Kiyoshi Terao

Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method

In 1987 a cyanobacterium (blue-green alga) Umezakia natans was isolated from Lake Mikata, Fukui, Japan, as a new member of the family of Stigonemataceae. The crude extract of U. natans showed hepatotoxicity to mice, from which a toxic compound was isolated. The toxin was identical in all respects to a recently reported hepatotoxin, cylindrospermopsin, isolated from an Australian tropical cyanobacterium Cylindrospermopsis raciborskii. Because cylindrospermopsin causes fatty liver and central necroses in mice and is suspected of being an agent causing human hepatoenteritis, its monitoring in drinking water supplies has been required. So a rapid screening method including four steps, extraction, clean-up, separation, and determination, has been proposed for cylindrospermopsin. A combination of a clean-up using HP-20 and C18-cartridge, and HPLC with photodiode array detector made it possible to establish a screening method for the toxin. The established method was applied to five samples and cylindrospermopsin was traced in one of them.

Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue-green alga Umezakia natans.

The effects of cylindrospermopsin isolated from a blue-green alga Umezakia natans on mice were examined morphologically and biochemically. The main target of the phycotoxin was the liver. The thymus, kidneys and heart were also affected. There were four consecutive phases of the pathological changes in the liver. The initial phase was that of inhibition of the protein synthesis, the second phase of membrane proliferation followed, and then the third phase of fat droplet accumulation and finally the phase of cell death. Using globin synthesis in the rabbit reticulocytes system, it was clearly demonstrated that cylindrospermopsin is a potent inhibitor of the protein synthesis. Protein in microsomes from the mouse livers treated by cylindrospermopsin decreased in amount more significantly than that of phospholipid in microsomes. Furthermore, the amount of total P450 was extensively diminished in the toxin treated with hepatic microsomes.

Neoplastic nodular formation in mouse liver induced by repeated intraperitoneal injections of microcystin-LR

Neoplastic nodules were observed in mice liver treated with microcystin-LR (MCLR) by the intraperitoneal (i.p.) route over 28 weeks. After 100 i.p. injections of a sublethal dose (20 micrograms/kg) of MCLR, neoplastic nodules were observed without the use of an initiator. Multiple neoplastic nodules up to 5 mm in diameter were observed in the liver of mice in both groups, i.e. those injected 100 times i.p. and those injected 100 times with a 2 month withdrawal. The cysteine conjugate of MCLR was detected mainly in the affected livers. In contrast, when 80 micrograms/kg was orally administered 100 times, characteristic chronic injuries such as fibrous changes and nodule formation were not observed.

Histopathological studies on experimental marine toxin poisoning--5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative.

The histopathological response of male ICR mice to yessotoxin, isolated from the digestive organ of scallops Patinopecten yessoensis, was compared with that of desulfated yessotoxin. The target organ of the former was the heart and those of the latter were the liver and pancreas. Electron microscopically, marked intracytoplasmic edema in cardiac muscle cells was seen within 3 hr after the i.p. injection of over 300 micrograms/kg of yessotoxin. In contrast, desulfated yessotoxin at the same dose caused within 24 hr of i.p. injection severe fatty degeneration and intracellular necrosis in the liver and pancreas but not in the heart. Biochemically, the content of triglycerides in the liver of mice treated with desulfated yessotoxin increased about 60 times, and phospholipids two-fold more than the control levels of those of mice treated with yessotoxin.

Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1

Sequential ultrastructural changes were studied in mouse digestive organs after i.p. injections of dinophysistoxin-1 and pectenotoxin-1, causative agents of diarrhetic shellfish poisoning. Dinophysistoxin-1, a diarrheagenic substance, produced severe mucosal injuries in the small intestine within 1 hr after the administration of the toxin. The injuries were divided into 3 consecutive stages: extravasation of villi vessels, degeneration of absorptive epithelium and desquamation of the degenerated epithelium from the lamina propria. In contrast to dinophysistoxin-1, pectenotoxin-1, a non-diarrheagenic toxin from diarrhetic shellfish poisoning causative mussels, resulted in no abnormalities in the small intestine, but did cause characteristic liver injuries. Within 1 hr after the injection of pectenotoxin-1 numerous non-fatty vacuoles appeared in the hepatocytes around the periportal regions of the hepatic lobules. Electron microscopic observations with colloidal iron demonstrated that these vacuoles originated from invaginated plasma membranes of the hepatocytes.

Effects of intra-arterially infused biodegradable microspheres containing mitomycin C

We prepared biodegradable microspheres containing about 5% mitomycin C (MMC) and of 45 ± 8 μm in diameter. These preparations were infused into the rat hepatic artery as a preclinical model of intra‐arterial infusion treatment for patients with inoperable hepatic tumor. The leaked MMC levels in the hepatic vein decreased below the assay limitation 2 hours after conventional MMC injection, whereas in the case of MMC microsphere the leaked drug levels were maintained at almost the same concentration for over 2 hours after infusion. The entrapped period of MMC microspheres within the hepatic artery was at least 2 weeks, and the necrobiotic foci due to antitumor effects of the condensed MMC released from the microspheres were observed in the area fed by these entrapped arterioles. This phenomenon was never observed in the case of conventional MMC and placebo microspheres. Intra‐arterial infusion of MMC microspheres may be a promising clinical treatment for patients with malignant hepatic tumor.

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.

Light and electron microscopic studies of pathologic changes induced in mice by ciguatoxin poisoning

Acute poisoning induced by ciguatoxin or ciguatoxin-4c in male ICR mice was examined by light and electron microscopy. Target organs were the heart, medulla of adrenal glands, autonomic nerves and penis. There were no significant differences between the toxicity of ciguatoxin and ciguatoxin-4c. Either i.p. injection or oral administration (0.7 micrograms/kg) resulted in marked swelling and focal necrosis of cardiac muscle cells and effusion into the interstitial space of the heart. Degeneration of cells in the medulla of the adrenal glands was also observed. Continuous erection of the penis was observed in about 15% of the mice suffering from ciguatoxicosis. Although severe diarrhea was brought about by the administration of these phycotoxins, no morphological alterations were seen in the mucosa and muscle layers of the small intestine except in autonomic nerve fibers and synapses. Atropine suppressed the symptoms of diarrhea but had no effect on the injury to the cardiac muscle. Reserpine aggravated the clinical signs and pathological findings. Guanethidine and 5-hydroxy dopamine as well as those undergoing bilateral adrenalectomy had no significant effects on the ciguatoxicosis.

Sterigmatocystin-a Masked Potent Carcinogenic Mycotoxin

AbstractSterigmatocystin (stg), a related compound of aflatoxin B1 (af B1) is a secondary metabolite of a wide variety of fungi species.Stg is a compound in which a substituted anthraquinone is fused to the bisdihydrofuran ring. An unsaturated 2, 3 bond in the bis-dihydrofuran ring is closely related to the biological activities of stg. Activated stg is covalently bound with DNA at the N7-−guanyl position in the target cells. Acute toxicity of stg is infact, rather weak. The lower toxicity in in vivo systems may be due to the poor absorption rate of stg from the digestive tracts. The data obtained from in vitro systems show that stg is as toxic as af El when the inycotoxin contacts directly with the cells. In spite of its low acute toxicity, stg shows a potent mutagenicity as well as carcinogenicity. As for its carcinogenicity, the target organ of stg in rats is the liver, and those in mice are the lung and blood capillaries. Stg induced malignant tumors not only in the target organs, but also at the site...

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.