Development of an analytical method for the unambiguous structure elucidation of cyclic peptides with special appliance for hepatotoxic desmethylated microcystins

The periodical occurrence of harmful algal blooms (HABs) in freshwater lakes requires the determination of potential cyanobacterial toxins, especially microcystins (MCs). On demand of an adequate risk assessment, the high diversity of these hepatotoxic cyclic heptapeptides implicates the need of an unambiguous detection of their specific structural variants. Therefore, LC-MS and LC-MS/MS methods are the approaches of choice for determination of MCs. In contrast, even tandem mass spectromic fragmentation patterns are not even sufficient in any kind of structural determination requirements, whereas NMR methods require very high amounts of MCs. In this study, we present a novel method for chromatographic separation of desmethylated microcystins (dm-MCs). Based on the isolation of the specific structural variants using semi-preparative HPLC, a method was developed for the structure elucidation of cyclic peptides with special appliance for the determination of dm-MCs via analysis of the specific amino acid composition after peptide hydrolysis followed by stereospecific detection of the amino acids and resulting keto acids. On the basis of this method it is demonstrated that dm-MC-RR with the structure [Dha⁷]MC-RR represented the major compound in the microcystin pattern of Microcystis aeruginosa bloom events in 2005 and 2006 in Lake Senftenberg, Germany.     

Hepatotoxin-induced hypertaurinuria: a proton NMR study

The urinary excretion of taurine by rats after dosing with various hepatotoxins has been investigated by¹H NMR spectroscopy. After single hepatotoxic doses of hydrazine, carbon tetrachloride, 1-naphthylisothiocyanate, or thioacetamide there was biochemical and histopathological evidence of hepatic damage. Proton NMR spectroscopy of the urine collected for 24 h after dosing from these animals revealed a marked elevation in taurine (control 11.9 mole/h/kg) after dosing with thioacetamide (42.2 mole/h/kg), carbon tetrachloride (52.5 gmmole/ h/kg), 1-naphthyl-isothiocyanate (80.4 mole/h/kg) and hydrazine (52.9 mmole/h/kg). After allyl alcohol administration there was no increase in taurine excretion (7.5 mol/h/kg). The excretion of taurine after hydrazine administration was dose related. High resolution proton NMR spectroscopic analysis of urine also revealed resonances from several metabolites of hydrazine, an N-acetylcysteine conjugate of allyl alcohol, and acetamide as a metabolite of thioacetamide after dosing with the respective compounds. Changes in endogenous substances that may be related to the pathological events were also detected, such as a decrease in the excretion of 2-oxoglutarate and citrate after both hydrazine and carbon tetrachloride administration. The results confirm that proton NMR spectroscopic analysis of urine is a powerful analytical tool for the evaluation and study of toxic substances. Furthermore, measurement of urinary taurine may provide a non-invasive indicator of acute hepatic damage with certain classes of hepatotoxins.     

Problems associated with the presence of cyanobacteria in recreational and drinking waters

Eutrophication of waters can lead to the development of blooms of cyanobacteria (blue-green algae) and consequent health and environmental problems. The presence of these organisms in recreational and drinking waters is generally undesirable or even hazardous, although nitrogen fixing blue-green algae can be beneficially used as biofertilisers for plantation crops. This paper reviews the characteristics of cyanobacteria and particularly their toxins. The mechanisms of toxic algal blooms are discussed, as are the factors influencing toxin production. The nuisance and health hazards associated with freshwater bluegreen algae are discussed and the options for public health control are evaluated. The problems associated with statutory control of toxic algae problems is also considered.     

Isolation and structural characterization of microcystin-LR and three minor oligopeptides simultaneously produced by Radiocystis feernandoi (Chroococcales, Cyanobacteriae): a Brazilian toxic cyanobacterium.

