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.     

Methods for correlation analysis of toxins and species present in random samples of phytoplankton.

We developed and tested two new procedures to find which species present in samples of phytoplankton is responsible for the production of a toxin. The procedures represent a different form of correlation analysis that uses information on the presence or absence of the toxin, and on the relative abundance of each species of plankton in the samples. The efficiency of the algorithms is tested by random process simulation. The algorithms were clearly superior to known techniques dealing with correlations between binary variables to show the toxin producing microorganism. We used experimental toxin isolation from phytoplankton as an example of practical success using the most efficient of the algorithms tested.     

Cell-based assay coupled with chromatographic fractioning: A strategy for marine toxins detection in natural samples

Cell-based assays (CBA) have been proposed for the evaluation of toxicity caused by marine toxins in natural samples (fish, shellfish and microalgae). However, their application has been hindered due to the interferences present in biological matrices that may cause cellular response and interfere in toxicity evaluation. This work reviews in an extensive introduction the use of CBA for toxicity evaluation of marine toxins. Afterwards, the coupling of chromatographic fractioning with neuroblastoma Neuro-2a CBA is presented to enhance the applicability of CBA for complex matrices. Examples of application are provided for mussel samples (Mytilus galloprovincialis) and microalgae (Gambierdiscus sp.), and the results demonstrated the great potential of the combined strategy for reliable toxicological evaluation without ethical concern. Fractioning of an equivalent of 72 mg eq mL^?1 of mussel sample allowed the identification of non-toxic and toxic fractions whereas only 2.5 mg eq mL^?1 of non-purified mussel sample was responsible for 20% of cell mortality. Furthermore, the application of CBA allowed selectively distinguishing between ciguatoxin-like and other unspecific toxicity in Gambierdiscus sp. extract.     

Properties of two toxins isolated from the blue-green alga Microcystis aeruginosa

Attempts were made to characterize the two toxins (P-1 and P-2) isolated from the blue-green alga Microcystis aeruginosa, by amino acid analysis, mass spectrometry, 1H- and 13C-NMR. P-2, the major toxin, had a molecular weight of 1044, and consisted of one molecule each of beta-methylaspartic acid, D-Glu, D-Ala, L-Arg, L-Tyr, N-methyldehydroalanine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). P-1, with a molecular weight of 994, appeared to have almost the same composition except that it contained L-Leu instead of L-Tyr in P-2. Mass spectrometric studies, along with a negative ninhydrin reaction, indicated that each toxin was a cyclic peptide. P-2 showed an LD99 of 70 micrograms/kg mice when injected i.p. and [alpha]D24 of -72.42 degrees (c = 0.095 in methanol), and was decomposed at around 218 degrees C.     

Characterization of heptapeptide toxins extracted from Microcystis aeruginosa (Egyptian isolate)

Four toxic peptides from local fresh water cyanobacterium Microcystis aeruginosa were purified and identified by high performance liquid chromatography (HPLC) and ion spray mass spectroscopic studies as: RR; YR; LR and LA with molecular weights of 1006.8, 1073, 984.8 and 910.6 respectively. Amino acid analysis indicated the presence of equimolar amounts of aspartic acid, glutamic acid, arginine, leucine and tyrosine, in addition to both alanine and dehydroalanine. Mouse assay toxicity indicated that the first two peptides, at the peak area of RR, YR. were highly toxic with LD₅₀ 20, 18.2 μg/kg body weight; however, the latter two, at the peak areas LR and I.A. have a lesser toxicity with LD₅₀ 36 and 40 μg/kg body weight respectively. Three linear peptide analogs to those naturally found devoid of Adda were synthesized using the continuous flow technique. HPLC pure synthesized analog products were tested for toxicity using male mice (i.p. injection). None of them induced toxic activity.     

