James S. Metcalf

Cyanobacterial toxins, exposure routes and human health

The production of potent toxins by bloom-, scum- and mat-forming cyanobacteria, in fresh-, brackish and marine waters, appears to be a global phenomenon. Cyanobacterial toxins can also be produced by cyanobacteria from terrestrial sources. The range and number of known cyanobacterial toxins are increasing apace as associated poisoning incidents are investigated, and increasingly powerful analytical methods are applied to complement toxicity-based studies on both natural samples and laboratory isolates of cyanobacteria. Water quality management to reduce toxic cyanobacterial mass developments, and schemes to mitigate the potential effects of cyanobacterial toxins, require an understanding of the occurrence and properties of the toxins and of the exposure routes via which the toxins present risks to health. Here, we review advances in the recognition of cyanobacterial toxins and their toxicity, and of the exposure routes with reference to human health, namely via skin contact, inhalation, haemodialysis and ...

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

ABSTRACT Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m−2 s−1 on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell−1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m−2 s−1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m−2 s−1 than at 10 μmol of photons m−2 s−1 and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.

Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya

Cyanobacterial mats at hot springs on the shore of the alkaline Lake Bogoria, Kenya, were investigated regarding species community and cyanobacterial toxin content. The hepatotoxins microcystin-LR, -RR, -LF and -YR, and the neurotoxin anatoxin-a were present. The mats were dominated by Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. The concentration of microcystins in mat samples, ranged from 221 to 845 microg microcystin-LR equivalents g(-1) DW of mat. Anatoxin-a concentrations ranged from 10 to 18 microg g(-1) DW of mat. A contribution of the cyanobacterial toxins from the hot spring mats to the mass mortalities of Lesser Flamingos is suggested by: (a), the presence of hot spring cyanobacterial cells and cell fragments, and high concentrations of the cyanobacterial hepato- and neurotoxins in flamingo stomach contents and faecal pellets; (b), observations of neurological signs of bird poisoning at the lake. Cyanobacterial toxins in stomach contents, intestine and fecal pellets were 0.196 microg g(-1) fresh weight (FW) for the microcystins and 4.34 microg g(-1) FW for anatoxin-a. Intoxication with cyanobacterial toxins could occur by uptake of detached cyanobacterial cells from the mats, as the flamingos need to drink fresh or brackish water, and to wash their feathers daily, which they do in the vicinity of the hot springs, where salinity is lower than in the main body of water of the lake.

Co-occurrence of β-N-methylamino-l-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990–2004

The neurotoxic amino acid, beta-N-methylamino-L-alanine, was found to be present in all of 12 analysed samples of cyanobacterial blooms, scums and mats, which had been collected in seven years between 1990 and 2004 inclusive and stored at -20 degrees C. BMAA identification was by high performance liquid chromatography with fluorescence detection and by triple quadrapole mass spectrometry after derivatization. The samples originated from 11 freshwater lakes and 1 brackish waterbody, used either for drinking water, recreation, or both. BMAA was present at between 8 and 287 microg g(-1) cyanobacterial dry weight and was present as both the free amino acid and associated with precipitated proteins. Ten of the samples contained additional cyanotoxins (including microcystins, anatoxin-a, nodularin and saxitoxin) at the time of sample collection. Five of the samples were associated with animal deaths, attributable at the time of sample collection, to microcystins, nodularin or anatoxin-a. The data demonstrate the presence of BMAA by high performance liquid chromatography and mass spectrometry in a diverse range of cyanobacterial bloom samples from high resource waterbodies. Furthermore, samples collected over several years shows that BMAA can co-occur with other known cyanotoxins in such waterbodies. Health risk assessment of cyanobacterial BMAA in waterbodies is suggested.

Effects of enteric bacterial and cyanobacterial lipopolysaccharides, and of microcystin-LR, on glutathione S-transferase activities in zebra fish (Danio rerio).

Cyanobacteria (blue-green algae) can produce a variety of toxins including hepatotoxins e.g. microcystins, and endotoxins such as lipopolysaccharides (LPS). The combined effects of such toxins on fish are little known. This study examines the activities of microsomal (m) and soluble (s) glutathione S-transferases (GST) from embryos of the zebra fish, Danio rerio at the prim six embryo stage, which had been exposed since fertilisation to LPS from different sources. A further aim was to see how activity was affected by co-exposure to LPS and microcystin-LR (MC-LR). LPS were obtained from Salmonella typhimurium, Escherichia coli, a laboratory culture of Microcystis CYA 43 and natural cyanobacterial blooms of Microcystis and Gloeotrichia. Following in vivo exposure of embryos to each of the LPS preparations, mGST activity was significantly reduced (from 0.50 to between 0.06 and 0.32 nanokatals per milligram (nkat mg(-1)) protein). sGST activity in vivo was significantly reduced (from 1.05 to between 0.19 and 0.22 nkat mg(-1) protein) after exposure of embryos to each of the cyanobacterial LPS preparations, but not in response to S. typhimurium or E. coli LPS. Activities of both m- and sGSTs were reduced after co-exposure to MC-LR and cyanobacterial LPS, but only mGST activity was reduced in the S. typhimurium and E. coli LPS-treated embryos. In vitro preparations of GST from adult and prim six embryo D. rerio showed no significant changes in enzyme activity in response to the LPS preparations with the exception of Gloeotrichia bloom LPS, where mGST was reduced in adult and embryo preparations. The present study represents the first investigations into the effects of cyanobacterial LPS on the phase-II microcystin detoxication mechanism. LPS preparations, whether from axenic cyanobacteria or cyanobacterial blooms, are potentially capable of significantly reducing activity of both the s- and mGSTs, so reducing the capacity of D. rerio to detoxicate microcystins. The results presented here have wide ranging implications for both animal and human health.

