Journal of Nuclear Medicine, published on November 9, 2011 as doi:10.2967/jnumed.110.087072 PET of Aromatase in Gastric Parietal Cells Using 11C-Vorozole Makoto Ozawa1, Kayo Takahashi1, Ko-hei Akazawa1, Tadayuki Takashima1, Hiroko Nagata2, Hisashi Doi2, Takamitsu Hosoya3, Yasuhiro Wada1, Yilong Cui4, Yosky Kataoka4, and Yasuyoshi Watanabe1 1Molecular Probe Dynamics Laboratory, RIKEN Center for Molecular Imaging Science, Minatojima minamimachi, Chuo-ku, Kobe, Hyogo, Japan; 2Molecular Imaging Labeling Chemistry Laboratory, RIKEN Center for Molecular Imaging Science, Minatojima minamimachi, Chuo-ku, Kobe, Hyogo, Japan; 3Graduate School of Biomedical Science, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda-ku, Tokyo, Japan; and 4Cellular Function Imaging Laboratory, RIKEN Center for Molecular Imaging Science, Minatojima minamimachi, Chuo-ku, Kobe, Hyogo, Japan Key Words: positron emission tomography; gastric aromatase; Aromatase is a rate-limiting enzyme for estrogen biosynthesis 11C-vorozole; fatigue and has been implicated in pathophysiological states of various J Nucl Med 2011; 52:1–6 diseases via estrogen production. This enzyme is known to be DOI: 10.2967/jnumed.110.087072 widely distributed in extragonadal and gonadal tissues includ- ing the stomach. In contrast to circulating estrogen, the func- tional role of gastric aromatase/estrogen has not been elucidated in detail, because there is no efficient methodology to investigate spatiotemporal changes of gastric aromatase/ estrogen in vivo. Recently, (S)-11C-6-[(4-chlorophenyl)(1H- Aromatase is a key enzyme for the production of sex 1,2,4-triazole-1-yl)methyl]-1-methyl-1H-benzotriazole (11C- steroids and catalyzes the final step of the biosynthetic labeled vorozole), based on a potent nonsteroidal aromatase pathway of estrogens. Aromatase has been reported to inhibitor, has been developed as a tracer to investigate aro- be widely distributed in extragonadal and gonadal tissues matase distribution in living animals and humans using a including the ovaries (1), testis (2), placenta (3), adipose noninvasive PET technique. In the present study, we inves- tissues (4), skin (5), and brain (6). Aromatase is well tigated gastric aromatase expression by means of PET with 11C-vorozole. Methods: After bolus injection of 11C-vorozole known to take part in various important functions, such into the tail vein, emission scans were obtained for 90 min as cell proliferation and differentiation, through estrogen on male and female rats under isoflurane anesthesia. Dis- biosynthesis in an endocrine, autocrine, or paracrine fash- placement studies with unlabeled vorozole and autora- ion (7). Recently, Ueyama et al., using morphological and diographic analysis were conducted for demonstration of biochemical methods, determined that parietal cells in rat specific binding. Immunohistochemistry was performed to gastric mucosa express aromatase and substantially se- confirm aromatase expression. Results: PET scans revealed crete estrogen into the portal vein (8,9). Though gastric that 11C-vorozole highly accumulated in the stomach and adrenal glands. Displacement studies and autoradiography aromatase/estrogen has been implicated in several pathophy- demonstrated that aromatase was expressed in the stomach siological states, such as liver regeneration (10) and femini- but that the accumulation of 11C-vorozole in the adrenal zation (11), by acting as a local regulator of the gastrohepatic glands might be through nonspecific binding. Immunohisto- axis, the functional role of gastric aromatase/estrogen has chemical analysis revealed that aromatase is expressed in still to be elucidated. One of the obstacles to clarification gastric parietal cells but not in adrenal glands. Moreover, of its role is that gastric estrogen is thought to be trapped and the accumulation of 11C-vorozole in the stomach was signifi- cantly increased in fatigued rats. Conclusion: These results metabolized in the liver and rarely overflows into the sys- suggest that the 11C-vorozole PET technique is a useful tool temic circulation (9). Therefore, an adequate method for for evaluation of gastric aromatase dynamics in vivo, which evaluation of the spatiotemporal changes in gastric aroma- may provide important information for understanding the tase/estrogen in vivo is necessary. molecular mechanisms of gastric aromatase/estrogen– Noninvasive molecular imaging techniques, such as PET, related pathophysiological processes and for the develop- provide powerful tools for quantitative investigation of ment of new drugs. the tissue-distribution and dynamic changes of functional molecules in vivo, because of their high sensitivity and spatiotemporal resolution. 11C-labeled vorozole, (S)-11C- Received Dec. 25, 2010; revision accepted Aug. 8, 2011. 