procedures as outlined above and then analyzed by the same procedure described above. Quantification was conducted using a standard curve for authentic THP-dG concentrations of 5−500 μM. The limit of detection was 5 pmol/50 μg of DNA with this analytical method. For confirmation analysis, we used pooled DNA samples (combined 16 samples from esophageal cancer in the high-incidence area, ∼1600 μg) and enzymatically digested using the same procedures as outlined above. Because the existing level of THP-dG in the human tissues was thought to be low, we enriched the DNA adduct by collecting the fraction with the same elution position of authentic [15N5]THP-dG (tR = 23−24 min) and then analyzed it by a Simadzu Prominence LC system interfaced with a Triple TOF6600 C


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Figure 1. Comprehensive DNA adduct analysis. (a) Map views of the DNA adducts detected in esophageal cancer patients living in high-incidence (n = 7) and low-incidence (n = 8) areas. (b) 2D PCA scores and variable loading plots for the DNA adducts obtained from the adductome analysis. The first two principal components (PC1 and PC2) explained 75.01% of the total variance and discriminated between the esophageal cancer patients living in high- and low-incidence areas. Analysis of Mutation Patterns and Signatures. The number of each of 96 possible somatic substitution types (C > A/G > T, C > G/G > C, C > T/G > A, T > A/A > T, T > C/A > G, and T > G/A > C with the bases immediately 5′ and 3′ to each substitution in targeted exon capture regions) was counted for each sample. The frequency of each of these substitutions was determined by dividing each count by the total number of substitutions. Negative matrix factorization (NMF) was applied to the 96-substitution pattern using published software24 by performing 1000 iterations of NMF in each run and repeating each NMF run until convergence was achieved (10000 iterations without a change), or the maximum number of 1000000 iterations was reached. We trialed two to eight signatures in a cycle of NMF runs and determined that four was the optimal number because this preserved the signature stability at a high level while allowing the reconstruction error to become converged at a low value. To validate the signatures that were identified in this study, they were compared with the current COSMIC signature studies (https://cancer.sanger.ac.uk/cosmic/signatures) by calculating their cosine similarity. All samples were clustered on the basis of the number of somatic mutations contributed by each signature in each sample using unsupervised hierarchical clustering with the cosine distance and Ward’s linkage. Statistical Analysis. Principal component analysis (PCA) was used to model the results from the DNA adductome analysis. The data obtained from the gpt mutation assay were expressed as means ± the standard deviation (SD) and compared with those of the corresponding solvent control using the F test followed by a Student’s t test. Mutational spectra were compared using Fisher’s exact test.25 The data on THP-dG formation in high- and low-incidence areas for

sonicator (Covaris Inc., Woburn, MA) to generate an average size of 150−200 bp, following which end repair, A-tailing, and ligation of SureSelect adapter oligos were performed. Five to six cycles of PCR amplification were then carried out on the adapter-ligated library, and 750 ng of the amplified libraries was hybridized with the SureSelect capture library for 24 h. Then, 11 cycles of postcapture amplification were performed using Herculase II polymerase with SureSelect Indexing Post-Capture PCR primers. DNA purification was performed with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). The exon capture libraries were sequenced using a HiSeq 2000 system (Illumina, San Diego, CA) with a 2 × 100 bp paired-end run to obtain a sequencing coverage of more than 150 times for the tumors and 80 times. Identification of Somatic Mutations. Paired-end reads from the tumor and peripheral blood samples were aligned to human reference genome GRCh37 using the Burrows−Wheeler Aligner (BWA).21 Any probable PCR duplications where the paired-end reads aligned to the same genomic positions were removed, and pileup files were generated using SAMtools22 and a program developed in house. Somatic point mutations [single-nucleotide variations (SNVs) and short indels] were located by first comparing variants in matched pairs (tumor/normal samples from each patient) and removing personal germline variants. All normal samples were then grouped together to produce a so-called “normal panel”, and false positive variants that had originated from sequence errors were removed. This strategy is very effective for removing false positives because sequence-specific errors occur with a particular frequency rather than randomly, resulting in increased error rates at specific genomic positions. The details of these filtering conditions have been previously reported.23 D


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Figure 2. Heat maps and clustering dendrogram for the major adducts. Hierarchical clustering was performed using 21 major contributors selected by PCA. The nine major contributors are indicated by red arrows (specific to the high-incidence area) and blue arrows (specific to the lowincidence area). esophageal cancer were compared using a Student’s t test. The correlation between the THP-dG level and the proportion of each of four identified mutation signatures (Sigs. A−D) was analyzed by calculating the Pearson product moment correlation coefficient, which represents the strength of the linear association between two variables. All analyses were performed in the statistical package R with a P < 0.05 significance level.

