Homoanatoxin-a Research Articles

Multi-class secondary metabolites in cyanobacterial blooms from a tropical water body: Distribution patterns and real-time prediction

Cyanobacterial blooms that produce toxins occur in freshwaters worldwide and yet, the occurrence and distribution patterns of many cyanobacterial secondary metabolites particularly in tropical regions are still not fully understood. Moreover, predictive models for these metabolites by using easily accessible water quality indicators are rarely discussed. In this study, we investigated the co-occurrence and spatiotemporal trends of 18 well-known and less-studied cyanobacterial metabolites (including [D-Asp3] microcystin-LR (DM-LR), [D-Asp3] microcystin-RR (DM-RR), microcystin-HilR (MC-HilR), microcystin-HtyR (MC-HtyR), microcystin-LA (MC-LA), microcystin-LF (MC-LF), microcystin-LR (MC-LR), microcystin-LW (MC-LW), microcystin-LY (MC-LY), microcystin-RR (MC-RR) and microcystin-WR (MC-WR), Anatoxin-a (ATX-a), homoanatoxin-a (HATX-a), cylindrospermospin (CYN), nodularin (NOD), anabaenopeptin A (AptA) and anabaenopeptin B (AptB)) in a tropical freshwater lake often plagued with blooms. Random forest (RF) models were developed to predict MCs and CYN and assess the relative importance of 22 potential predictors that determined their concentrations. The results showed that 11 MCs, CYN, ATX-a, HATX-a, AptA and AptB were found at least once in the studied water body, with MC-RR and CYN being the most frequently occurring, intracellularly and extracellularly. AptA and AptB were detected for the first time in tropical freshwaters at low concentrations. The metabolite profiles were highly variable at both temporal and spatial scales, in line with spatially different phytoplankton assemblages. Notably, MCs decreased with the increase of CYN, possibly revealing interspecific competition of cyanobacteria. The rapid RF prediction models for MCs and CYN were successfully developed using 4 identified drivers (i.e., chlorophyll-a, total carbon, rainfall and ammonium for MCs prediction; and chloride, total carbon, rainfall and nitrate for CYN prediction). The established models can help to better understand the potential relationships between cyanotoxins and environmental variables as well as provide useful information for making policy decisions.

Quantification of cylindrospermopsin, anatoxin-a and homoanatoxin-a in cyanobacterial bloom freshwater using direct injection/SPE coupled with UPLC-MS/MS

Analytical methods based on direct injection (DI) and solid phase extraction (SPE) coupled with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) were developed for the determination of anatoxin-a (ATX-a), cylindrospermopsin (CYN), and homoanatoxin-a (HATX-a) in freshwater samples impacted with cyanobacterial blooms. The presence of CYN in freshwater samples was detected and quantified based on direct injection method, while ATX-a and HATX-a could be determined by both DI and SPE-based methods. Matrix effects (ME) on the signal intensity of the cyanotoxins were systematically evaluated for both direct injection and SPE extract samples. CYN, ATX-a, and HATX-a suffered a significant suppression during UPLC-MS/MS. The selection of internal standards (ISs) for compensating/correcting the losses of target cyanotoxins during sample preparation and matrix effects in UPLC-MS/MS analyses were systematically evaluated. Acetaminophen-d4 (an isotopically labelled acetaminophen) is a suitable internal standard for correcting the ME on the signal intensity of ATX-a and HATX-a, while the use of L-phenylalanine-d5 or caffeine-d9 as IS for correcting ME of these toxins was not efficient, as expected. The method detection limit (MDL) for the target cyanotoxins ranged from 0.6 to 15 ng/L, which is sensitive enough to detect the presence of these toxins in cyanobacterial bloom freshwater. The developed methods were successfully applied for routine monitoring of the occurrence of these cyanotoxins in a local water body. Monitoring results depicted that ATX-a, CYN and HATX-a were ubiquitously detected in water samples, at concentrations ranging from 70 to 24,600 ng/L.

Benthic periphyton from Pennsylvania, USA is a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a)

In 2016, the Pennsylvania Department of Environmental Protection conducted a limited survey of streams in the Susquehanna River basin in Pennsylvania, USA, to screen for microcystins/nodularins, anatoxin-a (ATX) and homoanatoxin-a (HTX). Testing revealed the presence of HTX in samples collected from the Pine Creek basin, with ATX present at lower levels. Microcystins/nodularins (MCs/NODs) were also tested and found to be concomitant, with NOD-R confirmed present by LC-MS/MS.

Planktic Tychonema (Cyanobacteria) in the large lakes south of the Alps: phylogenetic assessment and toxigenic potential.

