ORIGINAL ARTICLE Annals of Nuclear Medicine Vol.9, No.1, 1-5, 1995 Differential@mechanism of retention of Cu-pyruvaldehyde-bis(N4methylthiosemicarbazone) (Cu-PTSM) by brain and tumor: A novel radiopharmaceutical for positron emission tomography imaging Yasuhisa FUJIBAYASHI,** Hideyuki TANIUCHI,* Kouichi WADA,* Yoshiharu YONEKURA,*** Junji KONISHI**** and Akira YOKOYAMA* *Department of Radiopharmaceutical Chemistry, **Department of Genetic Biochemistry, Faculty of Pharmaceutical Sciences, Kyoto University ***Department of Brain Pathophysiology, ****Department of Nuclear Medicine, Kyoto University Hospital The reductive retention of 62Cu-PTSM was comparatively studied in the brain and Ehrlich ascites tumor cells by electron spin resonance spectrometry and nonradioactive Cu-PTSM. In the brain, only the mitochondrial fraction showed the ability to reduce Cu-PTSM, and the other subcellular fractions did not. In contrast, the cytosolic fraction of Ehrlich ascites tumor cells was the specific site of Cu-PTSM reduction. It was therefore considered that the retention of Cu-PTSM in the brain is closely related to mitochondrial reduction, most probably involving the mitochondrial electron transport system. Key words: Cu-PTSM, reduction, metabolism, brain, tumor INTRODUCTION GENERATOR-PRODUCED positron-emitting 62Cu-labeled copper(II)-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) has been proposed as a radiopharmaceutical for positron emission tomography imaging of the brain, heart kidneys and tumors,1,2 but, the mechanism of its retention is still unclear. Green et al. postulated that prolonged tissue retention of copper i s due to the reductive decomposition of Cu-PTSM by reaction with ubiquitous intracellular sulthydryl (SH) groups.3 Their proposal is based on the findings of Petering et al, regarding the reaction of Cu(II)-a-diketone-bis(thiosemicarbazone) (DTS) complexes in Ehrlich ascites tumor cells.4,5 On the other hand, Fujibayashi et al. have reported the selective mitochondrial reduction in Cu-PTSM in the murine brain, presumably by an enzymatic reaction.6 It is therefore likely that there are several mechanisms involved in the retention of Cu-PTSM. In the present study, Cu-PTSM reduction in brain and tumor tissues was investigated. With subcellular fractions as well as tissue homogenates, the metabolism of Cu-PTSM was studied, and the activity of marker enzymes of the fractions and the SH concentration of each fraction were determined. MATERIALS AND METHODS Cu-PTSM was prepared as described previously.6,7 BCA protein assay reagent was obtained from Pierce Ltd. (Illinois, USA). Reagents for enzyme assays and DTNB were purchased from Nacarai Tesque (Japan). DdY male mice weighing 25 g were used for the metabolic studies. These mice were fed a commercial diet and tap water ad libitum. Subcellular fractionation Brain: Subcellular fractionation of the murine brain was performed by the method reported previously.8 Brains were isolated, weighed and homogenized with an isolation medium (1.5g wettissue/ml, 0.25 M sucrose buffered to pH 7.4 with 10 mM HEPES) in a Potter-Elvehjem type homogenizer. Then the homogenate was centrifuged at l,000 x g for 5 min at 4'C. The supernatant (Sl) was removed and the precipitate (PI , crude nuclear fraction) was resuspended in the medium. The S1 fraction was centrifuged at 10,000 x g for 10 min at 4'C, and the supernatant (S2, crude microsomal and soluble fractions) was isolated, and the precipitate was resuspended in the medium (P2, crude mitochondrial fraction). The volumes of the Pl , P2, and S2 fractions were adjusted to the initial of the brain homogenate volume. Ehrlich cells: Ehrlich ascites tumor cells were maintained in male ddY mice and were withdrawn from the abdominal cavity 7 days after inoculation. Subcellular fractionation was done by a modification of the method reported previously.4 Three milliliters of the ascites was centrifuged at 500 x g and the cells were washed twice with the isolation medium. The cell pellet was suspended in 10 ml of a lysis buffer (0.005% sodium dodecyl sulfate buffered to pH 7.4 with 10 mM HEPES). The suspension was homogenized with a Dounce homogenizer, after which the Pl fraction was obtained by centrifugation at l,000 x g for 5 min at 4'C. The supernatant was centrifuged at 8,000 x g for I O min at 4'C to yield the P2 and S2 fractions. The volumes of the Pl and P2 fractions resuspended in the isolation medium and that of the S2 fraction were adjusted to the initial homogenate volume. In vitro metabolic studies One hundred microliters of the Cu-PTSM solution (200 mM in 0.25 M sucrose buffered to pH 7.4 with 10 mM HEPES containing 10% DMSO) was mixed with 900 ml of the homogenate or subcellular fraction, and then the mixture was incubated for 15 min at 37'C. After incubation, 300 ml of the mixture was placed in a sample tube for electron spin resonance spectrometry (ESR) and was frozen in liquid nitrogen so that the ESR signal could be measured at 77'K. Samples without incubation were used as controls. Assays for protein content and marker enzymes Fractions were diluted with 10 mM phosphate buffer (pH 7.5), if necessary, and the protein concentration was measured with BCA Protein Assay Reagent. The activities of succinate dehydrogenase, NADH-dehydrogenase and lactate dehydrogenase were measured as markers of mitochondria,9 microsome10 and cytosol,11 respectively. SH assay Fractions were diluted with 1% SDS, and then the SH concentration was determined by the DTNB method.12 ESR spectrometry The ESR spectrum of Cu-PTSM was obtained with an X-band spectrometer (JES-FE3Xg, Japan Electron Optic Laboratory, Japan) under the following conditions; microwave power, 5mW; modulation amplitude, 6.3 gauss; modulation frequency, 100 kHz; microwave frequency, 9.25 GHz; magnetic field, 3300 +- 500 