Furthermore, hierarchical clustering analysis of the data set consisting of those DNA adducts that strongly contributed to the incidence of esophageal cancer showed a clear separation of the subjects between the high- and low-incidence areas (Figure 2). Among these major adducts, A10 was highly specific to the high-incidence area, and A26 and A72 also demonstrated a clear relationship with this area, though they were present at an abundance much lower than that of A10. Other adducts, such as A13, A15, and A11, did not appear to be highly correlated with the high-incidence area. By contrast, A130, A410, and A605 tended to be correlated with the lowincidence area. The DNA adducts that may have contributed to the esophageal cancer risk in high- and low-incidence areas are listed in Table 1. To determine the chemical structures of the major DNA adducts that were specific to esophageal cancer in the high-incidence area, we compared the m/z [M + H]+ values of the detected DNA adducts with those of known DNA adducts listed in the in-house database that we compiled (Table S1). The results showed that the m/z value of A10 (m/ z [M + H]+ 352.1818), which was exclusively detected in the high-incidence area, was almost identical to that of N2-(3,4,5,6tetrahydro-2H-pyran-2-yl)deoxyguanosine [THP-dG; m/z [M + H]+ 352.1622 (Figure 3a)], which is derived from the nitroso compound N-nitrosopiperidine (NPIP) (Figure 3b). To screen candidate adducts properly, the mass tolerance for

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RESULTS PCA of the Adductome Data and Inferred Chemical Structure of the Detected DNA Adducts. The characteristics of the study subjects, including their age, gender, and family history, are listed in Table S2. Because the formation of DNA adducts is known to be influenced by cigarette smoking and alcohol consumption, we selected subjects who had no history of smoking or drinking. DNA samples were prepared from surgical specimens of apparently nontumorous lesions collected from subjects inhabiting areas with high (Cixian; n = 7) and low (Shijiazhuang; n = 8) incidences of esophageal cancer. Multiple DNA adducts were detected in samples from both areas but appeared to be more abundant in samples from the high-incidence area (Figure 1a). The two-dimensional (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- or lowincidence area based on their PCA significance (Figure 1b). E


