Yoshitaka Matsushima

Synthesis and evaluation of new imaging agent for central nicotinic acetylcholine receptor α7 subtype.

INTRODUCTION The nicotinic acetylcholine receptor (nAChR) alpha7 subtype (alpha(7) nAChR) is one of the major nAChR subtypes in the brain. We synthesized C-11 labeled alpha(7) nAChR ligands, (R)-2-[(11)C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([(11)C](R)-MeQAA) and its isomer (S)-[(11)C]MeQAA, for in vivo investigation with positron emission tomography (PET). Then, the potential of (R)- and (S)-[(11)C]MeQAA for in vivo imaging of alpha(7) nAChR in the brain was evaluated in mice and monkeys. METHODS The binding affinity for alpha(7) nAChR was measured using rat brain. Biodistribution and in vivo receptor blocking studies were undertaken in mice. Dynamic PET scans were performed in conscious monkeys. RESULTS The affinity for alpha(7) nAChR was 41 and 182 nM for (R)- and (S)-MeQAA, respectively. The initial uptake in the mouse brain was high ([(11)C](R)-MeQAA: 7.68 and [(11)C](S)-MeQAA: 6.65 %dose/g at 5 min). The clearance of [(11)C](R)-MeQAA was slow in the hippocampus (alpha(7) nAChR-rich region) but was rapid in the cerebellum (alpha(7) nAChR-poor region). On the other hand, the clearance was fast for [(11)C](S)-MeQAA in all regions. The brain uptake of [(11)C](R)-MeQAA was decreased by methyllycaconitine (alpha(7) nAChR antagonist) treatment. In monkeys, alpha(7) nAChRs were highly distributed in the thalamus and cortex but poorly distributed in the cerebellum. The high accumulation was observed in the cortex and thalamus for [(11)C](R)-MeQAA, while the uptake was rather homogeneous for [(11)C](S)-MeQAA. CONCLUSIONS [(11)C](R)-MeQAA was successfully synthesized and showed high uptake to the brain. However, since the in vivo selectivity for alpha(7) nAChR was not enough, further PET kinetic analysis or structure optimization is needed for specific visualization of brain alpha(7) nAChRs in vivo.

Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer

BackgroundThe rediscovery of 5-hydroxymethylcytosine, the ten-eleven translocation (TET) family, thymine-DNA glycosylase (TDG) and isocitrate dehydrogenase (IDH) have opened new avenues in the study of DNA demethylation pathways in gastric cancer (GC). We performed a comprehensive and robust analysis of these genes and modified cytosines in gastric cancer.MethodsLiquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was used to assess 5-methyldeoxycytidine (5-mC), 5-hydroxymethyldeoxycytidine (5-hmC), 5-formyldeoxycytidine (5-fC) and 5-carboxyldeoxycytidine (5-caC) quantitatively in tumorous and non-tumorous regions of GCs; [D2]-5-hmC was used as an internal standard. Expression levels of the genes TET1, TET2, TET3, TDG, IDH1 and IDH2 were measured using a real-time reverse transcription polymerase chain reaction (RT-PCR) and were compared to the clinical attributes of each case. Using HEK293T cells the effects of introducing plasmids containing full-length TET1,TET2, and TET3 and 7 variants of the TET2 catalytic domain were evaluated in terms of their effect on cytosine demethylation.ResultsLC-MS/MS showed that 5-hmC was significantly decreased in tumorous portions. 5-mC was also moderately decreased in tumors, while 5-fC and 5-caC were barely detectable. The expressions of TET1, TET2, TET3, TDG and IDH2, but not IDH1, were notably decreased in GCs, compared with the adjacent non-tumor portion. TET1 expression and the 5-hmC levels determined using LC-MS/MS had a significantly positive correlation and TET1 protein had a greater effect on the increase in 5-hmC than TET2 and TET3 in HEK293T cells.ConclusionsThe loss of 5-hmC and the down-regulation of TET1-3, TDG and IDH2 were found in GCs. The loss of 5-hmC in GCs was mainly correlated with the down-regulation of TET1.

Human DNA glycosylase enzyme TDG repairs thymine mispaired with exocyclic etheno-DNA adducts

