Daniel Dietrich | University of Konstanz, Germany (original) (raw)

Papers by Daniel Dietrich

Research paper thumbnail of Chapter 35: Risk Assessment Workgroup Report

Research paper thumbnail of ABSTRACT Keyword&quot

uptake and toxicity of microcystins in primary murine whole brain cells

Research paper thumbnail of Human MRP2 exports MC-LR but not the glutathione conjugate

Chemico-Biological Interactions, 2019

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as li... more Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters-MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and-RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.

Research paper thumbnail of Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

Toxins, 2019

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increa... more Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuter...

Research paper thumbnail of Toxin mixture in cyanobacterial blooms – a critical comparison of reality with current procedures employed in human health risk assessment

Advances in Experimental Medicine and Biology

Cyanobacteria are the oldest life forms on earth known to produce a broad spectrum of secondary m... more 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, however, 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 production of so called blue green algae supplements (BGAS), supposedly efficacious 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 example for human exposure to a mixture of cyanobacterial toxins in a complex 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.

Research paper thumbnail of Molecular cloning and functional characterization of a rainbow trout liver Oatp

Toxicology and Applied Pharmacology

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.... more Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, r...

Research paper thumbnail of Zebrafish Oatp-mediated transport of microcystin congeners

Archives of toxicology, Jan 9, 2015

Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard f... more Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard for aquatic species. As MC congeners vary in their toxicity, the congener composition of a bloom primarily dictates the severity of adverse effects and appears primarily to be governed by toxicokinetics, i.e., whether transport of MCs occurs via organic anion-transporting polypeptides (Oatps). Differences in observed MC toxicity in various fish species suggest differential expression of Oatp subtypes leading to varying tissue distribution of the very same MC congener within different species. The objectives of this study were the functional characterization and analysis of the tissue distribution of Oatp subtypes in zebrafish (Danio rerio) as a surrogate model for cyprinid fish. Zebrafish Oatps (zfOatps) were cloned, and the organ distribution was determined at the mRNA level. zfOatps were transiently expressed in HEK293 cells for functional characterization using the Oatp substrates estr...

Research paper thumbnail of Enzymatic analysis of liver samples from rainbow trout for diagnosis of blue-green algae-induced toxicosis

American journal of veterinary research, 1995

Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent an... more Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent and selective inhibitors of protein phosphatases 1 and 2A. We adapted existing enzymatic techniques to analyze the liver of rainbow trout after oral administration of hepatotoxic cyanobacteria. Liver tissue was removed 3 and 12 hours after treatment, and phosphatase activity was determined in liver extracts, using a specific phosphoprotein substrate. In all samples from fish exposed to toxic cyanobacteria, phosphatase activity was suppressed, whereas the control enzyme, lactate dehydrogenase, present in the same liver extract, was not affected by cyanobacteria. Thus, experimental poisoning by hepatotoxic cyanobacteria resulted in an abnormally low ratio of phosphatase to lactate dehydrogenase activity in the liver extracts. These results indicate that specific inhibition of phosphatases 1 and 2A may provide a useful diagnostic tool to determine the early effects of cyanobacteria toxic pept...

Research paper thumbnail of Molecular cloning and functional characterization of a rainbow trout liver Oatp

Toxicology and Applied Pharmacology, 2014

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.... more Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K m value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K m value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications.

Research paper thumbnail of Comparison of two ELISA-based methods for the detection of microcystins in blood serum

Chemico-biological interactions, Jan 7, 2014

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC co... more Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC-MS, GC-MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC-MS methodology would appear to allow detection of these MC levels. However, LC-MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could ...