Development of standardized methodology for identifying toxins in clinical samples and fish species associated with tetrodotoxin-borne poisoning incidents

Tetrodotoxin (TTX) is a naturally occurring toxin in food, especially in puffer fish. TTX poisoning is observed frequently in South East Asian regions. In TTX-derived food poisoning outbreaks, the amount of TTX recovered from suspicious fish samples or leftovers, and residual levels from biological fluids of victims are typically trace. However, liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods have been demonstrated to qualitatively and quantitatively determine TTX in clinical samples from victims. Identification and validation of the TTX-originating seafood species responsible for a food poisoning incident is needed. A polymerase chain reaction-based method on mitochondrial DNA analysis is useful for identification of fish species. This review aims to collect pertinent information available on TTX-borne food poisoning incidents with a special emphasis on the analytical methods employed for TTX detection in clinical laboratories as well as for the identification of TTX-bearing species.     

Persistence of cyclic peptide toxins in dried Microcystis aeruginosa crusts from lake Mokoan,Australia

Dried Microcystis aeruginosa Kuetzing emend. Elenkin crusts estimated to be 5–6 months old from the shore of Lake Mokoan were toxic by mouse bioassay (LD₁₀₀ 100–140 mg dry wt/kg mouse). Fresh bloom material from the lake was also highly toxic (LD₁₀₀ 25–35 mg dry wt/kg mouse). Microcystin high performance liquid chromatography (HPLC) profiles of the crust and fresh material were very similar, with 24 compounds having UV spectra consistent with microcystin LR. Five of the major microcystins were purified and analysed by electrospray/mass spectrometry. The molecular weights of these microcystins [910, 924, 982, 982 (two compounds), and 986] do not correspond with known microcystins. All five compounds were hepatotoxic to mice with LD₁₀₀ values ranging from 85 to 140 μg microcystin/kg mouse). Total microcystin contents (expressed as microcystin LR aquivalents) determined by HPLC correlated with the mouse bioassay analyses (crust 2.1 μg microcystin/mg dry wt; fresh 4.1 μg microcystin/mg dry wt). These results suggest that microcystin is protected from degradation while encapsulated within the dried Microcystis crusts. Leaching experiments demonstrated that re-wetting of the crust material leads to rapid release of microcystins into the surrounding water. These observations have important management implications for lakes and reservoirs where crusts of cyanobacterial material form on the shoreline. © by John Wiley & Sons, Inc.     

Microcystin-like toxins in different freshwater species of Oscillatoria.

In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tenuis and Oscillatoria mougetii were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms.     

Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products

Terpenoids form a large and structurally diverse family of natural products and are ingredients of various herbal medicines. We have investigated possible interactions between herbal medicines and conventional medicines, and recently reported that monoterpenoids contained in Zanthoxyli Fructus can be potent inhibitors of P-glycoprotein (P-gp). In the present study, the influence of 70 kinds of terpenoids present in natural products on P-gp-mediated efflux transport was investigated. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used to screen the terpenoids. Large increases in the intracellular accumulation of [³H]digoxin were observed in the presence of (R)-(+)-citronellal, (S)-(−)-β-citronellol, -terpinene, terpinolene, (−)-β-pinene, abietic acid, ophiobolin A, cucurbitacin I, and glycyrrhetic acid. A study of the concentration-dependency revealed that the IC₅₀ of ophiobolin A, glycyrrhetic acid, (R)-(+)-citronellal, abietic acid, and cucurbitacin I was smaller than that of verapamil. The transcellular transport of [³H]digoxin across Caco-2 cell monolayers was then examined in the presence of (R)-(+)-citronellal, abietic acid, and glycyrrhetic acid. Significant increases in the apical-to-basolateral transport and decreases in the basolateral-to-apical transport and efflux ratio were demonstrated. These findings suggest that some natural products containing these terpenoids may inhibit P-gp-mediated transport and interact with P-gp substrates in the intestinal absorption process.     

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.     

Isolated rat liver perfusion studies with cyclic heptapeptide toxins of Microcystis and Oscillatoria (freshwater cyanobacteria)