6-[(4-chlorophenyl)(1H-1,2,4-triazole-1-yl)methyl]-1-methyl- For correspondence or reprints contact: Yasuyoshi Watanabe, Molecular 1H-benzotriazole (12), was developed as a PET tracer to Probe Dynamics Laboratory, RIKEN Center for Molecular Imaging Science, 6-7-3 Minatojima minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. investigate the in vivo distribution of aromatase in living ani- E-mail: [email protected] 13,14 Published online nnnn. mals and humans ( ). Consistent with previous reports, COPYRIGHT ª 2011 by the Society of Nuclear Medicine, Inc. we have shown that the accumulation of 11C-vorozole was PET OF GASTRIC AROMATASE • Ozawa et al. 1 jnm087072-pm n 11/9/11 Copyright 2011 by Society of Nuclear Medicine. increased in several brain regions of rats and monkeys treated 108 frames). For the displacement studies with the other aroma- with anabolic steroids (15–18), indicating that 11C-vorozole is tase inhibitor, 11C-vorozole was injected into a tail vein as a bolus a useful molecular probe for aromatase imaging. In the present in coadministration with letrozole (1 mg/kg) or vehicle. 11 study, we used C-vorozole PET to evaluate gastric aroma- Radiometabolite Analysis tase under physiological and pathological conditions. Radiometabolite analysis of rat stomach and adrenal gland was performed. At 30 min after intravenous injection of 11C-vorozole, MATERIALS AND METHODS the blood flow was terminated, and then the tissues were quickly removed and homogenized. Sample preparation and chromato- Animals graphic separation was performed according to previously de- Male and female Sprague–Dawley rats, 8–9 wk old and weigh- scribed procedures with slight modification (18). ing approximately 300 g, were purchased from Japan SLC, Inc. During the experiment, body temperature was maintained at 37°C. Autoradiography All experimental protocols were approved by the Ethics Commit- Frozen rat brains (n 5 3), stomachs, and adrenal glands were tee on Animal Care and Use of RIKEN and were performed in sliced into serial coronal sections (25 mm) using a cryostat micro- accordance with the Guide for the Care and Use of Laboratory tome, and the sections were thaw-mounted onto glass slides. Con- Animals (19). secutive sections were preincubated at room temperature (;22°C) for 10 min in phosphate-buffered saline (pH 7.4) and incubated Chemicals with 2 nM 11C-vorozole in the presence or absence of unlabeled 11 (S)- C-6-[(4-chlorophenyl)(1H-1,2,4-triazole-1-yl)methyl]-1- vorozole (1 mM) at room temperature for 30 min. The sections 11 methyl-1H-benzotriazole ( C-vorozole) was synthesized from were washed for 2 min in the buffer 3 times and rapidly air-dried. its N-demethylated derivative via a reaction of N-methylation with The sections were exposed on imaging plates (BAS-SR-2040; 11 11 C-methyl iodide (20). Synthesized C-vorozole was purified Fujifilm) at room temperature for 40 min to generate the auto- using reversed-phase high-performance liquid chromatography. radiograms. FLA-7000 (Fujifilm) was used for scanning the imag- 11 m Specific radioactivity of C-vorozole was 32–49 GBq/ mol, ing plates. and radiochemical purity was greater than 99.5%. The solvent was removed in vacuo, and the residue was dissolved in saline for Immunohistochemistry injection. Injected volume was 0.5 mL. Unlabeled vorozole was a Rats (n 5 3) were perfused with 4% formaldehyde buffered kind gift from the Janssen Research Foundation. Letrozole was with 0.1 M phosphate-buffered saline under deep anesthesia. The purchased from LKT Labs, Inc. stomach and adrenal glands were removed and further fixed in the same fixative at 4°C for 24 h. Frozen sections (10 mm thick) were PET Scans prepared in a cryostat microtome and thaw-mounted on Superfrost The rats (n 5 6) were anesthetized and maintained using a glass slides (Matsunami Glass). The immunohistochemical stain- mixture of 1.5% isoflurane and nitrous oxide/oxygen (7:3) and ing procedure was described in the previous reports (17). After positioned in the PET scanner gantry (microPET Focus220; Sie- immunohistochemical detection, the slides were stained with hema- mens Co., Ltd.). After a bolus injection of 11C-vorozole (injected toxylin and eosin. radioactivity, 95–98 MBq; chemical mass, 0.7–1.5 mg per animal) into the tail vein, a 90-min emission scan was obtained. The Fatigue-Loading Procedure abdominal region was scanned for up to 5 min and sorted into The fatigued condition in rats was induced using the method 60 · 5 s dynamic signograms. Whole-body scans were then previously established by our group (21). Briefly, rats (male, n 5 obtained with 180-s duration for 1 bed pass. Emission data were 4) were transferred to cages filled with water (23°C 6 1°C) to a acquired in the list mode. For whole-body scans, the scanner bed height of 2.2 cm and kept in the water cage for 5 d. The water was was moved continuously in a reciprocating motion to ensure scan- changed once a day. Compared with the control group, rectal ning of the entire rat body, and the data were sorted into dynamic temperatures remained within the normal range in these rats dur- signograms for every 1 bed pass. Emission data were recon- ing fatigue-loading, and no gastric ulcers were observed up to the structed by Fourier rebinning and maximum likelihood expecta- end of fatigue-loading.