Research paper thumbnail of Risk Assessment Workgroup Report

Advances in Experimental Medicine and Biology

Research paper thumbnail of Chapter 35: Risk Assessment Workgroup Report

Research paper thumbnail of Biochemical characterization of microcystin toxicity in rainbow trout (Oncorhynchus mykiss)

Research paper thumbnail of Toxin production in cyanobacterial mats from ponds on the McMurdo Ice Shelf, Antarctica

Toxicon, 2000

Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of ... more Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of these toxins, however, remains largely unclear. Toxic properties of cyanobacteria collected in Antarctica were investigated to determine whether toxin-producing species can also be found under these environmental conditions. Samples were collected from meltwater ponds on the McMurdo Ice Shelf, Antarctica in the summers of 1997 to 1999. These ponds are colonized by benthic algae and cyanobacterial mats. Oscillatoriales, Nodularia sp., and Nostoc sp. constituted the major taxa in freshwater ponds, while Nostoc sp. was missing from brackish and saline ponds. Samples were taken from either¯oating, submerged or benthic mats, and extracted for in vitro toxicity testing. The presence of toxins was determined by the phosphatase-inhibition assay and by high performance liquid chromatography. The cytotoxic properties of the extracts were investigated in hepatocytes determining 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism and trypan blue dye exclusion. The results show that all cyanobacterial extracts display phosphatase-inhibiting activity, of which approximately half had signi®cantly greater than 50% inhibiting activity. The presence of nodularin and microcystin-LR was established by high performance liquid chromatography. Cytotoxic properties, independent of the phosphatase inhibiting activity, were also detected. Toxic strains of cyanobacteria can therefore also be found in Antarctica and this ®nding may lead to further insight into potential ecological roles of cyanobacterial phosphatase inhibiting toxins.

Research paper thumbnail of Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?

Toxicology and Applied Pharmacology, 2005

This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcys... more 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.

Research paper thumbnail of Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

Toxicology and Applied Pharmacology, 2009

Keyword" Cyanobacteria Toxin Microcystin Protei n phosphatase Oatp/OATP Neurotoxicity Primary who... more Keyword" Cyanobacteria Toxin Microcystin Protei n phosphatase Oatp/OATP Neurotoxicity Primary whole brain cells Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro-and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brainbarrier (BBB) and blood-cerebrospinal-f1uid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses. the presence of murine Oatp (rnOatp) in primary murine whole brain cells (mWBC) was invt'stigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR,-LW,-LFJ with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by-LW and-LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 pM) and BSP (50 pM). MC-Western blot analys is demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vieTO data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

Research paper thumbnail of Toxin content and cytotoxicity of algal dietary supplements

Toxicology and Applied Pharmacology, 2012

Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially dis... more Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph.flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in AS49 cells as well as fortoxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELlSA and (3) LC-MSjMS. In addition, all samples were analyzed by PeR for the presence of the mcyE gene, a part of the microcystin and nodulatin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were s 1 IJg MC-LR equivalents g-l dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of AplL flos-aquae products, whether pure or mixed formuations' for human consumption appear highly questionable.

Research paper thumbnail of Quantitative assessment of aerosolized cyanobacterial toxins at two New Zealand lakes

Journal of Environmental Monitoring, 2011

The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular... more The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular protein phosphatases. Toxicity primarily evolves following ingestion of cyanobacterial material or toxins and results in liver and renal pathology. Ingestion is the main route of exposure in the World Health Organizations current risk assessment of nodularin and microcystins. Nasally applied microcystin appears to have a 10-fold higher availability and toxicity than orally ingested toxins, suggesting that aerosolized toxins could represent a major risk for human populations close to lakes with cyanobacterial blooms. In this study, nodularin and microcystin levels in aerosols were assessed using high and low volume air samplers for 4, 12 and 24 h periods at lakes Forsyth and Rotorua (South Island, New Zealand). These lakes were experiencing blooms of Nodularia spumigena and Microcystis sp., respectively. Using the high volume samplers up to 16.2 pg m À3 of nodularin and 1.8 pg m À3 of microcystins were detected in the air. Aerosolized nodularin and microcystins do not appear to represent an acute or chronic hazard to humans. The latter was concluded based on calculations using average human air intakes, the highest nodularin or microcystin concentrations measured in the air in this study, and assuming inhalatory toxicities comparable to toxicological data obtained following intraperitoneal applications in mice. However, as the toxin concentrations in the air were calculated over extended sampling periods, peak values may be underestimated. Aerosolized toxins should be considered when developing risk assessments particularly for lakeside populations and recreational users where inhalation of cyanotoxins may be a secondary exposure source to a primary oral exposure.

