Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain.

To evaluate the oxidative stress-related parameters and to determine their order of appearance in the brain aging process, radionuclide experiments were carried out on male DBF1 mice at 3, 12, 24 and 30 months of age. The content of nonprotein sulfhydryl compounds, mainly glutathione, was estimated with technetium-99m meso-hexamethyl propyleneamine oxime ([99mTc]meso-HMPAO) tissue sampling. Glucose transport and metabolism was examined with [1-14C]2-deoxy-D-glucose (2-DG) tissue sampling. Mitochondrial electron transport function was estimated with [15O]O2 gas-tissue ARG. [99mTc]Meso-HMPAO uptake in brain expressed as standardized uptake value (SUV), (radioactivity in brain tissue/tissue weight)/(total administered radioactivity/body weight), reached maximum at 12 months of age and decreased at 24 and 30 months of age in every region examined. The pattern of 2-DG, expressed as SUV, showed a tendency to increase rather than decrease with aging. [15O]O2 fixation in brain slices remained constant until 24 months, while it decreased significantly at 30 months of age. The results suggested the possibility of using imaging techniques in vivo for longitudinal evaluation of the aging process and indicated reduction of nonprotein sulfhydryl compounds including GSH at the early stages of aging may also accelerate the dysfunction of mitochondrial electron transport and neurodegeneration.

Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas-tissue autoradiography using.

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas-tissue autoradiography technique with [15O]O2. Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O2 for 30 min at 37 degrees C. (1) Control culture was incubated in the same medium bubbled with 100% O2 for 5-40 min at 37 degrees C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N2 for 5-40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N2 for 5-40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [15O]O2 fixation by brain slices reflect to mitochondrial electron transport function was determined using gas-tissue autoradiography technique with [15O]O2. The fixation of [15O]O2 by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [15O]O2 fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [15O]O2 fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [15O]O2 fixation between ischemia-treated young and senescent brain slices, reduction of 15O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas-tissue autoradiography using [15O]O2 is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.

Usefulness of (99m)Tc-d,l-HMPAO for estimation of GSH content in tumor tissues.

To investigate whether [(99m)Tc]-hexamethyl propyleneamine oxime ([(99m)Tc]-HMPAO) is applicable for evaluating glutathione (GSH) localization in tumor, the difference of distribution between [(99m)Tc]-d,l- and meso-HMPAO was studied using a mouse tumor model. Biodistribution of [(99m)Tc]-d,l- or meso-HMPAO was studied in GSH-depleted and control Ehrlich tumor-bearing mice. GSH levels in tumors in GSH-depleted and control mice were measured in another set of mice. The uptake of [(99m)Tc]-d,l-HMPAO in tumor was significantly decreased by the diethyl maleate (DEM) treatment. On the other hand, the DEM treatment increased the accumulation of [(99m)Tc]-meso-HMPAO in tumor. Meanwhile, the content of GSH was lowest in tumor among the tissues tested and decreased in a manner similar to other tissues on preloading of DEM. [(99m)Tc]-d,l-HMPAO may be useful for estimating the GSH status in a certain tumor and thereby contribute to the diagnosis of anticancer therapy.

A new radiopharmaceutical for the cerebral dopaminergic presynaptic function: 6-radioiodinated L-meta-tyrosine.

Geometric isomers of radioiodinated L-meta-tyrosine, 6-[I-125]iodo-and 4-[1-125]iodo-L-meta-tyrosine (6-I-L-mTyr, 4-I-L-mTyr) were separated by high-performance liquid chromatography . Both 6-I- and 4-I-L-mTyr had high energy-dependent brain accumulation. 6-I- and 4-I-L-mTyr were also metabolically stable and were rapidly excreted through the urine. 6-I-L-mTyr had a predilection for the cerebral aromatic L-amino acid decarboxylase (DOPA decarboxylase), the final enzyme of dopamine biosynthesis. 6-Radioiodinated L-mTyr is a new radiopharmaceutical that can be both useful in assessing cerebral amino acid transport mechanism and quantifying metabolically active DOPA decarboxylase.

Synthesis of [11C] coenzyme Q-related compounds for in vivo estimation of mitochondrial electron transduction and redox state in brain.

We have studied the synthesis of [11C]2,3-dimethoxy-5-methyl-6-(10-hydroxy)-decyl-1,4-benzoquinone (idebenone) and [11C]2,3-dimethoxy-5-methyl-1,4-benzoquinone (CoQo) by methylation of their respective desmethyl precursors using [11C]CH3I for in vivo measurement of mitochondrial electron transfer and redox state. The [11C]idebenone was more lipophilic than [11C]CoQo; the latter became hydrophilic by reduction. Clearance of [11C]idebenone from mouse brain was more rapid than that of [11C]CoQo. The results indicated that modification of the isoprenoid side chain in [11C]CoQ is necessary to develop more suitable radiopharmaceuticals.

