Cylindrospermopsin- and Deoxycylindrospermopsin-Producing Raphidiopsis raciborskii and Microcystin-Producing Microcystis spp. in Meiktila Lake, Myanmar.

Meiktila Lake is a shallow reservoir located close to Meiktila city in central Myanmar. Its water is used for irrigation, domestic purposes and drinking water. No detailed study of the presence of cyanobacteria and their potential toxin production has been conducted so far. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Meiktila Lake, water samples were collected in March and November 2017 and investigated for physico-chemical and biological parameters. Phytoplankton composition and biomass determination revealed that most of the samples were dominated by the cyanobacterium Raphidiopsis raciborskii. In a polyphasic approach, seven isolated cyanobacterial strains were classified morphologically and phylogenetically as R. raciborskii, and Microcystis spp. and tested for microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins and anatoxins by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). ELISA and LC-MS analyses confirmed CYNs in three of the five Raphidiopsis strains between 1.8 and 9.8 µg mg-1 fresh weight. Both Microcystis strains produced MCs, one strain 52 congeners and the other strain 20 congeners, including 22 previously unreported variants. Due to the presence of CYN- and MC-producing cyanobacteria, harmful effects on humans, domestic and wild animals cannot be excluded in Meiktila Lake.

Oxidative Release of Thiol-Conjugated Forms of the Mycotoxin 4-Deoxynivalenol.

Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by several species of Fusarium, which may infect grain crops. DON, as well as other type-B trichothecenes, contain an α,ß-unsaturated carbonyl group that may react with sulfhydryl groups in, for example, amino acids and peptides. Such conjugates have been shown to occur in plants. Nucleophilic addition of thiols to the conjugated double bond in DON afforded several isomeric reaction products, and the thermodynamically favored isomers of DON-10-cysteine and DON-10-glutathione have been prepared and characterized previously. This study reports the preparation and characterization of the kinetically favored DON-10-cysteine isomer. We subsequently studied and compared the rate of the deconjugation reaction of the two DON-10-cysteine isomers and the thermodynamically favored DON-10-glutathione adduct. The deconjugation rate of the thermodynamically favored thiol conjugates was slow with half-lives of weeks even at pH 10.7, while the kinetically favored DON-10-cysteine isomer deconjugated within a few hours, affording free DON. We adapted a simple and rapid oxidation protocol in which the sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with the base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate to afford free DON. The deconjugation reactions of the sulfoxides and sulfones of thermodynamically favored DON-10-thiols were complete within hours or minutes at pH 10.7, respectively. The increase in deconjugation rates for the kinetically favored DON-10-cysteine were less dramatic. Oxidation of sulfides to sulfoxides is known to occur in vivo, and thus, our data show that thiol-conjugated DON might become bioavailable via sulfide oxidation followed by elimination to regenerate DON. The oxidation-elimination approach could also be useful for the indirect quantification of DON-10-thiol conjugates in plant and animal tissues.

Novel Microcystins from Planktothrix prolifica NIVA-CYA 544 Identified by LC-MS/MS, Functional Group Derivatization and 15N-labeling.

Microcystins are cyclic heptapeptides from cyanobacteria that are potent inhibitors of protein phosphatases and are toxic to animals and humans. At present, more than 250 microcystin variants are known, with variants reported for all seven peptide moieties. While d-glutamic acid (d-Glu) is highly-conserved at position-6 of microcystins, there has been only one report of a cyanobacterium (Anabaena) producing microcystins containing l-Glu at the variable 2- and 4-positions. Liquid chromatography-mass spectrometry analyses of extracts from Planktothrix prolifica NIVA-CYA 544 led to the tentative identification of two new Glu-containing microcystins, [d-Asp3]MC-ER (12) and [d-Asp3]MC-EE (13). Structure determination was aided by thiol derivatization of the Mdha7-moiety and esterification of the carboxylic acid groups, while 15N-labeling of the culture and isotopic profile analysis assisted the determination of the number of nitrogen atoms present and the elemental composition of molecular and product-ions. The major microcystin analog in the extracts was [d-Asp3]MC-RR (1). A microcystin with an unprecedented high-molecular-mass (2116 Da) was also detected and tentatively identified as a sulfide-linked conjugate of [d-Asp3]MC-RR (15) by LC-HRMS/MS and sulfide oxidation, together with its sulfoxide (16) produced via autoxidation. Low levels of [d-Asp3]MC-RW (14), [d-Asp3]MC-LR (4), [d-Asp3,Mser7]MC-RR (11), [d-Asp3]MC-RY (17), [d-Asp3]MC-RF (18), [d-Asp3]MC-RR-glutathione conjugate (19), and [d-Asp3]MC-RCit (20), the first reported microcystin containing citrulline, were also identified in the extract, and an oxidized derivative of [d-Asp3]MC-RR and the cysteine conjugate of 1 were partially characterized.