Research paper thumbnail of Abundance and toxicity of Planktothrix rubescens in the pre-alpine Lake Ammersee, Germany

Harmful Algae, 2009

Regular occurrences of the cyanobacterium Plankcotlllix rubescens have been observed in several l... more Regular occurrences of the cyanobacterium Plankcotlllix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothn",~ species are known to produce microcystins, potent pllOsphatase inhibitors that have been associated with morbidities and mortalities in hUinans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of r. 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 cm) 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-' (summer). The results demonstrate that r. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the mNalimnion. Moreover, the r. 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-EUSA measurements, respectively. The main microcystin congeners in the seston samples were ]Asp I]-MC-RR and [Asp),Dhb 7 ]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the JJO Table 1 Size and nutritional status in (pre-) alpine lakes with documented P. rubescens abundance during the last decade Country/lake Size (km 2) Trophic status Abundallce Referellces

Research paper thumbnail of Presence of Planktothrix sp. and cyanobacterial toxins in Lake Ammersee, Germany and their impact on whitefish (Coregonus lavaretus L.)

Environmental Toxicology, 2001

Due to the increasing oligotrophy of Lake Ammersee, southern Germany, metalimnic Planktothrix hav... more Due to the increasing oligotrophy of Lake Ammersee, southern Germany, metalimnic Planktothrix have become one of the dominant planktonic species causing regular blooms. Whitefish (Coregonus lavaretus) is the dominant local fish species with great importance for the fishing industry. Recently, whole age groups of this fish species have disappeared and since 1991, average body‐weight has decreased. The causes for this remain unclear. Planktothrix species produce the cyclic peptide toxin desmethyl microcystin‐RR, which inhibits glycogen metabolism and has detrimental effects on the development of aquatic organisms. During blooms, gut contents of whitefish displayed a blue discoloration, possibly representing phycobiliproteins typical for cyanobacteria. This study aimed to elucidate the impact of Planktothrix blooms on fish population dynamics. Planktothrix cell counts, performed by epifluorescence microscopy, showed blooms to contain up to 80,000 cells/ml. Microcystin levels of 1−5μg/m...

Research paper thumbnail of Chapter 35: Risk Assessment Workgroup Report

Research paper thumbnail of ABSTRACT Keyword&quot

uptake and toxicity of microcystins in primary murine whole brain cells

Research paper thumbnail of Human MRP2 exports MC-LR but not the glutathione conjugate

Chemico-Biological Interactions, 2019

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as li... more Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters-MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and-RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.

Research paper thumbnail of Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

Toxins, 2019

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increa... more Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuter...

Research paper thumbnail of Toxin mixture in cyanobacterial blooms – a critical comparison of reality with current procedures employed in human health risk assessment

Advances in Experimental Medicine and Biology

Cyanobacteria are the oldest life forms on earth known to produce a broad spectrum of secondary m... more 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, however, 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 production of so called blue green algae supplements (BGAS), supposedly efficacious 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 example for human exposure to a mixture of cyanobacterial toxins in a complex 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.

Research paper thumbnail of Molecular cloning and functional characterization of a rainbow trout liver Oatp

Toxicology and Applied Pharmacology

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.... more Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, r...

Research paper thumbnail of Zebrafish Oatp-mediated transport of microcystin congeners

Archives of toxicology, Jan 9, 2015

Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard f... more Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard for aquatic species. As MC congeners vary in their toxicity, the congener composition of a bloom primarily dictates the severity of adverse effects and appears primarily to be governed by toxicokinetics, i.e., whether transport of MCs occurs via organic anion-transporting polypeptides (Oatps). Differences in observed MC toxicity in various fish species suggest differential expression of Oatp subtypes leading to varying tissue distribution of the very same MC congener within different species. The objectives of this study were the functional characterization and analysis of the tissue distribution of Oatp subtypes in zebrafish (Danio rerio) as a surrogate model for cyprinid fish. Zebrafish Oatps (zfOatps) were cloned, and the organ distribution was determined at the mRNA level. zfOatps were transiently expressed in HEK293 cells for functional characterization using the Oatp substrates estr...