Brain uptake of iodinated L-meta-tyrosine, a metabolically stable amino acid derivative.

The possibility of using L-meta-tyrosine (L-mTyr) with high metabolic stability and amino acid transport affinity was evaluated. mTyr was first separated into D- and L-isomers with high-performance liquid chromatography and both were labelled with non-carrier-mediated 125I. Biodistribution and pharmacological studies of radioiodinated mTyr in mice and rats were then performed. 125I-L-mTyr showed greater accumulation in the brain and the pancreas. It accumulated in the brain stereospecifically in the in vivo studies and by the L-tyrosine competitive energy dependent transport system in the in vitro studies. It was resistant to deiodination, appeared to have no retention mechanism and was rapidly excreted. 123I-L-mTyr has the potential of an amino acid transport marker, especially in the brain and the pancreas.

Bridge-heterologous chemiluminescence enzyme-linked immunosorbent assay of estriol 3-sulfate in pregnancy plasma.

A facile and sensitive chemiluminescence enzyme-linked immunosorbent assay (ELISA) of estriol 3-sulfate using a bridge-heterologous system was established. 6 alpha-Hydroxyestriol 3-sulfate 6-hemisuccinate was synthesized as a novel hapten. Antisera were raised in male guinea-pigs against 6 alpha-hydroxyestriol 3-sulfate 6-hemisuccinate-bovine serum albumin (BSA) and 6-oxoestriol 3-sulfate O-carboxymethyloxime-BSA conjugates. Both haptens were coupled to horseradish peroxidase as an enzyme-label reagent. For separation of free and estriol 3-sulfate bound to the antibody, the crude globulin fractions of these antisera were immobilized to CNBr-activated Sepharose-4B. The enzyme activity was measured by chemiluminescent reaction using amino-butylethylisoluminol and hydrogen peroxide as a substrate. The immobilized antibody raised against 6 alpha-hydroxyestriol 3-sulfate 6-hemisuccinate-BSA exhibited a high affinity and an excellent specificity for estriol 3-sulfate. The two bridge-heterologous ELISAs were more sensitive than the homologous systems. The specificity and sensitivity (10 pg) of the bridge-heterologous chemiluminescence ELISAs was comparable to those of the radioimmunoassays (RIAs). Results obtained by the ELISA and the RIA in pregnancy plasmas, showed excellent correlation between ELISA and RIA (r = 0.96).

Localization of glutathione and induction of glutathione synthesis-related proteins in mouse brain by low doses of gamma-rays.

First, we determined the cerebral localization of reduced glutathione (GSH) in normal mice by means of autoradiography using 99mTc-meso-hexamethyl propylene oxime. A highly specific localization of GSH in the cerebellum and hippocampus was observed. Secondly, we measured the elevation of GSH level in the brain after low-dose gamma-irradiation. The cerebral GSH levels increased soon after irradiation with 50 cGy of gamma-rays, reaching a maximum at 3 h post-treatment, then remaining significantly higher than that of the non-irradiated control until 12 h and returning to the control level by 24 h. Thirdly, we examined the induction of the activities and the mRNAs of proteins involved in the synthesis and regeneration of GSH in the brain of mice subjected to low-dose gamma-ray irradiation. The level of mRNA for gamma-glutamylcysteine synthetase was significantly increased at 0.5 h, and remained high until 2 h post-irradiation (50 cGy). The level was transiently lowered to the non-irradiated control level at 3 h and slightly increased again after 6 h post-irradiation. gamma-Glutamylcysteine synthetase activity was significantly increased 3 h after irradiation, and remained high up to 24 h post-irradiation. As for glutathione reductase, the mRNA level was increased at 0.5 h, and peaked strongly at 2 h, while the enzyme activity was significantly increased at 6 h after irradiation, and continued to increase up to 24 h. The level of mRNA for thioredoxin, which contributes to GSH biosynthesis by supplying cysteine to the de novo pathway, peaked between 0.5 h and 2 h post-irradiation, and rapidly declined thereafter. The content of thioredoxin showed a transient decrease immediately after irradiation, but was then remarkably elevated, reaching a maximum at 3 h, and thereafter declining sharply. These results indicate that the increase in endogenous GSH in mouse brain soon after low-dose gamma-ray irradiation is a consequence of the induction of GSH synthesis-related proteins and occurs via both the de novo synthesis and the regeneration pathways.

