2010, Fischer, Andreas, Höger, Stefan J., Stemmer, Kerstin, Feurstein, Daniel, Knobeloch, Daniel, Nussler, A., Dietrich, Daniel R.

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

2007, Höger, Stefan J., Schmid, Daniela, Blom, Judith F., Ernst, Bernhard, Dietrich, Daniel R.

The microcystin (MC) producing P. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, and raw water quality. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants [Asp3]MC-RR and [Asp3,Dhb7]MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability to quantify [Asp3]MC-RR, [Asp3,Dhb7]MC-RR, and MC-RR was compared to HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, and [Asp3]MC-RR were in the same range (1.9-3.8 and 0.45-0.75 nM). A 50-fold higher concentration of [Asp3,Dhb7]MC-RR (29.8 nM) was necessary to inhibit the PP2A by 50%. The PP1-IC50 of [Asp3,Dhb7]MC-RR was 22-fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capacity. Western blots demonstrated that [Asp3,Dhb7]MC-RR does not covalently bind to PP1. [Asp3,Dhb7]MC-RR has comparable in vivo LD50 values to MC-RR, despite a far lower PP-inhibiting capacity, suggesting that toxicodynamic and toxicokinetic characteristics of [Asp3,Dhb7]MC-RR are responsible for its high in vivo toxicity. The data demonstrate that cPPIA analysis of [Asp3,Dhb7]MC-RR-containing samples prevent reliable MC determination and lead to underestimation of potential toxicity.

2006, Ernst, Bernhard, Neser, Stephan, O'Brien, Evelyn, Höger, Stefan J., Dietrich, Daniel R.

Cyanobacteria occur in surface waters worldwide. Many of these produce peptides and/or alkaloids, which can present a risk for animal and human health. Effective risk assessment and management requires continuous and precise observation and quantification of cyanobacterial cell densities. In this respect, quantification of filamentous Planktothrix species is problematic. The aim of this study was to develop an automated system to count filamentous Planktothrix rubescens using image processing. Furthermore, this study aimed to assess optimum sample volumes and filament density for measurement precision and to validate image processing measurement of P. rubescens for an effective risk assessment.
Three environmental samples and one cultured sample of P. rubescens were collected by filtration onto nitrocellulose filters. Filament lengths were determined using fluorescence microscopy combined with an image processor. Cell density could be calculated from the resulting images. Cyanobacteria could easily be discriminated from algae via their fluorescence properties. The results were found to be independent of the mode of image acquisition. The precision of total filament length determination was dependent on the total filament length on the filter, i.e. analyses of highest precision could be expected for filters containing 2000 20,000 μm filaments per mm2. When using suitable filtration volumes, the detection limits of the described method are sufficient for an effective risk assessment. To summarise, this procedure is a fast, easy and accurate method to determine cell densities of filamentous P. rubescens in water samples without costly and tedious manual handling.

2005, Höger, Stefan J., Hitzfeld, Bettina C., Dietrich, Daniel R.

Toxin-producing cyanobacteria (blue-green algae) are abundant in surface waters used as drinking water resources. The toxicity of one group of these toxins, the microcystins, and their presence in surface waters used for drinking water production has prompted the World Health Organization (WHO) to publish a provisional guideline value of 1.0 μg microcystin (MC)-LR/l drinking water. To verify the efficiency of two different water treatment systems with respect to reduction of cyanobacterial toxins, the concentrations of MC in water samples from surface waters and their associated water treatment plants in Switzerland and Germany were investigated. Toxin concentrations in samples from drinking water treatment plants ranged from below 1.0 μg MC-LR equiv./l to more than 8.0 μg/l in raw water and were distinctly below 1.0 μg/l after treatment. In addition, data to the worldwide occurrence of cyanobacteria in raw and final water of water works and the corresponding guidelines for cyanobacterial toxins in drinking water worldwide are summarized.

2009, Ernst, Bernhard, Höger, Stefan J., O´Brien, Evelyn, Dietrich, Daniel R.

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.
P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m‾² were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml‾¹ (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion. possibly additionally benefiting from high nitrogen loads.
Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb7]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.
In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.

2007, Dietrich, Daniel R., Fischer, A., Michel, C., Höger, Stefan J.

Cyanobacteria are the oldest life forms on earth known to produce a broad spectrum of secondary metabolites. The functions/advantages of most of these secondary metabolites (peptides and alkaloids) are unknown, how-ever, some of them have adverse effects in humans and wildlife, especially when ingested, inhaled or upon dermal exposure. Surprisingly, some of these cyanobacteria are ingested voluntarily. Indeed, for centuries mankind has used cyanobacteria as a protein source, primarily Spirulina species. However, recently also Aphanizomenon flos aquae are used for the pro-duction of so called blue green algae supplements (BGAS), supposedly ef-ficacious for treatment of various diseases and afflictions. Unfortunately, traces of neurotoxins and protein phosphatases (inhibiting compounds) have been detected in BGAS, making these health supplements a good ex-ample for human exposure to a mixture of cyanobacterial toxins in a com-plex matrix. The discussion of this and other possible exposure scenarios, e.g. drinking water, contact during recreational activity, or consumption of contaminated food, can provide insight into the question of whether or not our current risk assessment schemes for cyanobacterial blooms and the toxins contained therein suffice for protection of human health.