Several blooms of cyanobacteria naturally occurring in freshwater reservoirs have been associated to numerous fatalities and cases of livestock and human poisoning. Microcystins (Mcs) are the most frequently found cyclic heptapeptide toxins in the cyanobacterial extracts. In previous work, Radiocystis fernandoi (strain SPC 714) lyophilized extracts were found to be hepatotoxic to mice with LD100 of about 60 mg kg(-1) and Mc LR was suggested as responsible for that toxicity. Here, we describe the isolation of four oligopeptides from R. fernandoi methanol extract by reversed-phase high performance liquid chromatography (RP-HPLC). The major component, which eluted with 65% acetonitrile from acetonitrile/water gradient, was identified as Mc-LR and its structure was confirmed by the presence of molecular related ion species [M+H]+ at m/z 996.3, ([M+H-Adda])+ at m/z 861.5, [Arg-Adda-Glu+H]+ at m/z 599.8, and [PhCH2CH(OMe)]+ at m/z 135.1 in the ESI spectra. Two components corresponding to small signals eluted from C18 column, respectively, with 44 and 45% acetonitrile had their structures proposed as isomers of aeruginosin derivatives showing molecular ions at m/z 651.7 and a [CHOI]+ immonium at m/z 140.1. Finally, the structure of the third minor and most hydrophobic component (68% acetonitrile elution) isolated from R. fernandoi extract seemed to correspond to a cyclic cyanopeptolin like micropeptin K139, a trypsin inhibitor firstly isolated from Microcystis aeruginosa, showing similar ions fragmentation pattern and [M+H]+ at m/z 987.6 in its ESI spectra.     

Toxicological aspects of treatment to remove cyanobacterial toxins from drinking water determined using the heterozygous P53 transgenic mouse model

The presence of toxic cyanobacteria in drinking water reservoirs renders the need to develop treatment methods for the ‘safe’ removal of their associated toxins. Chlorine has been shown to successfully remove a range of cyanotoxins including microcystins, cylindrospermopsin and saxitoxins. Each cyanotoxin requires specific treatment parameters, particularly solution pH and free chlorine residual. However, currently there has not been any investigation into the toxicological effect of solutions treated for the removal of these cyanotoxins by chlorine. Using the P53^def transgenic mouse model male and female C57BL/6J hybrid mice were used to investigate potential cancer inducing effects from such oral dosing solutions. Both purified cyanotoxins and toxic cell-free extract cyanobacterial solutions were chlorinated and administered over 90 and 170 days (respectively) in drinking water. No increase in cancer was found in any treatment. The parent cyanotoxins, microcystins, cylindrospermopsin and saxitoxins were readily removed by chlorine. There was no significant increase in the disinfection by-products trihalomethanes or haloacetic acids, levels found were well below guideline values. Histological examination identified no effect of treatment solutions except male mice treated with chlorinated cylindrospermopsin (as a cell free extract). In this instance 40% of males were found to have fatty vacuolation in their livers, cause unknown. It is recommended that further toxicology be undertaken on chlorinated cyanobacterial solutions, particularly for non-genotoxic carcinogenic compounds, for example the Tg. AC transgenic mouse model.     

A Review of the Hepatotoxic Plant Lantana camara

Lantana (Lantana camara Linn) is a noxious weed that grows in many tropical and subtropical parts of the world. Ingestion of lantana foliage by grazing animals causes cholestasis and hepatotoxicity. Both ruminants and nonruminant animals such as guinea pigs, rabbits, and female rats are susceptible to the hepatotoxic action of lantana toxins. The hepatotoxins are pentacyclic triterpenoids called lantadenes. Molecular structure of lantadenes has been determined. Green unripe fruits of the plant are toxic to humans. Lantana spp. exert allelopathic action on the neighboring vegetation. The allelochemicals have been identified as phenolics, with umbelliferone, methylcoumarin, and salicylic acid being the most phytotoxic. In addition to phenolics, a recent report indicates lantadene A and B as more potent allelochemicals. Management of lantana toxicosis in animals is achieved by drenching with activated charcoal and supportive therapy. Recent reports on the bilirubin clearance effect of Chinese herbal tea Yin Zhi Huang (decoction of the plant Yin Chin, Artemisia capillaries, and three other herbs) or its active ingredient 6,7-dimethylesculetin, in jaundice are very exciting and warrant investigations on its, possible, ameliorative effects in lantana intoxicated animals. Research is being conducted on new drug discovery based on natural products in different parts of the lantana plant.     

Experimental models used for the study of antihepatotoxic agents

Both in vitro and in vivo liver models have been developed in the past years to study the hepatoprotective agents. These systems measure the ability of the test drug to prevent or cure liver toxicity (induced by various hepatotoxins) in experimental animals. In in vitro models fresh hepatocytes are treated with hepatotoxin and the effect of the test drug on the same is evaluated. In in vivo models, a toxic dose or repeated doses of a known hepatotoxin are administered to induce liver damage in experimental animals. The test substance is administered along with, prior to and/or after the toxin treatment. Various chemical agents normally used to induce hepatotoxicty in experimental animals for the evaluation of hepatoprotective agents include carbon tetrachloride, paracetamol, Acrylamide, adriamycin, alcohol, antitubercular drugs etc. The present article explains the mechanism of action of various hepatotoxic chemical/drugs, their dosage and route of administration.