Research paper thumbnail of Enzymatic analysis of liver samples from rainbow trout for diagnosis of blue-green algae-induced toxicosis

American journal of veterinary research, 1995

Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent an... more Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent and selective inhibitors of protein phosphatases 1 and 2A. We adapted existing enzymatic techniques to analyze the liver of rainbow trout after oral administration of hepatotoxic cyanobacteria. Liver tissue was removed 3 and 12 hours after treatment, and phosphatase activity was determined in liver extracts, using a specific phosphoprotein substrate. In all samples from fish exposed to toxic cyanobacteria, phosphatase activity was suppressed, whereas the control enzyme, lactate dehydrogenase, present in the same liver extract, was not affected by cyanobacteria. Thus, experimental poisoning by hepatotoxic cyanobacteria resulted in an abnormally low ratio of phosphatase to lactate dehydrogenase activity in the liver extracts. These results indicate that specific inhibition of phosphatases 1 and 2A may provide a useful diagnostic tool to determine the early effects of cyanobacteria toxic pept...

Research paper thumbnail of Molecular cloning and functional characterization of a rainbow trout liver Oatp

Toxicology and Applied Pharmacology, 2014

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.... more Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K m value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K m value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications.

Research paper thumbnail of Comparison of two ELISA-based methods for the detection of microcystins in blood serum

Chemico-biological interactions, Jan 7, 2014

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC co... more Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC-MS, GC-MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC-MS methodology would appear to allow detection of these MC levels. However, LC-MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could ...

Research paper thumbnail of Risk Assessment Workgroup Report

Advances in Experimental Medicine and Biology

Research paper thumbnail of Chapter 35: Risk Assessment Workgroup Report

Research paper thumbnail of Biochemical characterization of microcystin toxicity in rainbow trout (Oncorhynchus mykiss)

Research paper thumbnail of Toxin production in cyanobacterial mats from ponds on the McMurdo Ice Shelf, Antarctica

Toxicon, 2000

Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of ... more Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of these toxins, however, remains largely unclear. Toxic properties of cyanobacteria collected in Antarctica were investigated to determine whether toxin-producing species can also be found under these environmental conditions. Samples were collected from meltwater ponds on the McMurdo Ice Shelf, Antarctica in the summers of 1997 to 1999. These ponds are colonized by benthic algae and cyanobacterial mats. Oscillatoriales, Nodularia sp., and Nostoc sp. constituted the major taxa in freshwater ponds, while Nostoc sp. was missing from brackish and saline ponds. Samples were taken from either¯oating, submerged or benthic mats, and extracted for in vitro toxicity testing. The presence of toxins was determined by the phosphatase-inhibition assay and by high performance liquid chromatography. The cytotoxic properties of the extracts were investigated in hepatocytes determining 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism and trypan blue dye exclusion. The results show that all cyanobacterial extracts display phosphatase-inhibiting activity, of which approximately half had signi®cantly greater than 50% inhibiting activity. The presence of nodularin and microcystin-LR was established by high performance liquid chromatography. Cytotoxic properties, independent of the phosphatase inhibiting activity, were also detected. Toxic strains of cyanobacteria can therefore also be found in Antarctica and this ®nding may lead to further insight into potential ecological roles of cyanobacterial phosphatase inhibiting toxins.

Research paper thumbnail of Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?

Toxicology and Applied Pharmacology, 2005

This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcys... more 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.

Research paper thumbnail of Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

Toxicology and Applied Pharmacology, 2009

Keyword" Cyanobacteria Toxin Microcystin Protei n phosphatase Oatp/OATP Neurotoxicity Primary who... more Keyword" Cyanobacteria Toxin Microcystin Protei n phosphatase Oatp/OATP Neurotoxicity Primary whole brain cells Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro-and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brainbarrier (BBB) and blood-cerebrospinal-f1uid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses. the presence of murine Oatp (rnOatp) in primary murine whole brain cells (mWBC) was invt'stigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR,-LW,-LFJ with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by-LW and-LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 pM) and BSP (50 pM). MC-Western blot analys is demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vieTO data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

Research paper thumbnail of Toxin content and cytotoxicity of algal dietary supplements

Toxicology and Applied Pharmacology, 2012

Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially dis... more Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph.flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in AS49 cells as well as fortoxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELlSA and (3) LC-MSjMS. In addition, all samples were analyzed by PeR for the presence of the mcyE gene, a part of the microcystin and nodulatin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were s 1 IJg MC-LR equivalents g-l dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of AplL flos-aquae products, whether pure or mixed formuations' for human consumption appear highly questionable.