Lipid peroxidation directly reacts with DNA and produces various exocyclic etheno-base DNA adducts, some of which are considered to contribute to carcinogenesis. However, the system for repairing them in humans is largely unknown. We hypothesized that etheno-DNA adducts are repaired by base excision repair initiated by DNA glycosylase. To test this hypothesis, we examined the activities of the DNA glycosylase proteins OGG1, SMUG1, TDG, NEIL1, MUTYH, NTH1, MPG, and UNG2 against double-stranded oligonucleotides containing 1,N(6)-ethenoadenine (εA), 3,N(4)-ethenocytosine (εC), butanone-ethenocytosine (BεC), butanone-ethenoguanine (BεG), heptanone-ethenocytosine (HεC), or heptanone-ethenoguanine (HεG) using a DNA cleavage assay. We found that TDG is capable of removing thymine that has mispaired with εC, BεC, BεG, HεC, or HεG in vitro. We next examined the effect of TDG against etheno-DNA adducts in human cells. TDG-knockdown cells exhibited the following characteristics: (a) higher resistance to cell death caused by the induction of etheno-DNA adducts; (b) lower repair activity for εC; and (c) a modest acceleration of mutations caused by εC, compared with the rate in control cells. All these characteristics suggest that TDG exerts a repair activity against etheno-DNA adducts in human cells. These results suggest that TDG has novel repair activities toward etheno-DNA adducts.

Influence of GSH S-transferase on the mutagenicity induced by dichloromethane and 1,2-dichloropropane

It has been suggested that dichloromethane (DCM) and 1,2-dichloropropane (DCP) are responsible for occupational cholangiocarcinoma. Dihaloalkanes are metabolically activated by GSH S-transferase theta1 (GSTT1) to yield products such as episulfonium ions. However, whether the GSTT1-mediated step of these dihaloalkanes is related to occupational cholangiocarcinoma is not known. In the present study, we investigated the influence of GSTT1 activation on the mutagenicity of DCM and 1,2-DCP using GSTT1-expressing Salmonella typhimurium TA100 (TA100-GST). Since the mutagenicity of DCM was significantly increased in TA100-GST compared with mock control (TA100-pCTC), GSTT1 is thought to be involved in the mutagenicity of DCM. Mutation spectrum analysis on the hisG gene revealed that C:G to A:T transversions were the predominant form observed in DCM-treated TA100-pCTC. However, C:G to T:A transitions were dramatically increased in TA100-GST. We also analysed the DCM-DNA adduct, N2-GSH-Me-dG, and formation of N2-GSH-Me-dG was increased in TA100-GST compared with TA100-pCTC. On the other hand, 1,2-DCP did not increase the numbers of revertants in TA100-GSTT1. In mutation spectrum analysis, C:G to T:A transitions was predominant in both TA100-pCTC and TA100-GSTT1. These findings suggest that GSTT1 has little involvement in DCP mutagenicity, and other mechanisms might be more important for bioactivation and consequent genotoxicity. Clarification of the mechanisms underlying the development of DCM- and/or 1,2-DCP-related human cholangiocarcinoma may help establish risk assessment and prevention strategies against occupational cancer.

Abstract 4619: DNA adductome analyses at multiple sites of human gastric mucosa, resected for gastric cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA DNA adducts are recognized as an initiative step of mutagenesis, but the measurement of them is still a technical challenge. So-called adductome approach, based on LC-MS/MS, is a popular procedure to identify multiple DNA adducts in various tissues at a single experiments. Recently we reported 7 lipid peroxidation-induced DNA adducts in human gastric mucosa resected for gastric cancer in two countries for the first time, and showed the profile of these DNA adducts could indicate geo-pathological background of gastric mucosa (Matsuda 2013). Though DNA adductome approach posesses the high throghput potential in terms of finding numerous specis of DNA adducts having different molecular weights, there are still challenges to overcome. First of all, the numbers of the standards of DNA adducts molecules available are less than the species with identified molecular weight. This implies there are much more DNA modifications in human tissues than previously characterized. The other challaenge is that the absolute quantifications of these adducts requires isotopic standard chemicals. In this study, we strengthened DNA adductome method by including as many as known adducts in the assay system. Mass/Charge value and liquid chromatography retention time of known 254 DNA addcuts were collected and incoporated into LC-MS/MS machine (4000-QTRAP; Thermo). By this procedure we could measure the 254 DNA adducts at one time in MRM mode. Using this system, we measured 254 DNA adducts in the following human gastric mucosa. Written informed consents were obtained from the patients who took gastrectomy for gastric cancer. Under the hypothesis of field cancerization of gastric mucosa, in order to analyze the spatial influence of the exposure of the potential carcinogens toward cancer site, muocsal tissue at multiple sites with different distance to cancer, (10cm and more distant from the tumor site in the stomach to the site adjacent to the tumor) were taken. Three to 10 sites/case were investigated for DNA adducts measurements. Totally 11 cases and 77 sites are analyzed. Considerable numbers of known DNA adducts were present in human gastric mucosa in different quantities. Among them, tobacco-related and lipid-peroxidation-induced DNA adducts were noted. We also did the same measurements in proximal, middle, and distal gastric mucosa from autospy cases without gastric cancer and compared them to those of the cancer cases. Among them, etheno-dA was able to be quantitatively measured using isotopic standard. The mean level of etheno dA in the gastric mucosa of autopsy cases without gastric cancer was 8.02/107 nucleotides, while that of gastric cancer patients mucosae was 4.80/107 nucleotides. These comprehensive identifications in adducts in human gastric mucosa will provide the basic information for dosimetry and risk assessment of environmental chemicals toward gastric cancer. Citation Format: Nobuya Kurabe, Ippei Ohnishi, Masako Suzuki, Yusuke Inoue, Tomoaki Kahyo, Moriya Iwaizumi, Yoshitaka Matsushima, Yukari Totsuka, Hitoshi Nakagama, Masako Kasami, Hideto Ochiai, Keigo Matsumoto, Shioto Suzuki, Fumihiko Tanioka, Haruhiko Sugimura. DNA adductome analyses at multiple sites of human gastric mucosa, resected for gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2015-4619