This work allowed assessing a widespread occurrence of Tychonema bourrellyi in the largest lakes south of the Alps (Garda, Iseo, Como and Maggiore). The taxonomy of the species was confirmed adopting a polyphasic approach, which included microscopic examinations, molecular (16S rRNA and rbcLX sequences) and (Lake Garda) ecological characterisations. Over 70% of the 36 isolates of Tychonema sampled from the four lakes tested positive for the presence of genes implicated in the biosynthesis of anatoxins (anaF and/or anaC) and for the production of anatoxin-a (ATX) and homoanatoxin-a (HTX). A detailed analysis carried out in Lake Garda showed strong ongoing changes in the cyanobacterial community, with populations of Tychonema developing with higher biovolumes compared to the microcystins (MCs) producer Planktothrix rubescens Moreover, the time × depth distribution of Tychonema was paralleled by a comparable distribution of ATX and HTX. The increasing importance of Tychonema in Lake Garda was also suggested by the opposite trends of ATX and MCs observed since 2009. These results suggest that radical changes are occurring in the largest lakes south of the Alps. Their verification and implications will require to be assessed by extending a complete experimental work to the other large perialpine lakes.

Effects of nitrogen and phosphorus on anatoxin-a, homoanatoxin-a, dihydroanatoxin-a and dihydrohomoanatoxin-a production by Phormidium autumnale

Anatoxins are powerful neuromuscular blocking agents produced by some cyanobacteria. Consumption of anatoxin-producing cyanobacterial mats or the water containing them has been linked to numerous animal poisonings and fatalities worldwide. Despite this health risk, there is a poor understanding of the environmental factors regulating anatoxin production. Non-axenic Phormidium autumnale strain CAWBG557 produces anatoxin-a (ATX), homoanatoxin-a (HTX) and their dihydrogen-derivatives dihydroanatoxin-a (dhATX) and dihydrohomoanatoxin-a (dhHTX). The effects of varying nitrogen and phosphorus concentrations on the production of these four variants were examined in batch monocultures. The anatoxin quota (anatoxin per cell) of all four variants increased up to four fold in the initial growth phase (days 0–9) coinciding with the spread of filaments across the culture vessel during substrate attachment. Dihydroanatoxin-a and dhHTX, accounted for over 60% of the total anatoxin quota in each nitrogen and phosphorus treatment. This suggests they are being internally synthesised and not just derived following cell lysis and environmental degradation. The four anatoxin variants differed in their response to varying nitrogen and phosphorus concentrations. Notably, dhATX quota significantly decreased (P ≤ 0.03) when nitrogen and phosphorus concentrations were elevated (nitrogen = 21 mg L−1; phosphorus = 3 mg L−1), while HTX quota increased when the phosphorus concentrations were reduced (ca. < 0.08 mg L−1). This is of concern as HTX has a high toxicity and anatoxin producing P. autumnale blooms in New Zealand usually occur in rivers with low water column dissolved reactive phosphorus.

Spatial and temporal variability in Phormidium mats and associated anatoxin-a and homoanatoxin-a in two New Zealand rivers

Toxic benthic cyanobacterial proliferations in freshwater are becoming increasingly prevalent, and associated animal poisonings are being reported with greater regularity. Despite this, few studies have investigated spatial and temporal variations in freshwater mat-forming Cyanobac- teria and their associated toxin production. Some sections of the Hutt and Wainuiomata rivers (lower North Island, New Zealand) contain extensive Phormidium sp. proliferations that sometimes produce anatoxin-a (ATX) and homoanatoxin-a (HTX). The percentage coverage of Phormidium sp., ATX and/or HTX concentrations and a suite of physicochemical parameters were monitored at 8 sites for 12 mo. The percentage coverage of Phormidium mats was greater in the summer months and these correlated with warmer water temperatures and stable river flows. Flows in excess of 3 times the mean resulted in the removal of Phormidium mats. There was no correlation between the presence/absence of Phormidium mats and water-soluble nutrients. The presence and concentration of ATX and/or HTX and their degradation products, dihydroanatoxin-a and dihydrohomoanatoxin-a, was highly variable across all sites and over time. Anatoxin-a and HTX occurrence was restricted to periods of warm water temperatures (above 13.4°C) and below average river flows.

Fine-scale spatial variability in anatoxin-a and homoanatoxin-a concentrations in benthic cyanobacterial mats: implication for monitoring and management

The purpose of this study was to determine the variability in anatoxin-a (ATX) and homoanatoxin-a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR-based approach to determine ATX- and HTX-production potential. ATX and HTX variability was investigated by collecting 15 samples from 10 × 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography-mass spectrometry (LC-MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co-occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX-and-HTX- and non-ATX-and-HTX-producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC-MS data in only 58% of samples. Fine-scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR-based technique offers the potential to be a useful early warning technique.

Development of solid phase adsorption toxin tracking (SPATT) for monitoring anatoxin-a and homoanatoxin-a in river water

