Mariyo F. Watanabe

Inhibition of protein phosphatases by microcystis and nodularin associated with hepatotoxicity

SummaryMicrocystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent “activation” of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds,α-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

NAMING OF CYCLIC HEPTAPEPTIDE TOXINS OF CYANOBACTERIA (BLUE-GREEN-ALGAE)

UNIV ILLINOIS,COLL VET MED,URBANA,IL 61801; USA,MED RES INST INFECT DIS,DIV PATHOPHYSIOL,FREDERICK,MD 21701; UNIV NEW ENGLAND,DEPT BIOCHEM MICROBIOL & NUTR,ARMIDALE,NSW 2351,AUSTRALIA; UNIV ALBERTA,DEPT BOT,EDMONTON T6G 2E1,ALBERTA,CANADA; MEIJO UNIV,FAC PHARM,TEMPA KU,NAGOYA,AICHI 468,JAPAN; CHEM RES & DEV CTR,ABERDEEN,MD 21701; NATL BOT GARDENS,CLAREMENT,SOUTH AFRICA; ACAD SINICA,INST HYDROBIOL,WUHAN,PEOPLES R CHINA; NORWEGIAN INST WATER RES,OSLO 3,NORWAY; UNIV HAWAII MANOA,DEPT CHEM,HONOLULU,HI 96822; UNIV ILLINOIS,SCH CHEM SCI,URBANA,IL 61801; UNIV CALIF LOS ANGELES,DEPT MICROBIOL,LOS ANGELES,CA 90024; TOKYO METROPOLITAN RES LAB PUBL HLTH,SHINJUKU KU,TOKYO 160,JAPAN

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.

Stability of microcystins from cyanobacteria: effect of light on decomposition and isomerization.

Microcystins are potent hepatotoxins produced by cyanobacteria. Their geometrical isomers [6(Z)-Adda microcystin] do not essentially show hepatotoxicity and show weaker tumor-promoting activity than their parent toxins. The present study was undertaken to examine stability of microcystins during the analysis and purification and under photolysis conditions in connection with the detoxification. Microcystin LR was very stable because of limited decomposition and isomerization to its geometrical isomer during analysis and purification. While microcystins decomposed very limitedly by exposure with sunlight alone, the addition of pigments extracted from cyanobacteria accelerated their decompositions

In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts

Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.

Analysis and purification of toxic peptides from cyanobacteria by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable chemical analysis method for microcystins (cyanoginosins) has been studied. Three different mobile phases for high-performance liquid chromatography were selected and optimized. Also the adsorptive powers of three commercially available C18 cartridges were compared and the results successfully applied to the clean up of three of the toxins. Finally a total system for the analysis and isolation of microcystins was established.

Stability of microcystins from cyanobacteria—II. Effect of UV light on decomposition and isomerization

Microcystins are very potent hepatotoxins and strong liver tumor promoters produced by cyanobacteria, and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, we examined the stability of microcystins during photolysis with UV light. The toxins were easily decomposed by UV light at wavelengths around the absorption maxima of the toxins and the decomposition depended on the intensity of the light. The half-life of microcystin LR by 147 microW/cm2 UV irradiation was 10 min, and the toxin was completely decomposed by 2550 microW/cm2 UV after 10 min. When the toxins were irradiated with weaker UV light, isomerization was also observed by a different mechanism from that during photolysis by sunlight and pigment, and several products including three geometrical isomers of the conjugated diene of Adda were detected. Microcystin RR showed almost the same behavior as that of microcystin LR under the same conditions. Since no noxious products were formed in the present study, a water treatment including UV irradiation is very possible for removing microcystins from raw water.

Isolation and characterization of the minor components associated with microcystins LR and RR in the cyanobacterium (blue-green algae)

Two structurally similar analogues of microcystins LR and RR, cyclic peptide hepatotoxins from Microcystis, were isolated by chromatographic methods. Although they have the same mol. wt and amino acid compositions as those of the parent toxins, they do not possess similar toxicities. Ultraviolet and 1H-NMR spectral data for both components demonstrate clear structural difference of these cyclic peptides from the parent toxins, which are probably responsible for the marked decreases in their observed toxicities.

Toxins contained in Microcystis species of cyanobacteria (blue-green algae)

Cyclic peptide toxins were analyzed for three Microcystis species (M. aeruginosa, M. viridis and M. wesenbergii) using an ODS-silica gel cartridge and high performance liquid chromatography with ODS-silica gel. On strain of M. aeruginosa contained a high amount of microcystin (cyanoginosin) YR and a lesser amount of LR. Three toxins, microcystin-RR, -YR and -LR, were detected in two strains of M. aeruginosa and four of M. viridis. The main component of the toxins of these strains was microcystin-RR. M. wesenbergii showed no peak in the area where the three toxins were obtained in other Microcytisis species by HPLC analysis. LD50 values of the purified toxins of YR and LR were similar, while a lower toxicity was estimated for RR. This explains the relatively weak toxicity of M. viridis whose main component is microcystin-RR.