Induction of mRNAs for glutathione synthesis-related proteins in mouse liver by low doses of gamma-rays.

We examined the elevation of the reduced form of glutathione (GSH)level and the induction of MRNAs for proteins involved in the synthesis and regeneration of GSH in the liver of mice after low-dose gamma-ray irradiation. The liver GSH level increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at around 12 h post-treatment. The mRNA of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for de novo synthesis for GSH, showed a small increase that peaked at 6 h after gamma-ray irradiation at a dose of 50 cGy. Only a small increase in gamma-GCS activity was observed throughout the 24-h post-irradiation period. In the case of glutathione reductase (GR), which is involved in the regeneration of GSH from the oxidized form (GSSG), the mRNA level peaked strongly at 1 h, while the activity peaked at twice the control level 12 h after irradiation. The level of mRNA for thioredoxin (TRX), which contributes to GSH biosynthesis by supplying cysteine to the de novo pathway, peaked at 1 h and declined thereafter, while the activity peaked at 3 h and then declined sharply. These results indicate that the increase in endogenous GSH immediately following low-dose gamma-ray irradiation is predominantly due to operation of the regeneration cycle and not de novo synthesis. We also examined the dependence of mRNA induction on the gamma-ray dose.

Elevation of mouse liver glutathione level by low-dose gamma-ray irradiation and its effect on CCl4-induced liver damage.

We examined the elevation of glutathione (GSH) levels in the liver of C57BL/6 female mice after low-dose r-ray irradiation and its inhibitory effect on CClI4-induced liver damage. The liver GSH level increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at around 12 post-treatment, and returned almost to the control level by 24 h. The activities of glutathione reductase, and glutathione peroxidase also showed the same pattern of change, while the activity of gamma-glutamylcysteine synthetase showed a gradual increase up to 24 h. The effect of pre-irradiation on CCl4-induced liver damage was also investigated. The activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in serum were markedly increased 12 h post-treatment with CCl4. Both increases were significantly suppressed by a single low-dose pre-irradiation. Malondialdehyde, a marker of lipidperoxidation, was also greatly elevated after CCl4 treatment, and its increase was suppressed by irradiation. These results suggest low-dose gamma-ray irradiation might be effective for the prevention of and/or therapy of various reactive oxygen species-related diseases including cancer.

Effects of an inhibitor of protein kinases on the response to heat treatment in cultured mammalian cells.

Effects of H7, a protein kinase C inhibitor, on responses to hyperthermic treatment were investigated in relatively heat sensitive Chinese hamster V79 cells and resistant human glioma A7 cells. In V79 H7 (2-50 microM) enhanced cell killing of heat treatment of 42 or 44 degrees C. The magnitude of the heat sensitization was dependent on concentration and timing of H7 addition; addition of the inhibitor between 0 and 2 h before heat treatment was most effective. In A7 the inhibitor did not show such synergistic effect with heat treatment, but showed mere added toxicity. In split-heat experiments using V79 with addition of H7 (20 microM) before the initial heat treatment and thereon, the development of thermotolerance was partially inhibited. However, already thermotolerant cells were not sensitized when H7 was added before the test heat. In V79 there was a tendency for H7 to accelerate cell death and DNA ladder formation by heat. No significant change was detectable in HSP70 induction determined by Western analyses although H7 seemed to accelerate shifting of HSP70 out of nuclei back into cytoplasm. These results indicate that heat sensitizing effect of H7 may depend on cell type and that the effectiveness of H7 depends on timing of addition.

Purification of endothelin from a conditioned medium of cardiac fibroblastic cells using beating rate assay of myocytes cultured in a serum-free medium.

A conditioned medium from cardiac fibroblastic cells stimulated the beating of quiescent cardiac myocytes cultured in a serum-free medium. The aim of this study was to isolate and characterize the myocyte beat-stimulating activity of the conditioned medium of fibroblastic cells. Cardiac myocytes and fibroblastic cells were isolated individually from neonatal rats. The fibroblastic cells were grown in a growth medium until they became confluent, then serum-free conditioned medium was obtained from them. For the beating-rate assay, the cardiac myocytes were cultured in a completely serum-free medium. The beat-stimulating factor of myocytes in the conditioned medium was purified by reverse-phase liquid chromatographies and gel filtration, and was characterized by measuring the molecular weight of the activity and a pharmacological antagonistic study. The beat-stimulating activity in the conditioned medium was purified into two active fractions. Both of the activities have a molecular weight of 2.5 kDa, and the activities were abolished similarly by FR139317, an endothelin type-A receptor antagonist. These results indicate that cardiac fibroblastic cells secrete endothelin and that this may contribute in part to the functional abnormalities of the heart in patients with myocardial fibrosis.