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between the actual measured m/z value of THP-dG (352.1591) and that of A10 in the adductome analysis (352.1818) was considered to be due to the slightly loose mass tolerance (±0.05 Da) determined by adductome analysis. Moreover, we confirmed the presence of THP-dG in a blood sample collected from the high-incidence area. Similar elution patterns with distinctive double peaks with m/z 352.1 → 236.1 and m/z 352.1 → 152.05 transitions were also observed (Figure 3c). In Vitro and In Vivo Mutagenicity of NPIP. It has previously been shown that THP-dG is formed from NPIP via the cytochrome P450 (CYP) metabolic activation pathway (Figure S1)26 and that NPIP induces the formation of tumors in the esophagus and liver of rats.27 Therefore, we performed a series of experiments to confirm the mutagenicity of NPIP. NPIP exhibited mutagenic activity against S. typhimurium TA100 and TA1535 in a dose-dependent manner under the metabolic activation systems, with values of 9075 and 21250 revertants/μmol of NPIP toward TA100 and TA1535, respectively. Whole-genome sequencing analysis detected a total of 23 and 65 SNVs in 77 control clones (68 for TA100 and 9 for TA1535) and 50 NPIP-exposed clones (25 for TA100 and 25 for TA1535), respectively. Six types of mutation spectra were determined, among which the G:C to A:T transition was significantly higher in NPIP-exposed clones than in control clones (Figure 4). The mutation frequency also increased in a significant dosedependent fashion in the liver of gpt delta rats administered with NPIP (Figure 5a). Among the mutation profiles that were observed for the 6-TG coding sequence in the liver, the proportion of A:T to C:G transversion was significantly higher in NPIP-exposed clones than in control clones (Figure 5b). In addition, the proportions of G:C to A:T and A:T to G:C transitions were also higher in the NPIP-exposed clones, though these differences were not statistically significant [for G:C to A:T, P = 0.06; for A:T to G:C, P = 0.07 (Table S3)]. We also analyzed the mutation frequency in the esophagus of gpt delta rats. Although the amount of extracted DNA was not sufficient for a complete gpt mutation assay, 22 clones with gpt mutations were observed among 61500 surviving clones obtained from NPIP-treated rats, whereas only one clone with a gpt mutation was observed among 312000 surviving clones obtained from the control rats (Table S4). The mutational profile of the 22 clones obtained from the esophagus of gpt delta rats administered with NPIP was quite similar to that of the liver (Figure 5b). Somatic Mutational Profiles of Esophageal Tumor Samples. We performed a whole-exome analysis of surgical tissues obtained from patients with esophageal cancer living in the high- and low-incidence areas. We identified an average of 425 ± 239 somatic SNVs in the targeted exon capture regions of the 25 patients after removing variants that were found in the exome data of matched normal tissues [peripheral blood (Table S5)]. Patients in the high- and low-incidence areas had almost identical numbers of somatic SNVs [145−909, average of 422 ± 236 SNVs; 122−932, average of 430 ± 243 SNVs (data not shown)]. Recurrent mutations were observed in TP53, which is known to be one of the driver genes in esophageal squamous cell carcinoma, with 23 of 25 patients exhibiting mutations in this gene. Among these mutations, missense mutations (52%) were most common, followed by nonsense mutations (22%), splice site variations (9%), deletions (9%), insertions (4%), and multiple mutations (4%).

Table 1. List of the Major Adducts That May Have Contributed to the Risk of Esophageal Cancer in the Highand Low-Incidence Areasa adduct A10 A11 A13 A15 A26 A45 A59 A63 A72 A104 A107 A251 A269 A353 A378 A130 A410 A605 A1269 A1317 A1939

m/z [M + H]+

tR (min)

comparison with the DNA adduct database [M + H]+

Specific to the High-Incidence Area 352.1818 18.9 THP-dG (352.1622) 231.1665 18.8 NA 203.1364 17.8 NA 223.0983 8.6 NA 519.2866 18.3 NA 332.1793 11.0 NA 318.1705 8.4 NA 271.1405 28.2 NA 558.7676 27.6 NA 364.2014 6.7 NA 463.2098 8.4 NA 288.2051 17.8 NA 588.2637 9.5 NA 186.1146 14.8 NA 287.1793 21.7 NA Specific to the Low-Incidence Area 171.1161 24.6 NA 185.1280 24.1 NA 259.1576 7.8 NA 200.1314 14.5 NA 368.1796 14.3 NA 382.1622 11.9 NA

derivation NPIP unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown

a NA, not available; NPIP, N-nitrosopiperidine; tR, retention time; THP-dG, N2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)deoxyguanosine.

integration of individual adducts between samples has been set to slightly loose. Therefore, there is a slight difference in the m/z value between adduct A10 and the exact mass of THP-dG. We were unable to match the m/z [M + H]+ values of the other adducts that were highly correlated with the highincidence area to values in our DNA adduct database, however. Confirmation That Adduct A10 Was THP-dG. Our findings indicated that DNA adduct A10 may be a significant contributing factor to esophageal cancer in the high-incidence area of Cixian. To further explore its chemical structure, we synthesized authentic [15N5]THP-dG as well as THP-dG according to the procedure described in Experimental Procedures. To enrich human tissues with THP-dG, we collected the fraction with the same elution position of authentic [15N5]THP-dG and analyzed it by using a Simadzu Prominence LC system interfaced with a Triple TOF6600 mass spectrometer (SCIEX) in product ion (PI) mode (mass tolerance set to ±0.02 Da). We observed distinctive double peaks with m/z 352.1 → 236.1 and m/z 352.1 → 152.05 transitions, which correspond to loss of a deoxyribose moiety from the precursor and guanine base, respectively, with the same retention times of authentic [15N5]THP-dG (Figure 3c). To further characterize DNA adduct A10, high-resolution accurate mass (HRAM) MS/MS spectra obtained from a TT6600 mass spectrometer showed precursor and product ions with m/z values of 352.1591, 236.1124, and 152.0541, which correspond to a precursor, the loss of a deoxyribose moiety from a precursor, and a guanine base, respectively, and these m/z values were identical to those in authentic THP-dG [m/z 352.1585, 236.1122, and 152.0549, respectively (Figure 3d)]. On the other hand, the slightly different m/z value F


