Jun Kawada

Enhancement by streptozotocin of O−2 radical generation by the xanthine oxidase system of pancreatic β-cells

Spin‐trapping techniques and electron spin resonance (ESR) spectroscopy were used to study the relationship between the effect of streptozotocin (STZ) on pancreatic β‐cells and free radical formation by these cells. Results showed that STZ enhanced generation of the DMPO‐OH radical adduct, which is a degradation product of the superoxide anion (O− 2) in the presence of cellular components, in a hypoxanthine‐xanthine oxidase (XOD) system with a homogenate of β‐cells. This enhancing effect was also observed in a system without cellular components; STZ increased the signal height due to the O− 2 radical in a concentration‐dependent manner and caused a maximum of 150% enhancement at a concentration of 1.5 mM. Thus, STZ seemed to enhance the generation of the O− 2 radical in the XOD system, probably by some mechanism of its interaction with XOD. Pancreatic β‐cells exhibited a high XOD activity and a very low superoxide dismutase activity. Therefore, the present result supports the possibility that the cytotoxic effect of STZ is closely related to free radical generation in pancreatic β‐cells.

Generation of alloxan free radicals in chemical and biological systems: implication in the diabetogenic action of alloxan.

Electron spin resonance (ESR) studies that on reaction with NADPH, alloxan was reduced forming labile anion radicals giving a 7-line signal with g = 2.005. These radicals were also produced on incubation of alloxan with rat liver subcellular fractions and their production was greatly enhanced by NADPH. Alloxan effectively scavenged superoxide anion generated by a xanthine-xanthine oxidase (XOD) system in association with its reduction to these anion radicals. These radicals were also formed during incubation of alloxan with rat pancreatic beta-cells. These results suggest that the cytotoxicity of alloxan is related to the formation of alloxan anion radicals.

New Diabetogenic Streptozocin Analogue, 3-O-Methyl-2-{[(methylnitrosoamino) carbonyl]amino}-D-glucopyranose: Evidence for a Glucose Recognition Site on Pancreatic B-Cells

The nonmetabolizable glucose analogue 3-O-methylglucose is known to protect pancreatic B-cells against streptozocin (STZ) when injected with or just before STZ. If 3-O-methyl-glucose and the sugar moiety of STZ compete for a glucose recognition site on B-cells, it seemed likely that 3-O-methyl-2-deoxy-2-{[(methylnitrosoamino) carbonyl]amino}-D-glucopyranose, an analogue of STZ with a 3-O-methyl-glucosyl residue, would cause experimental diabetes. This possibility was tested by synthesis of this analogue (α-anomer) and comparison of its diabetogenic activity in Wistar rats with that of STZ. Results showed that the compound was diabetogenic and as potent as STZ. This new analogue is the first of the various STZ derivatives reported to show diabetogenic activity. Its activity supports the idea that 3-O-methyl-glucose and STZ bind competitively with a glucose recognition site on pancreatic B-cells.

Differential Effects of Methylmercuric Chloride and Mercuric Chloride on the Histochemistry of Rat Thyroid Peroxidase and the Thyroid Peroxidase Activity of Isolated Pig Thyroid Cells

This study was designed to characterize the interaction of CH3HgCl or HgCl2 with thyroid peroxidase (TPO). Two types of experiments were performed. First, the thyroids from rats that were given 5.6 mg/kg/day of either CH3HgCl or HgCl2 for 2 weeks by intubation were subjected to histochemical treatment and then to electron microscopy. TPO activities in all cell compartments were inhibited by HgCl2 but not by CH3HgCl. Morphological observation showed that taller epithelia were induced by HgCl2, whereas flattened epithelia forming large follicles were induced by CH3HgCl. The serum thyrotropin level was substantially lowered by CH3HgCl but was unchanged by HgCl2. Second, the guaiacol oxidation by TPO in isolated and ruptured pig thyroid cells was spectrophotometrically monitored in the presence of either CH3HgCl or HgCl2. The TPO was not inhibited by CH3HgCl but was inhibited by HgCl2. These results indicated that CH3HgCl induced a hypothyroid state without affecting TPO, whereas HgCl2 inhibited TPO and induced a hypertropic state owing to compensation for loss of enzyme activity, and that the lack of inhibitory activity of CH3HgCl was not due to the inability to penetrate the cells. Therefore, there appeared to be a differential interaction of organic and inorganic forms of mercurials with the thyroid.

Alteration of manganese distribution in organs of rats treated with streptozotocin

Etude de la distribution du manganese intrinseque et exogene chez le rat normal et chez le rat diabetique induit par la streptozotocine

Hormonal control of manganese transport in the mouse thyroid.