gauss; and temperature, 77'K. Figure 1 shows a typical ESR spectrum of Cu-PTSM. This spectrum was specific for Cu(II)-PTSM and any Cu(1) complexes were ESR inactive. The strength of the spectrum was used for determination of the Cu(II)PTSM concentration, as described previously.6 RESULTS Tables 1 and 2 show the marker enzyme activities in the subcellular fractions of the brain and Ehrlich ascites tumor cells. Although the reported procedures for subcellular fractionation were different, marker enzymes for the mitochondria, microsome and cytosol were rather similarly distributed. Succinate dehydrogenase, a mitochondrial enzyme, was found mainly in the mitochondrial fractions (ca. 80-90% of the total) of the tissues. In the case of lactate dehydrogenase, a cytosolic enzyme, almost 90% of its activity was found in the cytosolic fractions of Ehrlich ascites tumor cells, but the level fell to only 55% in the brain cytosolic fractions. In both tissues, microsomal NADH-cytochrome C reductase was distributed in both the cytosolic and mitochondrial fractions, but showed slightly higher levels in the cytosol. To obtain a similar 90-95% Cu-PTSM reduction under the conditions shown above, each homogenate was processed as indicated in Materials and Methods. The SH and protein concentrations of the homogenates were determined (Table 3). The SH concentration of brain homogenate was twice that found in the Ehrlich ascites tumor cells ( 1.28 and 0.68 mM, respectively). Accordingly, the reduction abilities normalized for the SH and protein concentrations were completely different, while the relative concentrations of SH (nmole SH/ mg protein) were rather similar. Figure 2 shows the reduction in Cu-PTSM due to the subcellular fractions of the brain tissue and Ehrlich ascites tumor cells. In the brain homogenate, most of the reducing activity was found in the mitochondrial fraction, rather than the nuclear or cytosolic fractions. On the other hand, Ehrlich ascites cells had more than 80% of their reducing activity in the cytosolic fraction. The mitochondrial fraction of the brain tissue had 9-fold higher levels of protein and SH than the same fraction of Ehrlich ascites tumor cells (Table 3). In contrast, the cytosolic SH concentrations of the brain and Ehrlich ascites cells were similar and the levels in the nuclei fraction were slightly lower. All fractions contained stoichiometrically excessive SH concentrations (final: 74 - 615 uM = 82 - 683 uM x 0.9) with regard to the reduction in Cu-PTSM added to the reaction mixture (final concentration: 20 uM). DISCUSSION Copper-DTS complexes were originally developed as anticancer agents and most of the studies on the reactions of these complexes have been performed with tumor cells.4,5,7 These studies have indicated that the reduction in Cu-DTS occurs by interaction with intracellular SH. The present findings in Ehrlich ascites tumor cells agree with this proposal. A large part of the intracellular SH was located in the cytosolic fraction and consequently Cu-PTSM was mostly reduced in the cytosol. Interestingly, no reduction in Cu-PTSM occurred in the mitochondrial fraction, although the SH concentration in the mitochondri a was one fifth of that in the cytosol. The nuclear fraction had a similar SH concentration to the mitochondrial fraction, but had a rather higher reducing ability. With our fractionation method, one tenth of the lactate dehydrogenase activity, a cytosolic enzyme, was found in the nuclear fraction, but little was noted in the mitochondrial fraction. Thus, SH derived from the cytosol contaminated the nuclear fraction to some extent and might have contributed to the reduction in Cu-PTSM. If the reduction data are correlated with the marker enzyme levels, then the reduction in Cu-PTSM attributed to various fractions can be corrected and reassigned as shown in Table 4. This analysis indicates that Cu-PTSM was specifically reduced in the cytosol of Ehrlich ascites cells. On the other hand, brain tissue showed a completely different pattern. The cytosolic fraction of the brain had no ability to reduce Cu-PTSM, although it contained the same SH concentration as that of Ehrlich ascites cells. This indicated that SH can be classified into Cu-PTSM-reducing and Cu-PTSM-nonreducing varieties . The former is found in the cytosol of Ehrlich ascites cells, and the latter is found in the cytosol of the brain. Interestingly, the reduction in Cu-PTSM in the brain occurred mainly in the mitochondrial fraction. This closely matched the subcellular distribution of succinate dehydrogenase, a mitochondrial marker, but not that of NADH-cytochrome c reductase or lactate dehydrogenase. From the corrected analysis shown in Table 4, it can be seen that the reduction in Cu-PTSM in the brain tissue was a mitochondria-specific phenomenon. The high affinity of Cu-PTSM for the mitochondria has been described by Petering et al. and the cytotoxicity of Cu-bisthiosemicarbazone complexes is primarily due to the inhibition of cellular respiration,13 especially at coupling site I between NADH dehydrogenase and coenzyme Q14 in Complex I. From these results, it appears that the reduction in Cu-PTSM in the brain was not caused by ubiquitous SH, but by mitochondriaspecific compound(s) and/or mechanism(s). The present studies were done with non-radioactive Cu-PTSM, but a rather low concentration of Cu-PTSM could be used when compared with the intracellular SH concentration because of the high sensitivity of ESR. The present results could not therefore be affected by carrier Cu, and might be able to be extrapolated to Cu-62 studies. In conclusion, it was shown that the mechanism of Cu-PTSM reduction varied according to the type of tissue, so that data obtained with one tissue cannot be extrapolated to other. 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