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Figure 3. Chemical structures of THP-dG and its precursor NPIP and typical LC−ESI-MS/MS chromatograms of authentic THP-dG and a human DNA sample obtained from nontumorous lesions of the esophagus and a blood sample measured in the PI mode: (a) THP-dG and (b) NPIP. (c) Product ion mass spectra at m/z 357.1 → 241.1 (THP-[15N5]G) and m/z 357.1 → 157.05 ([15N5]G) for the chemically synthesized standard [15N5]THP-dG (top) and at m/z 352.1 → 236.1 (THP-G) and m/z 352.1 → 152.05 (G) for the pooled human DNA from esophageal (middle) and blood DNA (bottom) samples. The peaks that correspond to THP-dG are indicated by arrows. (d) Fragmentation data for chemically synthesized authentic THP-dG (top) and the pooled human DNA sample (bottom) obtained from HRAM−MS/MS. G


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low-incidence area, and THP-dG was detected in all samples (Figure 7). However, the adduct levels were significantly higher in the high-incidence area than in the low-incidence area. Correlation between the THP-dG Level and the Mutational Signatures. The correlation between the THPdG level and the proportion of each identified mutational signature is shown in Figure 8. Sig. C, which was similar to signature 17 in the COSMIC database with an unknown exposure source, was weakly correlated with THP-dG levels among 19 subjects (r = 0.44; P < 0.05), 11 of whom were from the high-incidence area and eight of whom were from the lowincidence area. Other than signature C, negligible correlations were found for signatures A, B, and D, with correlation coefficients ranging from 0.10 to 0.27.

Figure 4. Mutation spectrum of NPIP. Analysis of global mutations was undertaken by performing whole-genome sequencing on 77 control clones and 50 NPIP-exposed clones of Salmonella strains, and six types of mutation spectra were determined. *P < 0.05 (χ2 test).

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DISCUSSION Using UPLC-QTOF MS-based DNA adductome analysis, we detected THP-dG adducts in nontumorous lesions obtained from esophageal cancer patients residing in Cixian and demonstrated that the precursor to THP-dG, NPIP, exhibited a strong mutagenic activity in gpt delta rats. Furthermore, blood THP-dG levels of patients were found to be much higher in the high-incidence area than those of patients in the low-incidence area. These findings indicate that NPIP is probably involved in the development of esophageal cancer in Cixian residents. The THP-dG adduct that we detected is formed from NPIP, which is a cyclic nitrosamine.14,28,29 In a series of follow-up experiments, we demonstrated that NPIP exhibits a strong mutagenic activity under metabolic activation in the Ames test that increases in a significant dose-dependent fashion in the liver of gpt delta transgenic rats. It has previously been demonstrated that NPIP induces both esophageal and liver cancer.27 Here we also found that NPIP induced mutations in the esophagus of gpt delta transgenic rats. The mechanisms underlying the carcinogenicity of NPIP in the human esophagus have not yet been elucidated, but one possible explanation is related to the exclusive activation of NPIP by rat esophageal microsomes to form DNA adducts, such as THPdG.26 Alternatively, the 1,N2-ethenodeoxyguanosine adduct may have been formed by α-acetoxyNPIP.28,30 Some ethenotype adducts are highly mutagenic and persist in the