Comprehensive multi-technique approach reveals the high diversity of microcystins in field collections and an associated isolate of Microcystis aeruginosa from a Turkish lake.

Microcystins (MCs) are hepatotoxic and potentially carcinogenic cyanotoxins. They exhibit high structural variability, with nearly 250 variants described to date. This variability can result in incomplete detection of MC variants during lake surveys due to the frequent use of targeted analytical methods and a lack of standards available for identification and quantitation. In this study, Lake Uluabat in Turkey was sampled during the summer of 2015. Phylogenetic analysis of the environmental mcyA sequences suggested Microcystis spp. were the major MC contributors. A combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography with UV detection and mass spectrometry (LC-UV-MS), and a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, together with thiol and periodate reactivity, revealed more than 36 MC variants in the lake samples and a strain of M. aeruginosa (AQUAMEB-24) isolated from Lake Uluabat. Only MCs containing arginine at position-4 were detected in the culture, while MC-LA, -LY, -LW and -LF were also detected in the lake samples, suggesting the presence of other MC producers in the lake. The previously unreported MCs MC-(H2)YR (dihydrotyrosine at position-2) (17), [epoxyAdda5]MC-LR, [DMAdda5]MC-RR (1) and [Mser7]MC-RR (8) were detected in the culture and/or field samples. This study is a good example of how commonly used targeted LC-MS methods can underestimate the diversity of MCs in freshwater lakes and cyanobacteria cultures and how untargeted LC-MS methods can be used to comprehensively assess MC diversity present in a new system.

Selectivity of commercial immunoaffinity columns for modified forms of the mycotoxin 4-deoxynivalenol (DON).

Commercial immunoaffinity columns (IACs) are today available for all major mycotoxins. However, manufacturers give usually no or very limited information on the epitope, i.e. the specific part of the toxin molecule that binds to the antibody. 4-Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by plant pathogenic field fungi and is regulated in many countries worldwide. DON was shown to be biotransformed via different metabolic pathways, and thus many different biotransformation products may be found in different products or organisms. In addition, several structurally similar mycotoxins may co-occur with DON. We compared five commercial IACs for their retention of a range of DON derivatives modified in the C-3, C-8, C-10, C-13 or C-15 positions, as well as nivalenol (NIV) and T-2 tetraol. The DON-derivatives were deepoxy-DON, DON 3-, 8- and 15-O-ß-d-glucuronides, 3- and 15-O-acetyl-DON, DON-3-O-ß-d-glucoside, 10- and 13-cysteinyl-adducts of DON, and the 13-mercaptoethanol and 10,13-dimercaptoethanol adducts of DON. The C-3 derivatives and deepoxy-DON were retained by most of the columns. Only one of the five IACs retained C-15 and C-8 derivatives, but it did not retain C-3 derivatives or deepoxy-DON. The antibodies in two of the IACs bound C-10 conjugates, but C-13 derivatives were not retained by any of the columns. This study shows that all of the antibodies in commercial IACs bind a range of DON derivatives, especially those that are modified at C-3. NIV was retained by three of the columns, and T-2 tetraol was partially retained by one IAC.