Retention of Microcystis aeruginosa and microcystin by salad lettuce (Lactuca sativa) after spray irrigation with water containing cyanobacteria

Colonies and single cells of Microcystis aeruginosa and the hepatotoxin microcystin were retained by salad lettuce after growth with spray irrigation water containing the microcystin-producing cyanobacteria. These findings are discussed in terms of crop spray irrigation with water containing cyanobacteria and potential human exposure to cyanobacterial toxins via plant foods grown in such circumstances.

Cyanobacteria and BMAA exposure from desert dust: A possible link to sporadic ALS among Gulf War veterans

Abstract Veterans of the 1990–1991 Gulf War have been reported to have an increased incidence of amyotrophic lateral sclerosis (ALS) compared to personnel who were not deployed. An excess of ALS cases was diagnosed in Gulf War veterans younger than 45 years of age. Increased ALS among Gulf War veterans appears to be an outbreak time-limited to the decade following the Gulf War. Seeking to identify biologically plausible environmental exposures, we have focused on inhalation of cyanobacteria and cyanotoxins carried by dust in the Gulf region, particularly Qatar. Cyanobacterial crusts and mats are widespread in the deserts of Qatar, occupying up to 56% of the available area in some microhabitats. These cyanobacterial crusts, which help bind the desert sands, are dormant throughout most of the year, but during brief spring rains actively photosynthesize. When disturbed by vehicular traffic or other military activities, the dried crusts and mats can produce significant dust. Using HPLC/FD, an amino acid analyzer, UPLC/MS, and triple quadrupole LC/MS/MS we find that the dried crusts and mats contain neurotoxic cyanobacterial toxins, including β-N-methylamino-L-alanine (BMAA) and 2,4 diaminobutyric acid (DAB). If dust containing cyanobacteria is inhaled, significant exposure to BMAA and other cyanotoxins may occur. We suggest that inhalation of BMAA, DAB, and other aerosolized cyanotoxins may constitute a significant risk factor for the development of ALS and other neurodegenerative diseases.

Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin

Abstract A novel conjugation method was developed by linking the hapten, the cyanobacterial hepatotoxin microcystin-LR, via 2-mercaptoethylamine to keyhole limpet haemocyanin. Polyclonal antisera were raised against this conjugate and an indirect competitive immunoassay (ELISA) developed which can detect purified microcystin-LR and the toxin in extracts of cyanobacteria from fresh-, brackish and marine waters. The cross-reactivity of the microcystin-LR antibodies was investigated with a range of purified microcystin variants (-LR, -LA, -LY, -LW, -LF, -D-Asp 3 -RR, and -Asp 3 ( Z )-Dhb 7 -HtyR) and nodularin. The antibodies cross-reacted well with all microcystin and nodularin variants. The microcystin-LA was the most readily detectable, followed by microcystins-LR, -LF, -LW, -D-Asp 3 -RR, -LY, nodularin and microcystin-Asp 3 ( Z )-Dhb 7 -HtyR. Extracts from several genera of cyanobacteria were investigated by the ELISA and the results compared to high performance liquid chromatography with diode array detection (HPLC–DAD). By the ELISA procedure, 60% were positive, compared to detection of microcystins in 45% of the samples by HPLC–DAD. Analysis of microcystin-LR equivalents by ELISA and HPLC–DAD showed good correlation ( R =0.96, p −10 ). The application of the anti-microcystin-LR antibodies for the detection and quantification of microcystins and nodularin in environmental water samples is discussed.

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.

Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain

Neurofibrillary tangles (NFT) and β-amyloid plaques are the neurological hallmarks of both Alzheimers disease and an unusual paralytic illness suffered by Chamorro villagers on the Pacific island of Guam. Many Chamorros with the disease suffer dementia, and in some villages one-quarter of the adults perished from the disease. Like Alzheimers, the causal factors of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) are poorly understood. In replicated experiments, we found that chronic dietary exposure to a cyanobacterial toxin present in the traditional Chamorro diet, β-N-methylamino-l-alanine (BMAA), triggers the formation of both NFT and β-amyloid deposits similar in structure and density to those found in brain tissues of Chamorros who died with ALS/PDC. Vervets (Chlorocebus sabaeus) fed for 140 days with BMAA-dosed fruit developed NFT and sparse β-amyloid deposits in the brain. Co-administration of the dietary amino acid l-serine with l-BMAA significantly reduced the density of NFT. These findings indicate that while chronic exposure to the environmental toxin BMAA can trigger neurodegeneration in vulnerable individuals, increasing the amount of l-serine in the diet can reduce the risk.