The analysis of the single-nucleotide substitution patterns in the esophageal cancer patients showed that C:G to T:A transitions were predominant, followed by C:G to A:T transversions, C:G to G:C transversions, T:A to C:G transitions, and T:A to G:C transversions. Non-NMF algorithm analysis identified four types of mutational signatures (Sigs. A−D) among the somatic SNVs observed in the esophageal cancer patients living in high- and low-incidence areas (Figure 6a), and the cosine similarity analysis showed that all of these signatures were highly similar to signatures in the COSMIC database (https://cancer.sanger.ac.uk/cosmic/ signatures): signature 13 for Sig. A (similarity score of 0.85), signature 1 for Sig. B (0.96), signature 17 for Sig. C (0.88), and signature 5 for Sig. D (0.92). On the basis of the contributions of each of these mutation signatures in individual samples, the samples could be separated into five clusters [C1−C5 (Figure 6b)]. However, no clear separation was observed between the high- and low-incidence areas, suggesting that there was no difference in the etiology of esophageal cancer between these areas. Quantitative Analysis of THP-dG Adduct Levels in Peripheral Blood Samples. To clarify the levels of exposure to THP-dG in high- and low-incidence areas for esophageal cancer, we examined THP-dG adduct levels in peripheral blood samples obtained from esophageal cancer patients living in these areas (Table S2). DNA samples were obtained from 32 subjects in the high-incidence area and 31 subjects in the

Figure 5. In vivo mutation analysis of NPIP. (a) gpt mutation frequency in the liver of rats treated with NPIP. Male rats were treated with five consecutive doses of 33 mg of NPIP/kg (NPIP-Low) or 66 mg of NPIP/kg (NPIP-High) per week for 4 weeks and then sacrificed at 6 weeks from the last NPIP administration. Mean ± SD mutation frequency (see Experimental Procedures for a description of the procedure). *P < 0.05, and **P < 0.01 (Student’s t test vs the corresponding vehicle control rats). (b) gpt mutation spectrum in the liver and esophagus of rats treated with NPIP. *P < 0.05 (χ2 test). H


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Figure 6. Mutational signatures of esophageal cancer in China. Mutational signatures were extracted from the trinucleotide mutational pattern of esophageal cancer collected from patients living in high- and low-incidence areas in China using non-NMF. (a) Four mutational signatures were identified (Sigs. A−D), which were similar to signatures 13, 1, 17, and 5, respectively, in the COSMIC database. The numbers in parentheses indicate the similarity scores obtained by cosine similarity analysis. (b) Hierarchical clustering and contributions of the four mutational signatures in individual subjects in terms of the proportion (top) and number (middle) of SNVs. The number of SNVs in each signature is shown in the bottom panel.

3,N4-ethenodeoxycytosine have been reported to date, similar etheno adducts may be formed by the reaction between 4-oxo2-pentenal and 2′-deoxyadenosine in the same way. Sources of NPIP exposure include drinking water, food, cigarette smoking, and occupation. Because we selected patients who were nonsmokers and nondrinkers, there is very little likelihood that the results were confounded by cigarette smoking or alcohol consumption. In addition to exogenous exposure, NPIP can be produced endogenously following the intake of nitrate and nitrites from a variety of sources, as supported by the observation that urinary excretion of NPIP occurs during exposure to high nitrate levels.35 Drinking water or vegetables may represent a major source of nitrate in Cixian on the basis of previous observations.5 Therefore, we conducted a pilot study in which we measured the nitrate content of vegetables and well water. We found that some vegetables and shallow well water contained higher nitrate contents in the high-incidence area than in the lowincidence area, indicating their possible involvement in the pathophysiology of esophageal cancer (data not shown). NPIP can also be formed endogenously by nitrosation of dietary piperidine or its precursor, piperine, in black pepper (Figure S2),36 and interestingly, spicy food intake has been shown to be associated with an increased risk of esophageal cancer in some high-incidence areas in China.37 Therefore, the exact source of NPIP exposure requires further exploration through epidemiological studies with detailed exposure measurements.

Figure 7. Quantitative analysis of THP-dG by LC−MS/MS. THP-dG levels were examined in peripheral blood samples collected from esophageal cancer patients living in high-incidence (n = 32) and lowincidence (n = 31) areas to determine the levels of exposure to NPIP. THP-dG was quantified using a standard curve constructed from authentic THP-dG with a detection limit of 5 pmol/50 μg of DNA. **P < 0.01 (Student’s t test).