Research paper thumbnail of Quantitative assessment of aerosolized cyanobacterial toxins at two New Zealand lakes

Journal of Environmental Monitoring, 2011

The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular... more The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular protein phosphatases. Toxicity primarily evolves following ingestion of cyanobacterial material or toxins and results in liver and renal pathology. Ingestion is the main route of exposure in the World Health Organizations current risk assessment of nodularin and microcystins. Nasally applied microcystin appears to have a 10-fold higher availability and toxicity than orally ingested toxins, suggesting that aerosolized toxins could represent a major risk for human populations close to lakes with cyanobacterial blooms. In this study, nodularin and microcystin levels in aerosols were assessed using high and low volume air samplers for 4, 12 and 24 h periods at lakes Forsyth and Rotorua (South Island, New Zealand). These lakes were experiencing blooms of Nodularia spumigena and Microcystis sp., respectively. Using the high volume samplers up to 16.2 pg m À3 of nodularin and 1.8 pg m À3 of microcystins were detected in the air. Aerosolized nodularin and microcystins do not appear to represent an acute or chronic hazard to humans. The latter was concluded based on calculations using average human air intakes, the highest nodularin or microcystin concentrations measured in the air in this study, and assuming inhalatory toxicities comparable to toxicological data obtained following intraperitoneal applications in mice. However, as the toxin concentrations in the air were calculated over extended sampling periods, peak values may be underestimated. Aerosolized toxins should be considered when developing risk assessments particularly for lakeside populations and recreational users where inhalation of cyanotoxins may be a secondary exposure source to a primary oral exposure.

Research paper thumbnail of Abundance and toxicity of Planktothrix rubescens in the pre-alpine Lake Ammersee, Germany

Harmful Algae, 2009

Regular occurrences of the cyanobacterium Plankcotlllix rubescens have been observed in several l... more Regular occurrences of the cyanobacterium Plankcotlllix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothn",~ species are known to produce microcystins, potent pllOsphatase inhibitors that have been associated with morbidities and mortalities in hUinans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of r. 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 cm) 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-' (summer). The results demonstrate that r. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the mNalimnion. Moreover, the r. 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-EUSA measurements, respectively. The main microcystin congeners in the seston samples were ]Asp I]-MC-RR and [Asp),Dhb 7 ]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the JJO Table 1 Size and nutritional status in (pre-) alpine lakes with documented P. rubescens abundance during the last decade Country/lake Size (km 2) Trophic status Abundallce Referellces

Research paper thumbnail of Presence of Planktothrix sp. and cyanobacterial toxins in Lake Ammersee, Germany and their impact on whitefish (Coregonus lavaretus L.)

Environmental Toxicology, 2001

Due to the increasing oligotrophy of Lake Ammersee, southern Germany, metalimnic Planktothrix hav... more Due to the increasing oligotrophy of Lake Ammersee, southern Germany, metalimnic Planktothrix have become one of the dominant planktonic species causing regular blooms. Whitefish (Coregonus lavaretus) is the dominant local fish species with great importance for the fishing industry. Recently, whole age groups of this fish species have disappeared and since 1991, average body‐weight has decreased. The causes for this remain unclear. Planktothrix species produce the cyclic peptide toxin desmethyl microcystin‐RR, which inhibits glycogen metabolism and has detrimental effects on the development of aquatic organisms. During blooms, gut contents of whitefish displayed a blue discoloration, possibly representing phycobiliproteins typical for cyanobacteria. This study aimed to elucidate the impact of Planktothrix blooms on fish population dynamics. Planktothrix cell counts, performed by epifluorescence microscopy, showed blooms to contain up to 80,000 cells/ml. Microcystin levels of 1−5μg/m...