Abstract 47: DNA adductome: An ultimate exposome of human tissues.

Abstract The formation of DNA adducts and the subsequent generation of mutations in the genome is believed to be a pivotal event in the pathogenesis of cancer, but several obstacles have hampered a concrete demonstration of the molecular species, quantity, and significance of DNA adducts in human tissues. For the last few years, we have attempted to demonstrate the existence of multiple DNA adducts in human tissues using a single experiment involving liquid chromatography tandem mass spectrometry. This procedure discloses a hundred to a few hundred peaks with distinct mass/charge and retention times in each sample. In previous experiments, we validated the presence of some tobacco-related and other DNA adducts in the lungs of a smoker and a non-smoker. Recently, we demonstrated the presence of lipid peroxidation-induced DNA adducts in several autopsy tissues including the lung, liver, pancreas, and spleen. In this presentation, we would like to show an adductome map for two regions of the human stomach in which the prevalence of gastric cancer differs. The DNA samples were obtained under anti-oxidation conditions from the gastric mucosa from regions adjacent to, but distant from, the cancer lesion in 22 patients with gastric cancer (12 cases in China and 10 cases in Japan) who underwent a gastrectomy. We identified 141 peaks in the Japanese sample and 159 peaks in the Chinese sample. Ninety-two of these peaks were present in both the Japanese and Chinese samples. Seven lipid peroxidation-related DNA adducts (1,N6-etheno-2′-deoxyadenosine [ϵdA], butanone-etheno-2′-deoxycytidine [BϵdC], butanone-etheno-2′-deoxy-5-methylcytidine [BϵmedC], butanone-etheno-2′-deoxyadenosine [BϵdA], heptanone-etheno-2′-deoxycytidine [HϵdC], heptanone-etheno-2′-deoxyadenosine [HϵdA], and heptanone-etheno-2′-deoxyguanosine [HϵdG]) were identified in a total of 22 gastric mucosa samples. The levels of these adducts ranged from 0 to 30,000 per 109 bases, and the levels of BedC, BedA, and HedA were higher in the Japanese gastric mucosa samples. Next, the mutation spectrum for p53 in gastric cancer tissues from the same hospital in China was investigated and compared with that of gastric cancer tissues obtained in Japan. The prevalence of a G-to-A transition in a non-GC area was higher in those derived from China; that is, G-to-A transitions in a GC rich area, which has been assumed to be related to endogenous inflammation, were more prevalent in the Japanese gastric cancer specimens. We interpreted these results as indicating that inflammation can mediate carcinogenesis via lipid peroxidation-induced adducts, and the mutation process that may consequently arise may play a greater role in Japanese gastric cancers than in Chinese gastric cancers. To delve into the mechanism of the cellular reactions to these adducts, oligonucleotides containing ϵdA, ϵdC, BϵdC, BϵdG, HϵdC, and HϵdG were synthesized and used as substrates for a gel retardation assay using the recombinant proteins of 8 different base excision repair genes. In contrast to their considerable capacity to repair smaller modified bases, such as ϵdA and ϵdC, the base excision repair gene products seemed to be less capable of repairing the larger modified bases BϵdC, BϵdG, HϵdC, and HϵdG, except for a few combinations. While a comprehensive understanding of the modifications of DNA bases, both endogenous and exogenous, and the corresponding host machineries might seem like a distant goal and an adductome approach is admittedly at a burgeoning stage, combing every un-annotated spot in an adductome map could help to realize a comprehensive understanding of the effects of exposures of various kinds. Citation Format: Haruhiko Sugimura, Hong Tao, Nobuya Kurabe, Masanori Goto, Yoshitaka Matsushima, Hidetaka Yamada, Kazuya Shinmura, Yohei Miyagi, Akira Tsuburaya, Takaki Yoshikawa, Yukari Totsuka, Hitoshi Nakagama, Yaping Wang, Tomonari Matsuda. DNA adductome: An ultimate exposome of human tissues. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 47.