DNA,31−34 so their production is thought to be important in the formation of NPIP-induced esophageal tumors. 4-Oxo-2pentenal is a reactive intermediate that is produced by the solvolysis of α-acetoxyNPIP and binds to DNA bases to form etheno adducts.28,30 Although the chemical structures of 7-(2oxopropyl)-1,N2-ethenodeoxyguanosine and 3-(2-oxopropyl)I


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Figure 8. Correlation between the level of THP-dG and the four types of mutational signatures.

through direct (conversion of the reactive form, αacetoxyNPIP) or indirect (inflammation related to NPIP exposure) routes and may induce mutations at A:T base pairs. We were unable to identify a distinctive mutation pattern that was associated with esophageal cancer in the highincidence area. Furthermore, the number of SNVs was almost comparable between the high- and low-incidence areas. These findings suggest that no single characteristic mutation pattern may exist for NPIP and that several mutational processes may operate in the development of esophageal cancer. A previous study conducted in Cixian found that p53 mutations occurred in approximately 50% of patients, with G:C to A:T transitions being the major mutation pattern.37 Interestingly, the same study also showed that the C to T (G to A) transition occurred more frequently in individuals who consumed spicy food.37 Consistent with this, approximately 90% of patients in our study had p53 mutations, and G:C to A:T transitions were also the dominant mutation (Figure S3). In the study presented here, NPIP induced G:C to A:T and A:T to G:C transitions and A:T to C:G transversion, predominantly in gpt delta rats, and similar patterns of mutations were observed in Sig. C, which demonstrated weak correlation with THP-dG levels. Because the mutation signature can be used to detect unknown carcinogenic exposure, as previously illustrated for aristolochic acid (AA),44 further studies are required to match the results of our DNA adductome analysis with genomic analyses. Our study also had several limitations. First, although the detection of THP-dG strongly suggested that past or present exposure to NPIP was related to esophageal cancer development, elucidating its causal role would require the supporting evidence from multidisciplinary studies that examine mutation signatures of NPIP, environmental sources of exposure, and associations between validated biomarkers of exposure and esophageal cancer risk. Second, information about the persistence of THP-dG adducts and the timing of their removal is lacking, which is important for understanding the exposure level and for facilitating the development of a biomarker. There is evidence that the rate of removal of the DNA adduct from human tissues may vary depending on the nature of the exposure and the structure of the DNA adduct;45

Whole-genome/-exome analysis provides a means of capturing the mutational imprints that remain in the cancer genome.38−40 All four signatures that were observed in this study (Sigs. A−D) were highly similar to signatures in the COSMIC database (signatures 1, 5, 13, and 17). Among these, signatures 1 and 13 are thought to be related to the age and overactivity of the APOBEC family of enzymes, respectively, and have generally been observed in various kinds of human cancers [COSMIC database (https://cancer.sanger.ac.uk/ cosmic/signatures)]. The etiology of signatures 5 and 17 is currently unknown, but signature 17, which was highly similar to Sig. C, has been observed in a limited number of cancers, such as esophageal cancer, breast cancer, liver cancer, lung adenocarcinoma, B-cell lymphoma, stomach cancer, and melanoma. Of the four signatures extracted from our human exome data, only Sig. C showed a weak correlation with THPdG levels (r = 0.44; P < 0.05), indicating that NPIP-induced DNA adducts are partly involved in esophageal carcinogenesis. The major DNA adduct produced from NPIP was reported as a guanine adduct; however, NPIP predominantly induced A:T to C:G transversions in the in vivo study using gpt delta rats, which is similar to the findings of a previous study of Nnitrosopyrrolidine (NPYR) (a structurally similar cyclic nitrosamine).41 NPYR is metabolically activated to convert reactive intermediates into exocyclic- and/or alkyl-guanine adducts, resulting in a predominance of mutations at G:C base pairs in vitro.41 However, mutations predominantly occurred at A:T base pairs in vivo, as shown in the study presented here. It has been suggested that this difference between in vitro and in vivo mutagenesis by NPYR could be explained by the formation of minor adenine adducts, such as 1,N6-ethenodeoxyadenosine (εdA), which is produced during lipid peroxidation42 and thus is considered to be a kind of inflammationrelated adduct. It has also been reported that εdA induces εdA → T, εdA → G, and εdA → C mutations in mammalian cells using the translational synthesis approach,43 which is consistent with the results of the study presented here. As mentioned above, another etheno-type adduct, propanoneεdA, can also be formed from α-acetoxyNPIP. Therefore, these etheno-deoxyadenosine adducts could be produced by NPIP J