Proton-donative antioxidant activity of fucoxanthin with 1,1-diphenyl-2-picrylhydrazyl (DPPH).

Reactions between a carotenoid, fucoxanthin and 1,1-diphenyl-2-picrylhydrazyl were investigated both under anoxic and aerobic conditions. Fucoxanthin equimolarly reacted with 1,1-diphenyl-2-picrylhydrazyl under anoxic conditions. Under aerobic conditions, only a part of fucoxanthin consumed 1,1-diphenyl-2-picrylhydrazyl and the degree of reaction fluctuated with repeated trials. beta-Carotene or other carotenoids, beta-cryptoxanthin, zeaxanthin, licopen and lutein, were also examined in the reaction with 1,1-diphenyl-2-picrylhydrazyl under anoxic conditions. All these compounds scarcely reacted with 1,1-diphenyl-2-picrylhydrazyl.

Does small-dose gamma-ray radiation induce endogenous antioxidant potential in vivo?

The induction of in vivo antioxidant potential following small doses of gamma-ray irradiation was investigated in C57BL/6 mice. The antioxidant capacity of various organs was assessed in terms of the scavenging activity of cytosol fractions against 1,1-diphenyl-2-picrylhydrazyl (DPPH), a chemically stable radical. Significant elevations in the scavenging activity were recognized in several organs, including the liver, pancreas and brain, soon after post-irradiation with 50 cGy of gamma-ray. These increases persisted for 24 h. gamma-Radiation of the liver at 25-50 cGy elevated its cytosolic antioxidant capacity, but this was lowered at 200 cGy. In order to assess which antioxidants underlie this phenomenon, the content of a reduced form of glutathione (GSH) in liver was assayed as a function of time after gamma-irradiation at a dose of 50 cGy. The GSH content was significantly increased from 6 h after irradiation, and this change was consistent with that of the total radical scavenging potency of the liver against DPPH. Further, the elevation of GSH content was accompanied by elevated GSSG reductase activity induced by gamma-irradiation.

[An improved automated synthesis and in vivo evaluation of PET radioligand for serotonin re-uptake sites: [11C]McN5652X].

Carbon-11 labeled serotonin (5-HT) re-uptake inhibitor, [11C]McN5652X [(6S, 10bR)-trans-(+)-1,2,3,5,6,10b-hexahydro-6-[4-(methylthio)phenyl] pyrrolo-[2,1-a]-isoquinoline], has recently been reported to be favorable for studying human 5-HT re-uptake site by positron emission tomography (PET) because of its rapid and high specific binding characteristics as radioligands. [11C]McN5652X has been synthesized by S-methylation of the corresponding des-methyl precursor A with [11Cliodemthane. One serious disadvantage of this procedure, however, is the lack of stability of A. The improved method for the synthesis of A has been desired. We have found that the decomposition of A is significantly reduced by adding a protecting agent for SH groups, dithiothreitol (DTT), into the reaction medium immediately after the demethylation of McN5652X. By using this stabilized precursor A, we have developed an automated procedure giving [11C]McN5652X with 98.6 +/- 0.4% radiochemical purity in high specific activity (181.3 +/- 7.4 GBq/mumol). Preclinical evaluation of the product was carried out by injecting the solution of [11C]-McN5652X obtained by this procedure into mice. [11C]McN5652X showed the high accumulation into mouse thalamus, striatum and cerebral cortex, organs known to have high level of 5-HT receptor density, after intravenous injection. Human PET studies also showed the high uptakes of this radioligand into the thalamus, striatum and midbrain.

Synthesis and evaluation of radioiodinated 6-iodo-L-DOPA as a cerebral L-amino acid transport marker.

Regioselective radioiodination of N-trifluoroacetyl 3,4-dimethoxy-6-trifluoroacetoxymercurio-L-phenylalanine ethyl ester 1 under no-carrier-added condition gave 6-[125I]iodo protected L-DOPA with a labeling efficiency of more than 85%, and no-carrier-added 6-[125I]I-L-DOPA was obtained with a radio-chemical purity of over 95% after hydrolysis and chromatography. A nonradioactive standard of 6-iodo protected L-DOPA was synthesized by the iododemercuration of 6-mercuric trifluoroacetate protected L-DOPA 1 using I2 in chloroform. 6-[125I]I-L-DOPA showed high brain accumulation and rapid blood clearance in mice. The rat brain slice studies indicated high affinity of 6-[125I]I-L-DOPA for carrier-mediated and stereoselective active transport systems. The tissue homogenate analysis revealed that most of the accumulated radioactivity was intact 6-[125I]I-L-DOPA. Thus, 6-[123I]I-L-DOPA appears to be a suitable single photon emission computed tomography (SPECT) tracer for the selective measurement of cerebral L-amino acid transport, having no affinity for dopamine metabolism.

