Wayne W. Carmichael

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR

SummaryCertain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the twol-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathioneS-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”

Increasingly, harmful algal blooms (HABs) are being reported worldwide due to several factors, primarily eutrophication, climate change and more scientific monitoring. All but cyanobacteria toxin poisonings (CTPs) are mainly a marine occurrence. CTPs occur in fresh (lakes, ponds, rivers and reservoirs) and brackish (seas, estuaries, and lakes) waters throughout the world. Organisms responsible include an estimated 40 genera but the main ones are Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, and Oscillatoria (Planktothrix). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins with biotoxins being responsible for acute lethal, acute, chronic and sub-chronic poisonings of wild/domestic animals and humans. The biotoxins include the neurotoxins; ana-toxin-a, anatoxin-a(s) and saxitoxins plus the hepatotoxins; microcystins, nodularins and cylindrospermopsins. Confirmations of human deaths from cyanotoxins are limited to exposure through renal dialysis at a haemodialysis center in Caruaru, Brazil, in 1996. A major effort to compile all available information on toxic cyanobacteria including issues of human health, safe water practices, management, prevention and remediation have been published by the World Health Organization. This paper will review our current understanding of CTPs including their risk to human health.

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.

A Drinking Water Crisis in Lake Taihu, China: Linkage to Climatic Variability and Lake Management

In late May, 2007, a drinking water crisis took place in Wuxi, Jiangsu Province, China, following a massive bloom of the toxin producing cyanobacteria Microcystis spp. in Lake Taihu, China’s third largest freshwater lake. Taihu was the city’s sole water supply, leaving approximately two million people without drinking water for at least a week. This cyanobacterial bloom event began two months earlier than previously documented for Microcystis blooms in Taihu. This was attributed to an unusually warm spring. The prevailing wind direction during this period caused the bloom to accumulate at the shoreline near the intake of the water plant. Water was diverted from the nearby Yangtze River in an effort to flush the lake of the bloom. However, this management action was counterproductive, because it produced a current which transported the bloom into the intake, exacerbating the drinking water contamination problem. The severity of this microcystin toxin containing bloom and the ensuing drinking water crisis were attributable to excessive nutrient enrichment; however, a multi-annual warming trend extended the bloom period and amplified its severity, and this was made worse by unanticipated negative impacts of water management. Long-term management must therefore consider both the human and climatic factors controlling these blooms and their impacts on water supply in this and other large lakes threatened by accelerating eutrophication.

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

Toxic peptides from freshwater cyanobacteria (blue-green algae). I: Isolation, purification and characterization of peptides from Microcystis aeruginosa and Anabaena flos-aquae

Toxic peptides from two European Microcystis aeruginosa and one Canadian Anabaena flos-aquae species of freshwater cyanobacteria (blue-green algae) were purified by high performance liquid chromatography (HPLC) and examined by amino acid analysis and mass spectrometry. A toxic fraction from a butanol/methanol extract of toxic lyophilized cells was separated by G-25 gel filtration and purified by HPLC using a C-18 semi-preparative Column. A toxic peak with the same elution time was detected for each of the three toxic cyanobacteria. The desalted purified toxins (i.p. LD50 in mice, 50 micrograms/kg) caused signs of poisoning identical with previous literature reports of hepatotoxic peptides from Microcystis. On hydrolysis and amino acid analysis all three toxins showed a similar profile, consisting of equimolar amounts of glutamic acid, alanine, arginine and leucine. beta-methyl aspartic acid was identified in all of the toxic peptides. The fast atom bombardment mass spectra of the toxins indicated the molecular weight to be 994 for all the peptides. The absence of sequence ions in their corresponding fast atom bombardment mass spectra indicated the peptides to be cyclic.

FIRST REPORT OF THE CYANOTOXINS CYLINDROSPERMOPSIN AND DEOXYCYLINDROSPERMOPSIN FROM RAPHIDIOPSIS CURVATA (CYANOBACTERIA)

A strain of Raphidiopsis (Cyanobacteria) isolated from a fish pond in Wuhan, P. R. China was examined for its taxonomy and production of the alkaloidal hepatotoxins cylindrospermopsin (CYN) and deoxy‐cylindrospermopsin (deoxy‐CYN). Strain HB1 was identified as R. curvata Fritsch et Rich based on morphological examination of the laboratory culture. HB1 produced mainly deoxy‐CYN at a concentration of 1.3 mg·g−1 (dry wt cells) by HPLC and HPLC‐MS/MS. CYN was also detected in trace amounts (0.56 μg·g−1). A mouse bioassay did not show lethal toxicity when tested at doses up to 1500 mg dry weight cells·kg−1 body weight within 96 h, demonstrating that production of primarily deoxy‐CYN does not lead to significant mouse toxicity by strain HB1. The presence of deoxy‐CYN and CYN in R. curvata suggests that Raphidiopsis belongs to the Nostocaceae, but this requires confirmation by molecular systematic studies. Production of these cyanotoxins by Raphidiopsis adds another genus, in addition to Cylindrospermopsis, Aphanizomenon, and Umezakia, now known to produce this group of hepatotoxic cyanotoxins. This is also the first report from China of a CYN and deoxy‐CYN producing cyanobacterium.

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.

Paralytic shellfish poisons produced by the freshwater cyanobacterium Aphanizomenon flos-aquae NH-5.

A single filament clonal isolate of Aphanizomenon flos-aquae was made from a water bloom sample taken at a small pond near Durham, New Hampshire, in 1980. When batch cultured the strain was toxic to mice and had an i.p. LD50 of about 5.0 mg/kg. Using an extraction procedure originally designed for paralytic shellfish poisons and other neurotoxins of freshwater cyanobacteria, a purification method was developed. The procedure involved acidified water/ethanol extraction of the cells followed by ultrafiltration, gel filtration, use of C18 cartridges to remove pigments, ion-exchange and high performance liquid chromatography using u.v. detection at 220 or 240 nm. Thin-layer chromatography and high performance liquid chromatography results indicate that Aphanizomenon flos-aquae NH-5 may produce paralytic shellfish poisons, mainly neo-saxitoxin and saxitoxin. Three labile toxins were also detected which were not similar to any of the known paralytic shellfish poisons.

Algal toxins and water‐based diseases

Waterborne diseases are usually caused by infectious microorganisms. However, an intermittent but widespread source of water‐based diseases are the exotoxins produced by several marine and freshwater algae species. Marine algal toxins come from dinoflagellates and cyanobacteria (blue‐green algae). The main di‐noflagellate genera involved are Gonyaulax, Protogonyaulax, Gymnodinium, and Gambierdiscus. Known toxic marine cyanobacteria include Lyngbya, Schizothrix, and Oscillatoria. Toxic signs of these marine toxins are varied but involve mainly neuro‐ and dermatoxicity. A few freshwater algal toxins are produced by dinoflagellates and haptophytes but the majority are formed by “water bloom”; species of cyanobacteria. Toxic blooms of these cyanobacteria can be found in several types of water bodies. The main toxic genera are Microcystis, Anabaena, Aphanizomenon, and Oscillatoria. Toxins produced are neurotoxic alkaloids and hepatotoxic peptides. Human poisonings from algal toxins can be from ingestion of con...