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Ministry of Education, Culture, Sports, Science, and Technology, Japan, by Research on Global Health Issues (U.S.-Japan Cooperative Medical Sciences Program) from the Japan Agency for Medical Research and Development, AMED (16jk0210009h001), Grant-in-Aid from the Third Term Comprehensive Control Research for Cancer, the Ministry of Health, Labour and Welfare, Japan, by the Japan China Medical Association, and by Princess Takamatsu Cancer Research Fund.

for example, the aristolactam-dA adduct of AA has been shown to persist for more than 9 years in the kidneys of affected individuals.44 Third, no data were available for variation in the metabolic activation of NPIP among individuals. Like other Nnitrosamines, NPIP undergoes simple cytochrome P450mediated metabolic activation before forming adducts (Figure S1).26 Therefore, additional studies that focus on genetic polymorphisms in the P450 pathways would complete our understanding of variations in the risk of esophageal cancer at various levels of NPIP exposure. In summary, our study indicated that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents. Therefore, as a next step, a well-designed longitudinal study with validated biomarkers should be performed to help in determining the precise nature of the relationship between NPIP exposure and esophageal cancer risk.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Mr. Naoaki Uchiya for his excellent technical assistance.

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ABBREVIATIONS AA, Aristolochic acid; BWA, Burrows−Wheeler Aligner; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; ESI, electrospray ionization source; εdA, 1,N6-ethenodeoxyadenosine; εdC, 3,N4-ethenodeoxycytidine; gpt, guanine phosphoribosyltransferase; HεdC, heptanone etheno-deoxycytidine; HPLC, high-performance liquid chromatography; HRAM, highresolution accurate mass; LC−MS/MS, liquid chromatography coupled with tandem mass spectrometry; MGTs, magnetite nanoparticles; MS, mass spectrometry; MSE, multiscale entropy; MRM, multiple-reaction monitoring; NMF, negative matrix factorization; NPIP, N-nitrosopiperidine; NPYR, Nnitrosopyrrolidine; PCA, principal component analysis; SNVs, single-nucleotide variations; THP-dG, N2-(3,4,5,6-tetrahydro2H-pyran-2-yl)deoxyguanosine; 6-TG, 6-thioguanine; 2D, twodimensional; UPLC-QTOF, ultraperformance liquid chromatography-quadrupole time of flight

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00017. Metabolic pathway of NPIP (Figure S1), natural occurrence of piperidine (Figure S2), and mutation patterns occurred in TP53 of esophageal cancer used in this study (Figure S3) (PDF) In-house database of the DNA adducts (Table S1), characteristics of the patients included in this study (Table S2), classification of gpt mutations detected in the liver of control and NPIP-treated rats (Table S3), and somatic SNVs in the targeted exon capture regions of the 25 patients used in this study (Table S4) (XLSX)

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AUTHOR INFORMATION

Corresponding Authors

REFERENCES

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*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yukari Totsuka: 0000-0003-3221-1796 Present Address □

K.I.: Department of Integrated Diagnostic Pathology, Graduate School of Medicine, Nippon Medical School, Tokyo 113-8602, Japan.

Author Contributions

Y. Totsuka, Y.L., and Y.H. contributed equally to this work. Y. Totsuka, Y.L., and Y.H. designed the study. Y. Totsuka, K.I., H.S., F.H., T.M., G.S., F.M., D.L., J.L., Y.Q., W.W., B.S., and Y.M. acquired the data. M.K., M.N., A.E., Y. Totoki, H. Nakamura, and T.S. analyzed and interpreted the data. Y. Totsuka, Y.L., Y.H., Y. Totoki, F.H., T.S., T.M., and Y.M. drafted the manuscript. Y. Totsuka, Y.L., Y.H., M.I., S.K., and H. Nakagama provided critical revisions to the manuscript. M.K., A.E., and Y. Totoki performed the statistical analyses. Y. Totsuka, Y.L., Y.H., and M.I. obtained funding. Funding

This study was supported by grants from the National Natural Scientific Foundation of China (81272682) and the National Natural Scientific Foundation of Hebei Province (C2011206058). This study was also supported by a Grantin-Aid for Scientific Research (B) (25305026) funded by the K


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