Synthesis of polymer-bound 6-mercuric carboxylate DOPA precursors and solid phase labelling method of 6-radioiodinated L-DOPA.

In order to avoid separating unreacted mercury precursor and other mercury-containing compounds after the halodemercuration of a 6-mercury DOPA precursor, we developed a polymer-bound mercury precursor for the preparation of 6-halogenated DOPA. In this study, polymer-bound 6-mercuric carboxylate DOPA derivatives were synthesized from ion-exchange resin and Merrifield-type resin. Iododemercuration of polymer-bound 6-mercuric carboxylate DOPA derivatives gave higher yields (49-54%) compared with monomeric 6-mercuric trifluoroacetate protected DOPA. The radioiodination of the resin with no-carrier added iodine-125 afforded protected 6-[125I]I-L-DOPA with labelling efficiency of 92-97% with both polymer-bound 6-mercuric carboxylate DOPA derivatives.

Specific imaging of hormone-dependent mammary carcinoma in nude mice with [131I]-anti-estriol 3-sulfate antibody.

We tried to put the estrogen metabolite to use in tumor imaging. The antibody against estriol 3-sulfate (E3 3-S), which was one of the major metabolites of estrogen in hormone-dependent mammary carcinoma, was prepared and the tissue distribution and imaging of human breast carcinoma with anti-E3 3-S antibody (Ab) were studied in nude mice. In hormone-dependent breast carcinoma, MCF-7,-bearing nude mice, [125I] anti-E3 3-S Ab localized in tumor with the percentage injected dose/g of 9.29 +/- 3.01 (mean +/- SD). This value was significantly high compared with that in hormone-independent breast carcinoma, MDA-MB-231,-bearing nude mice. At 72 h after the administration of [125I]anti-E3 3-S Ab to MCF-7 bearing mice, tumor/blood, tumor/liver and tumor/muscle ratios were 0.49, 5.02 and 6.83, respectively. These ratios were supposed to be enough for imaging. In radioimmunoscintigraphy, a MCF-7 tumor was clearly visualized at 120 or 168 h post-injection of [131I]anti-E3 3-S Ab.

Canine SPECT studies for cerebral amino acid transport by means of 123I-3-iodo-alpha-methyl-L-tyrosine and preliminary kinetic analysis.

We have already reported that 123I-3-iodo-alpha-methyl-L-tyrosine (123I-L-AMT) is superior as a single-photon emitter labeled radiopharmaceutical reflecting cerebral amino acid transport. In this study, we investigated the distribution of 123I-L-AMT in the canine head by means of SPECT and kinetically analyzed the data in the brain. As a result, clear SPECT images of the canine brain were obtained. Kinetic analysis with a 2-compartment model, including or expressing membrane transport of the amino acid, was performed with time-activity curves in the arterial blood and in the cerebral region. The results of the analysis coincided closely with the experimental data and the relevance of the model was strongly suggested. Therefore 123I-L-AMT is considered to be useful as a single photon radiopharmaceutical which enables us to measure the cerebral amino acid transport rate.

Synthesis and preclinical evaluation of technetium-99m-labeled hippurate analogues.

We have synthesized seven 99mTc-labeled hippurate analogues: 99mTc-hippurate. 99mTc-alpha-hydroxyhippurate, 99mTc-m-hydroxyhippurate. 99mTc-o-hydroxyhippurate [99m-salicylglycine (99mTc-SG)], 99mTc-p-hydroxyhippurate, 99mTc-salicylglycylglycine and 99mTc-salicylglycylglycylglycine. All of the 99mTc-hippurates were cleared rapidly from the rat blood and accumulated in the kidney. Of them 99mTc-SG has the desirable biological properties of two diagnostic agents. 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) and 99mTc-dimercaptosuccinate (99mTc-DMSA). A fraction of 99mTc-SG showed a transit time in the kidney and was excreted rapidly into the urine, being similar to 99mTc-MAG3. The binding ratio to the plasma proteins was 96.0% (91.1% in the albumin), being higher than that of 99mTc-DMSA, at 30 min. The lipophilicity revealed far less pH-dependent changes in a range of pH 4.0 to 7.4. 99mTc-SG distributed about 91% in the renal cortex, being similar to that of 99mTc-DMSA. From the present studies, the biological properties of 99mTc-SG suggest that it is a promising agent for measuring renal plasma flow and renal morphology.