Akiko Kubodera

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. β‐Carotene or other carotenoids, β‐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.

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

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

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.

Elevated uptake of 67Ga and increased heparan sulfate content in liver-damaged rats

Values of percent 67Ga binding with standard acid mucopolysaccharides (AMPS) were determined. The binding percent of 67Ga with heparan sulfate (HS) was especially high. The relation between 67Ga and HS content was investigated in liver-damaged rats. Elevated uptake of 67Ga occurred in two stages, corresponding to the early and the later stages during feeding with 2-acetylaminofluorene (AAF). Uptake of 67Ga in the liver was first observed at 5–6 weeks after the start of AAF feeding. There was a decrease at about week 7 almost to the control level, followed by a sustained increase. This changing pattern of 67Ga uptake was in good accord with that of liver HS content. A similar relation between 67Ga uptake and HS content was also observed in rat liver treated with a single dose of carbon tetrachloride (CCl4). The elevated 67Ga uptake and increased HS content induced by CCl4 were both significantly inhibited by aminoacetonitrile (AAN) or papain pretreatment.Moreover, we screened cations having a high affinity with HS from the exchangeability of 45Ca with those in 45Ca-HS complex. Fe3+ and other trivalent cations such as Al3+, La3+, Ce3+, Er3+, Sm3+, Yb3+, In3+, Ru3+, and Eu3+, and Le2+ represented strong affinity to HS. These cations also effectively decreased the 67Ga tissue distribution in normal rats. These results suggest that HS may be involved in the mechanism of 67Ga uptake in tumor and inflammatory tissues.

An artificial amino acid radiopharmaceutical for single photon emission computed tomographic study of pancreatic amino acid transports123I-3-iodo-alpha-methyl-L-tyrosine

Abstract123I-3-iodo-alpha-methyl-L-tyrosine (123I-L-AMT) was selected and its characteristics on pancreas accumulation, metabolic selectivity and metabolic stability of125I-L-AMT were studied. The studies on rat tissue slice as well as mouse biodistribution proved very high accumulation of125I-labeled L-AMT in the pancreas, which was remarkably inhibited by the active transport inhibitor, ouabain.125I-L-AMT does not enter into protein synthesis and general amino acid catabolism. Moreover,125I-L-AMT was very stable against enzymatic deiodination. Thus, the above studies indicated that the123I-labeled L-AMT was an “artificial amino acid” radiopharmaceutical to be used for the selective measurement of the membrane amino acid transport rate in the pancreas.

Development of an automated system for the quick production of 13N-labeled compounds with high specific activity using anhydrous [13N]NH3

Abstract An automated system was developed to synthesize 13 N-labeled compounds with high specific activity using anhydrous [ 13 N]NH 3 as a synthetic precursor. This system enabled (1) production of an aqueous solution of [ 13 N]NH 3 , (2) concentration and desiccation of the [ 13 N]NH 3 solution, (3) reaction of anhydrous [ 13 N]NH 3 with substrate, (4) purification and formulation. By the use of this system, [ 13 N]p-nitrophenyl carbamate ([ 13 N]NPC) ready for i.v. injection could be obtained in 5.1±0.1 min at the yield of 3.5±0.4 GBq, specific activity 460±55 GBq/μmol, and radiochemical purity >99% ( n =3) by irradiating a 10 mM ethanol solution with 18 MeV protons at 17 μA for 25 min. With special precautions, [ 13 N]NPC could be obtained at the extremely high specific activity of 1800±200 GBq/μmol.

67Ga accumulation and heparan sulfate metabolism in lysosomes

Abstract67Ga incorporation in the mitochondrial-lysosomal (Mt-Ly) fraction of rat liver increased sharply from 1 to 24 h after 67Ga IV injection, whereas that in the 105,000 g supernatant fraction decreased sharply from 6 to 48 h. Further, when 14C-labeled heparan sulfate (HS: biosynthesized by injection of [1-14C] glucosamine into a rat) was injected intravenously into a rat treated with Triton WR-1339, and the liver was isolated and fractionated, the radioactivity of 14C was concentrated in the lysosomal fraction.The effect of HS on 67Ga uptake in normal rat liver was also studied. On administration of 67Ga-HS complex, the peak uptake of administered radioactivity of 67Ga in rat liver occurred sooner than after the injection of 67Ga alone. The change was greatest in the Mt-Ly fraction among the subcellular fractions. Moreover, the 67Ga uptake in rat liver increased with increasing doses of HS.These results suggest that administered 67Ga may be bound to HS and that the concentration of 67Ga in lysosomes may be related to the in vivo metabolism of HS.

Renal accumulation and excretion of radioiodinated 3-iodo-α-methyl-L-ty rosine

Objective: We investigated mechanisms of renal accumulation of radioiodinated 3-iodo-α-methyl-L-tyrosine (IMT), which has been used clinically for tumor imaging and as an amino acid transport marker in studies of brain and pancreas function.Methods: In this study, we used125I- or123I-labeled IMT ([125I]IMT or [123I]IMT) as the transport marker. Partition coefficients of [125I]IMT were determined for hypothetic urine at pH ranging from 5 to 8. The examination of uptake and inhibition of [125I]IMT was performed using normal human renal proximal tubule epithelial cells (RPTEC), which are characteristic of the proximal convoluted tubule. The plasma protein binding ratio of [125I]IMT was determined using rats. In thein vivo experiments using mice, we examined biodistribution and excretion inhibition, and performed whole body autoradiography. Also, renal SPECT using [123I]IMT was performed using a normal canine.Results: Very low lipophilicity of [125I]IMT in hypothetic urine suggests that a carrier-mediated pathway contributes to its marked kidney accumulation. [125I]IMT uptake into RPTEC was significantly inhibited by 2-amino-bicyclo[2,2,l]heptane-2-carboxylic acid (BCH) in a sodium-dependent manner, suggesting reab-sorption mainly via system B° in apical membrane of proximal tubule. Plasma protein binding ratio of IMT was 45.4 ± 5.6%. At 6 hr after administration of IMT to mice, excretion via urinary tract was 77.51% of injected dose, and excretion into feces was 0.25%. Furosemide, ethacrynic acid and probenecid inhibited tubular secretion of [125I]IMT in mice. We obtained very clear autoradio-graphs of mouse renal cortex and a canine renal SPECT image (S2-like region).Conclusions: We believe that [123I]IMT is useful for kidney imaging. In future studies, we plan to examine the use of [123I]IMT in diagnosis of disease.

Usefulness of 99mTc-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.