The present study deals with a possible mechanism controlling the transport of manganese (Mn), an essential trace element, from the circulation to the thyroid. Mice were pretreated with propylthiouracil (PTU) or triiodothyronine (T3), and a measurement of the thyroid:serum concentration ratio (T/S) of radioactive manganese (54Mn) was carried out. The T/S of54Mn was greatly enhanced by PTU, but reduced by T3. Several methods were used to demonstrate that the T/S of54Mn depends upon the level of thyroid-stimulating hormone (TSH) in the serum. First, bovine TSH was injected into mice; an increase in the T/S resulted. Secondly, serum thyroxine and T3 levels measured by radioimmunoassay (RIA) suggested that PTU produced an increase in serum TSH and T3 a decrease. However, direct measurement of mouse TSH by RIA for rat TSH failed to produce proof of any changes in TSH level, owing to poor cross-reactivity. Taking all the information into account, it is concluded that Mn-transport into the thyroid is controlled by the thyroid state.

A signoidal relationship between liver stearoyl CoA desaturase activity and serum hormone concentrations caused by streptozocin and its antagonists

Stearoyl CoA desaturase activity in liver microsomes, and insulin, thyroxine, and triiodothyronine levels in serum were measured after administration of streptozocin (STZ) and its antagonists to rats. The effect of STZ, which caused hyperglycemia and inhibited the desaturase activity, was antagonized by 2-desoxyglucose and 3-O-methyl-glucose; 1-O-methyl-3-desoxyglucose and 1-O-methyl-3-O-methylglucose were without any effect. The enzyme activity plotted against insulin levels showed a broad sigmoidal curve, whereas the activities versus thyroid hormone levels showed steeper sigmoidal curves.

Muscle tissue reactions to implantation of bone matrix gelatin

Histologic changes of muscle tissue in the early stage of heterotopic osteogenesis induced by syngeneic insoluble bone matrix gelatin (BMG) with bone morphogenetic protein in rats was observed by light and electron microscopy. BMG induced cartilage in muscle tissue by Day 7 after its implantation, woven bone by Day 10, and lamellar bone with bone marrow by Day 14. The new findings in this work include (1) the disappearance of the basement membrane of muscle fibers; (2) the activation of the satellite cells of muscle fibers; (3) the appearance of fibroblastlike cells that closely resembled activated satellite cells among the degenerated muscle fibers or on the surface of the BMG; and (4) the change of fibroblastlike cells to chondroblasts or osteoblasts. These findings suggest that intramuscular implantation of BMG caused the conspicuous disappearance of the basement membrane of the muscle fiber and may play a part in osteogenesis induced by BMG.

Effect of Bupivacaine on Muscle Tissues and New Bone Formation Induced by Demineralized Bone Matrix Gelatin

Heterotopic bone formation induced by demineralized bone matrix gelatin (BMG) in bupivacaine-HCl-treated skeletal muscle was examined histologically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of male, 4-week-old Sprague-Dawley (SD) rats, cutting it into chips, and demineralizing and extracting the chips with various solutions. The BMG was implanted into the rectus abdominis muscle of male, 5-week-old SD rats, bupivacaine-HCl was injected at the same site, and the resulting plaques of tissues were examined histologically on days 5, 10, 15 and 20 after BMG implantation. Heterotopic bone formation occurred in all animals. The bupivacaine-treated group had more degenerated and injured muscle fibers, and more osteocytes than the control group. Electron microscopy showed that the basement membrane of muscle fibers was discontinuous and that many mononucleated cells resembling activated satellite cells were present on day 5. Many fibroblasts, undifferentiated mesenchymal cells and myogenic cells were seen in the area around the BMG. In new bones there were few osteocytes on day 10, but their numbers were increased on days 15 and 20 after implantation, especially in the bupivacaine-treated group. The population of osteocytes that increased rapidly may have included mononucleated cells similar to activated satellite cells.

Ethylidene glucose-substituted new analogue of streptozotocin cannot induce diabetes: Study on the basis of structure and activity relationship

4,6-O-Ethylidene glucose (ethylidene glucose), a specific inhibitor at the outer surface of a glucose transporter in the cell membranes, substituted analogue of streptozotocin was newly synthesized. This compound did not induce diabetes in rats and also did not show cytotoxic effect on pancreatic beta cells of neonatal rats in a monolayer culture system. The reasons why such a molecule was designed and why it showed no biological effects are discussed on the basis of a structure-activity relationship. Our results afford positive evidence for the presence of a glucose transport system or a glucose transporter on pancreatic beta cells and its involvement in the action of streptozotocin on beta cells.