Rapid and Convenient Oxidative Release of Thiol-Conjugated Forms of Microcystins for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in some cyanobacteria, and usually contain an α,ß-unsaturated carbonyl group that is readily conjugated to thiol-containing amino acids, peptides, and proteins in vivo and in vitro. Methods for deconjugating these types of adducts have recently been reported, but the reactions are slow or result in derivatized microcystins. Mercaptoethanol derivatives of a range of microcystins were therefore used as model compounds to develop deconjugation procedures in which the dialkyl sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate via ß-elimination to afford free microcystins with the α,ß-unsaturated carbonyl group intact. These free microcystins can be analyzed by LC/MS to determine the toxin profile of bound microcystins. The method was tested on Cys- and GSH-derivatives of [Dha7]MC-LR. In solution, the deconjugation reactions were complete within minutes at pH 10.7 and within a few hours at pH 9.2. Oxidation of sulfides to sulfoxides is easier and more rapid than oxidation to sulfones, allowing the use of milder oxidants and shorter reaction times. Oxidation of any methionine residues present in the microcystins occurs inevitably during these procedures, and interpretation of the microcystin profile obtained by LC/MS analysis needs to take this into account. Oxidation of tryptophan residues and degradation of microcystins by excess oxidant were circumvented by the addition of Me2SO as a sacrificial reducing agent. These methods may be useful for other compounds that undergo conjugation via thia-Michael addition, such as acrylamide and deoxynivalenol. Oxidation of sulfides to sulfoxides can occur in vivo and could affect the bioavailability of toxins and drugs conjugated via thia-Michael addition, potentially exacerbating oxidative stress by catalytically converting GSH to its sulfenate via conjugation, oxidation, and elimination to regenerate the free toxin.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group.

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes.

Characterization of Deoxynivalenol-Glutathione Conjugates Using Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass Spectrometry.

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

Preparation and Characterization of Cysteine Adducts of Deoxynivalenol.

Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON-cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,ß-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON-cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC-HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC-HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,ß-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,ß-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Nucleophilic Addition of Thiols to Deoxynivalenol.

Conjugation of deoxynivalenol (DON) with sulfur compounds is recognized as a significant reaction pathway, and putative DON-glutathione (DON-GSH) conjugates have been reported in planta. To understand and control the reaction of trichothecenes with biologically important thiols, we studied the reaction of DON, T-2 tetraol, and de-epoxy-DON with a range of model thiols. Reaction conditions were optimized for DON with 2-mercaptoethanol. Major reaction products were identified using HRMS and NMR spectroscopy. The results indicate that thiols react reversibly with the double bond (Michael addition) and irreversibly with the epoxide group in trichothecenes. These reactions occurred at different rates, and multiple isomers were produced including diconjugated forms. LC-MS analyses indicated that glutathione and cysteine reacted with DON in a similar manner to the model thiols. In contrast to DON, none of the tested mercaptoethanol adducts displayed toxicity in human monocytes or induced pro-inflammatory cytokines in human macrophages.

Biotransformation of zearalenone and zearalenols to their major glucuronide metabolites reduces estrogenic activity.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Once ingested, ZEN may be absorbed and metabolised to α- and ß-zearalenol (α-ZOL, ß-ZOL), and to a lesser extent α- and ß-zearalanol (α-ZAL, ß-ZAL). Further biotransformation to glucuronide conjugates also occurs to facilitate the elimination of these toxins from the body. Unlike ZEN and its metabolites, information regarding the estrogenic activity of these glucuronide conjugates in various tissues is lacking. ZEN-14-O-glucuronide, α-ZOL-14-O-glucuronide, α-ZOL-7-O-glucuronide, ß-ZOL-14-O-glucuronide and ß-ZOL-16-O-glucuronide, previously obtained as the major products from preparative enzymatic synthesis, were investigated for their potential to cause endocrine disruption through interference with estrogen receptor transcriptional activity. All five glucuronide conjugates showed a very weak agonist response in an estrogen responsive reporter gene assay (RGA), with activity ranging from 0.0001% to 0.01% of that of 17ß-estradiol, and also less than that of ZEN, α-ZOL and ß-ZOL which have previously shown estrogenic potencies of the order 17ß-estradiol>α-ZOL>ZEN>ß-ZOL. Confirmatory mass spectrometry revealed that any activity observed was likely a result of minor deconjugation of the glucuronide moiety. This study confirms that formation of ZEN and ZOL glucuronides is a detoxification reaction with regard to estrogenicity, serving as a potential host defence mechanism against ZEN-induced estrogenic activity.

Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues.

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC­WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N­formylkynurenine (Nfk). Investigation of one of these congeners (MC­NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

Semisynthesis of radiolabeled amino acid and lipid brevetoxin metabolites and their blood elimination kinetics in C57BL/6 mice.

Brevetoxin B (BTX-B), produced by dinoflagellates of the species Karenia, is a highly reactive molecule, due in part to an α,ß-unsaturated aldehyde group at the terminal side chain, leading to the production of metabolites in shellfish by reduction, oxidation, and conjugation. We have investigated in mice the blood elimination of three common bioactive brevetoxin metabolites found in shellfish, which have been semisynthesized from BTX-B in radioactive forms. BTX-B was reduced at C42 to yield [(3)H] dihydro-BTX-B. [(3)H] S-desoxy-BTX-B2 (cysteine brevetoxin B) was semisynthesized from BTX-B by the conjugation of cysteine at the C50 olefinic group then [(3)H] radiolabeled by C42 aldehyde reduction. [(14)C] N-Palmitoyl-S-desoxy-BTX-B2 was prepared using S-desoxy-BTX-B2 as the starting material with addition of the [(14)C] radiolabeled fatty acid via cysteine-amide linkage. The elimination of intravenously administered [(3)H] S-desoxy-BTX-B2, [(14)C] N-palmitoyl-S-desoxy-BTX-B2, or [(3)H] dihydro-BTX-B was measured in blood collected from C57BL/6 mice over a 48 h period. Each brevetoxin metabolite tested exhibited biexponential elimination kinetics and fit a two-compartment model of elimination that was applied to generate toxicokinetic parameters. The rate of transfer between the central compartment (i.e., blood) and the peripheral compartment (e.g., tissue) for each brevetoxin differed substantially, with dihydro-BTX-B exchanging rapidly with the peripheral compartment, S-desoxy-BTX-B2 eliminating rapidly from the central compartment, and N-palmitoyl-S-desoxy-BTX-B2 eliminating slowly from the central compartment. Toxicokinetic parameters were analyzed in the context of the unique structure of each brevetoxin metabolite resulting from a reduction, amino acid conjugation, or fatty acid addition to BTX-B.

Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4).

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.

A structural basis for regulation of actin polymerization by pectenotoxins.

(PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification of their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin, which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts, allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure-activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues.

Lactone ring of pectenotoxins: a key factor for their activity on cytoskeletal dynamics.

BACKGROUND: Pectenotoxins are a group of natural products from marine origin that can accumulate in shellfish and intoxicate humans. Recently, novel homologues such as pectenotoxin-11 (PTX-11) and pectenotoxin-2 seco acid (PTX-2SA) have been identified. Their toxic potential towards experimental animals has been evaluated however their interaction with cellular systems is almost unknown. This is the first report showing (i) the biological activity of PTX-11 and PTX-2SA on actin cytoskeleton and morphology of living cells and (ii) the structure- activity relationship for this family of toxic compounds. METHODS: Fluorescent phalloidin was utilized to quantify and visualize any modification in polymerized actin. Fluorescence values were obtained with laser-scanning cytometer and cells were imaged through confocal microscopy. For structure-activity evaluations, pectenotoxin-1 (PTX-1) and pectenotoxin-2 (PTX-2) was also analyzed. RESULTS: Data showed that PTX-11 triggered a remarkable depolymerizing effect on actin cytoskeleton and also modifications in the shape of cells. In contrast, PTX-2SA did not evidence the same effects. CONCLUSION: Our findings point out that (i) the actin cytoskeleton is a common target for PTX-11, PTX-2 and PTX-1, but not for PTX-2SA, and (ii) this difference in activity is related to the presence or absence of an intact lactone ring in their structures.