Sampling and monitoring for cyanotoxins can be problematic as concentrations change with environmental and hydrological conditions. Current sampling practices (e.g. grab samples) provide data on cyanotoxins present only at one point in time and may miss areas or times of highest risk. Recent research has identified the widespread distribution of anatoxin-producing benthic cyanobacteria in rivers highlighting the need for development of effective sampling techniques. In this study we evaluated the potential of an in situ method known as solid phase adsorption toxin tracking (SPATT) for collecting and concentrating anatoxin-a (ATX) and homoanatoxin-a (HTX) in river water. Fifteen different adsorption substrates were screened for efficiency of ATX uptake, nine of which retained high proportions (>70%) of ATX. Four substrates were then selected for a 24-h trial in a SPATT bag format in the laboratory. The greatest decrease in ATX in the water was observed with powdered activated carbon (PAC) and Strata-X (a polymeric resin) SPATT bags. A 3-d field study in a river containing toxic benthic cyanobacterial mats was undertaken using PAC and Strata-X SPATT bags. ATX and HTX were detected in all SPATT bags. Surface grab samples were taken throughout the field study and ATX and HTX were only detected in one of the water samples, highlighting the limitations of this currently used method. Both Strata-X and PAC were found to be effective absorbent substrates. PAC has the advantage that it is cheap and readily available and appears to continue to sorb toxins over longer periods than Strata-X. SPATT has the potential to be integrated into current cyanobacterial monitoring programmes and would be a very useful and economical tool for early warning of ATX and HTX contamination in water.

A non-radioactive ligand-binding assay for detection of cyanobacterial anatoxins using Torpedo electrocyte membranes

Anatoxin-a (ANTX) and homoanatoxin-a (HANTX), neurotoxins exclusively produced by cyanobacteria (LD 50 200–250 μg kg −1, i.p. mouse), are agonists of the nicotinic acetylcholine receptors (nAChRs) to which they tightly bind. We have exploited the high affinity of these neurotoxins for the nicotinic receptors to develop a non-radioactive ligand-binding assay using Torpedo electrocyte membranes and biotinylated α-bungarotoxin (Biotin-BgTx) as tracer for detection of this class of toxins. The affinity of the Torpedo nAChRs for Biotin-BgTx was determined by chemiluminescence ( K d = 1.2 × 10 −8 M Biotin-BgTx) or color development ( K d = 3.5 × 10 −8 M Biotin-BgTx). Binding of ANTX or HANTX to the nAChRs competitively inhibits the binding of Biotin-BgTx to the receptors in a concentration-dependent manner (chemiluminescence: IC 50: 6.2 × 10 −8 M ANTX; color development: IC 50: 1.7 × 10 −8 M ANTX). The proposed method was validated by HPLC/MS with detection in the single ion recording mode. The non-radioactive ligand receptor-binding assay was successfully applied to the analysis of extracts prepared from cyanobacteria in culture and from natural habitats, as well as from aqueous samples. This method is suitable for ANTX and HANTX early survey of environmental samples since it requires minimal manipulations, is highly sensitive and gives consistent signal-to-noise ratios.

MALDI-TOF-MS detection of the low molecular weight neurotoxins anatoxin-a and homoanatoxin-a on lyophilized and fresh filaments of axenic Oscillatoria strains

Anatoxin-a (ANTX) and homoanatoxin-a (HANTX) are low molecular weight neurotoxic secondary amines of 165 and 179 Da, respectively. We applied matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the detection of ANTX and HANTX directly on lyophilized and fresh filaments of axenic strains of the genus Oscillatoria, using 2,5-dihydroxybenzoic acid as matrix and purified ANTX and HANTX as references. To counteract the span of low molecular mass ions (< m/ z 1000) generated by the matrix, we induced the matrix-suppression effect to obtain high quality ANTX/HANTX MALDI signals. MALDI desorption/ionization of the matrix-ANTX and the matrix-HANTX generated protonated molecules [M+H] + at m/ z 166.12322 and 180.1372, respectively. The masses obtained from the analysis of lyophilized filaments of the ANTX-producer Oscillatoria sp. strain PCC 9240 ( m/ z 166.15) and of fresh filaments of the HANTX-producers Oscillatoria sp. strains PCC 6506 ( m/ z 180.1375), PCC 9029 ( m/ z 180.1334) and PCC 10111 ( m/ z 180.13996) corresponded to the protonated molecular ions of ANTX and HANTX, respectively. Therefore, the application of MALDI-TOF-MS for the detection of cyanobacterial anatoxins in clonal and axenic strains of the cyanobacterial culture collections worldwide may help to assess ANTX/HANTX incidence among cyanobacteria.

Anatoxins and degradation products, determined using hybrid quadrupole time‐of‐flight and quadrupole ion‐trap mass spectrometry: forensic investigations of cyanobacterial neurotoxin poisoning

The potent neurotoxins from cyanobacteria, anatoxin-a (AN), its methyl analogue, homoanatoxin-a (HMAN), and their degradation products, have been studied using nano-electrospray hybrid quadrupole time-of-flight mass spectrometry (QqTOF-MS). The anatoxin degradation products, which are readily produced in vivo by either reduction or epoxidation, were also examined in this study. The high mass accuracy QqTOF-MS data was used to confirm formula assignments for major product ions and quadrupole ion-trap (QIT)-MS was used to construct fragmentation pathways for anatoxins. Significant differences between these fragmentation pathways were observed. Comparisons between the spectra of compounds that differ in side-chain length (the AN and HMAN series) were used to identify ions that are characteristic of the homologues. The application to forensic samples in which the principal neurotoxin had undergone rapid biodegradation has been demonstrated and used to confirm anatoxin poisoning of dogs.

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water.

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.

Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC–FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2–84.9% at concentrations of 10 μg/l. The R.S.D. values were 1.7–3.9% ( n=8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.