2006, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

The microcystin-producing cyanobacterium Planktothrix is one of the most widespread genera amongst toxin producing cyanobacteria in European lakes. In particular, the metalimnic blooms of Planktothrix rubescens have been associated with growing problems in the professional freshwater fishery as a decrease in yearly yields in the important coregonids fishery often coincides with the appearance of P. rubescens. P. rubescens is a cyanobacterial species known to produce toxic compounds, e.g. microcystins. Although microcystins have been reported to affect fish health, behaviour, development and growth and have also been associated with feral fish kills, there is currently no specific information on the effects of toxic Planktothrix filaments in fish and especially coregonids. Therefore, the aim of this study was to investigate the effects of an environmentally relevant dose of P. rubescens filaments orally applied to coregonids and to discuss the findings in the context of microcystin toxicity previously reported in carp and trout.
A single dose of P. rubescens culture, at a density of 80,000 cells per 120 μl, was applied to coregonids thus corresponding to 0.6 0.9 μg microcystin-LRequiv./kg body weight. Behavioural changes and opercular beat rates, growth, hepatosomatic index, condition and plasma glucose were determined. Liver, kidney, gill and the gastrointestinal tract were assessed histopathologically and immunhistologically. Exposed fish showed behavioural changes, increased opercular beat rates and elevated plasma glucose levels, possibly representing a physiological stress response. Histopathological alterations in liver, gastrointestinal tract and kidney, also immunopositive for microcystin suggested causality of tissue damage and the in situ presence of microcystins.
The observed combination of stress and organ damage may explain the frequently reduced weight and thus the fitness noted in coregonids subjected to regular occurrences of stratified and dispersed P. rubescens blooms, e.g. in lake Ammersee, Bavaria, Germany.

2007, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

Planktothrix rubescens belongs to the most ubiquitous cyanobacterial species in mesotrophic and oligotrophic lakes in the pre-alpine regions. In most of these lakes, coregonids are among the dominant species of the ichthyofauna with great importance for the professional fishery. A possible link between the occurrence of toxic Planktothrix blooms and the recurrent slumps in coregonid yields has been suggested. Indeed, acute toxic effects of microcystins and other cyanobacterial toxins have been shown for various fish species. However, chronic exposure scenarios appear to be more common and thus more environmentally realistic than acute intoxications. The aim of this study was therefore to investigate the physiological stress response and organ pathology in coregonids sub-chronically exposed to ambient water containing low, medium and high P. rubescens densities, known to be typical of pre-alpine lakes. Coregonid hatchlings were exposed in four tanks containing 0 (sham-control) and approximately 1500 (low), 15,000 (medium) and 55,000 (high) P. rubescens cells/ml for up to 28 days. Temperature, oxygen concentration, pH-value, P. rubescens cell density and microcystin concentration were recorded and the fish were observed for behavioural changes and examined for parasite infestations. Gill ventilation rates, general condition factors and mortalities were determined and liver, kidney, gut and gill were assessed histopathologically and immunhistologically.
Depending on the cell density, exposed fish showed behavioural changes, including increased ventilation rates possibly representing a physiological stress response. Susceptibility to ectoparasitic infestation and increased mortality in exposed fish suggested P. rubescens associated effects on fish fitness. Histopathological alterations in liver, gastrointestinal tract and kidney, which were also immunopositive for microcystin suggested causality of tissue damage and the presence of microcystins. In contrast, observed gill pathology appeared to result primarily from mechanical abrasion and irritation due to ectoparasitic infestation. The current exposure experiment confirmed the hypothesis that subchronic and chronic exposure to low cyanobacterial cell densities and hence microcystins can exacerbate physiological stress and sustained pathological alterations in exposed coregonids. The study therefore supports the theory that P. rubescens blooms may be causal to the observed weight reduction and hence fitness of coregonids in pre-alpine lakes such as Lake Ammersee (Germany).

2006, Jungblut, Anne-Dorothee, Höger, Stefan J., Mountfort, Doug, Hitzfeld, Bettina C., Dietrich, Daniel R., Neilan, Brett A.

Cyanobacteria are well known for their production of non-ribosomal cyclic peptide toxins, including microcystin, in temperate and tropical regions, however, the production of these compounds in extremely cold environments is still largely unexplored. Therefore, we investigated the production of protein phosphatase inhibiting microcystins by Antarctic cyanobacteria. We have identified microcystin-LR and for the first time [d-Asp3] microcystin-LR by mass spectrometric analysis in Antarctic cyanobacteria. The microcystins were extracted from a benthic microbial community that was sampled from a meltwater pond (Fresh Pond, McMurdo Ice Shelf, Antarctica). The extracted cyanobacterial cyclic peptides were equivalent to 11.4 ng MC-LR per mg dry weight by semi-quantitative analyses using HPLC-DAD and the protein phosphatase inhibition assay. Furthermore, we were able to identify the presence of cyanobacterial non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes in total DNA extracts from the mat community.

2005, Dietrich, Daniel R., Höger, Stefan J.

This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcystins and compares this to the guidance values proposed for microcystins in water by the World Health Organization, and for blue-green algal food supplements by the Oregon State Department of Health. The basis of the risk assessment underlying these guidance values is viewed as being critical due to overt deficiencies in the data used for its generation: (i) use of one microcystin congener only (microcystin-LR), while the other presently known nearly 80 congeners are largely disregarded, (ii) new knowledge regarding potential neuro and renal toxicity of microcystins in humans and (iii) the inadequacies of assessing realistic microcystin exposures in humans and especially in children via blue-green algal food supplements. In reiterating the state-of-the-art toxicology database on microcystins and in the light of new data on the high degree of toxin contamination of algal food supplements, this review clearly demonstrates the need for improved kinetic data of microcystins in humans and for discussion concerning uncertainty factors, which may result in a lowering of the present guidance values and an increased routine control of water bodies and food supplements for toxin contamination. Similar to the approach taken previously by authorities for dioxin or PCB risk assessment, the use of a toxin equivalent approach to the risk assessment of microcystins is proposed.