Isolated perfused rat livers were used to study the dose-dependent effects of three cyclic heptapeptide toxins isolated from Norwegian freshwater bloom samples containing Microcystis aeruginosa, Oscillatoria agardhii var. and Oscillatoria agardhii var. isothrix. The high pressure liquid chromatography (HPLC) purified toxins had an i.p. LD50 in the rat and mouse of approximately 50, 500 and 1000 micrograms/kg, respectively. Hepatic insult of the toxins at concentrations of 0.5-4.0 times the rat i.p. lethal dose were assessed by monitoring bile flow, accumulation of total protein in the perfusate, release of intracellular enzymes and histopathologic examination of perfused liver tissue. One hundred micrograms of Microcystis toxin produced cessation of bile flow during a 1 hr perfusion period, while the two Oscillatoria toxins required 1000 and 2000 micrograms of toxin, consistent with their lower LD50 values. Hepatic cell membranes remained intact during the perfusion since release of enzymes and proteins into the perfusate was similar for toxin treated and control livers, and histopathologic examination of Trypan Blue infused livers revealed exclusion of the dye from the intracellular compartment of the parenchyma. Histopathologic findings for all three toxins showed hepatocellular disassociation that increased with toxin concentration. At the ultrastructural level, all three toxins caused dose-dependent vesiculation of rough endoplasmic reticulum, formation of concentric whorls composed of rough-ER, mitochondrial swelling, large cytoplasmic vacuoles and altered bile canaliculi. These changes were similar to those found for previous in vivo studies using Microcystis cyclic heptapeptides from Scotland and Australia. The Oscillatoria toxins required five to ten times more toxin to produce similar effects as the Microcystis toxin. At the higher concentrations, the Oscillatoria toxins also caused a proliferation of smooth-ER. The isolated perfused rat liver was found to be a good model for studying the hepatocellular effects of different cyclic peptide toxins from cyanobacteria.     

Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins

The protein serine/threonine phosphatases constitute a unique class of enzymes that are critical for cell regulation, as they must counteract the activities of thousands of protein kinases in human cells. Uncontrolled inhibition of phosphatase activity by toxic inhibitors can lead to widespread catastrophic effects. Over the past decade, a number of natural product toxins have been identified that specifically and potently inhibit protein phosphatase-1 and 2A. Amongst these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR and the polyether fatty acid okadaic acid from dinoflagellate sources. The molecular mechanism underlying potent inhibition of protein phosphatase-1 by these toxins is becoming clear through insights gathered from diverse sources. These include: 1. Comparison of structure-activity relationships amongst the different classes of toxins. 2. Delineation of the structural differences between protein phosphatase-1 and 2A that account for their differing sensitivity to toxins, particularly okadaic acid and microcystin-LR. 3. Determination of the crystal structure of protein phosphatase-1 with microcystin-LR, okadaic acid and calyculin bound. 4. Site-specific mutagenesis and biochemical analysis of protein phosphatase-1 mutants. Taken together, these data point to a common binding site on protein phosphatase-1 for okadaic acid, microcystin-LR and the calyculins. However, careful analysis of these data suggest that each toxin binds to the common binding site in a subtly different way, relying on distinct structural interactions such as hydrophobic binding, hydrogen bonding and electrostatic interactions to different degrees. The insights derived from studying the molecular enzymology of protein phosphatase-1 may help explain the different sensitivities of other structurally conserved protein serine/theonine phosphatases to toxin inhibition. Furthermore, studies on the binding of structurally diverse toxins at the active site of protein phosphatase-1 are leading to a clearer understanding of potential enzyme-substrate interactions in this important class of cell regulatory proteins.     

Pathophysiological mechanisms of TNF during intoxication with natural or man-made toxins

Intoxication with different natural toxins or man-made toxicants has been associated with the induction of tumor necrosis factor alpha (TNF). These include endotoxin, superantigens, Pseudomonas aeruginosa exotoxin A, bacterial DNA, T cell stimulatory agents such as agonistic anti-CD3 mAbs or concanavalin A, alpha-amanitin, paracetamol, ethanol, carbon tetrachloride, dioxin, and dimethylnitrosamine. In this paper we compile and discuss the current knowledge on the pathophysiological role of TNF during intoxication with all mentioned toxins and toxicants. A possible role of gut-derived endotoxin in several TNF-dependent toxic events has been considered. The development of pharmaceuticals that selectively interfere with the detrimental pathways induced by TNF during intoxication with bacteria, viruses, drugs, or other chemicals requires detailed knowledge of the signaling pathways originating from the two TNF receptors (TNFR1 and TNFR2). Major characteristics of these signaling pathways are described and put together.