Stephan Pflugmacher

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

Uptake and effects of microcystin-LR on detoxication enzymes of early life stages of the zebra fish (Danio rerio)

The effects of cyanotoxins on fish have been studied mainly in adults, rather than in early life stages which could be more sensitive or, in view of their immobility, more readily affected. The uptake of microcystin‐LR by different early life stages of the zebra fish (Danio rerio) was investigated using 14C‐labelled microcystin‐LR. The effects on the activity of the detoxication enzymes, microsomal and soluble glutathione S‐transferases (GST), and glutathione peroxidase (GP‐X) were examined. There was a detectable uptake of microcystin from the first day of embryonic development up to 5 day old larvae. On average, an absorption of 0.5 ng microcystin for eggs and eleuthero‐embryos was calculated over the entire exposure time. Because of the differences in volume of the eggs and eleuthero‐embryos, there was an increase in the microcystin‐LR concentration between these stages. In the eggs, approximately 25% of the medium concentration was found, and in eleuthero‐embryos an equilibrium between fish and medium was reached.

Exposure to human pharmaceuticals Carbamazepine, Ibuprofen and Bezafibrate causes molecular effects in Dreissena polymorpha

Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels.

Effects of the cyanobacterial toxin microcystin-LR on detoxication enzymes in aquatic plants

The enrichment of water bodies with plant nutrients often results in mass growths of cyanobacteria which can produce a range of toxins. Cyanobacterial toxins have adverse effects on fish, birds, and mammals, and are being increasingly recognized as animal and human health hazards. The effects of cyanobacterial toxins on plants, particularly aquatic macrophytes, are little understood. The uptake of microcystin‐LR (MC‐LR) by Ceratophyllum demersum was detected with approximately 11.2% of the applied 14C‐labelled microcystin being taken up after 7 days of exposure. The effects of various concentrations of this toxin on the detoxication enzyme glutathione S‐transferase (microsomal and soluble forms) were investigated. Microsomal glutathione S‐transferase activity was increased in C. demersum exposed to concentrations of MC‐LR above 0.12 μg/L, but reduced at concentrations above 1.0 μg/L, whereas soluble glutathione S‐transferase activity was increased by exposure to MC‐LR concentrations above 0.5 μg/L. No reduction of soluble GST activity was observed. The activity of both microsomal and soluble GST systems, freshly prepared from a range of plants, was inhibited in the presence of 0.5 μg/L MC‐LR. The results indicate a detoxication pathway for MC‐LR in aquatic plants. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 111–115, 1999

Accumulation of nodularin-like compounds from the cyanobacterium Nodularia spumigena and changes in acetylcholinesterase activity in the clam Macoma balthica during short-term laboratory exposure.

In this laboratory study the effects of the cyanobacterium Nodularia spumigena (strain AV1) that produces hepatotoxic nodularin (NODLN), non-toxic Nodularia sphaerocarpa (strain UP16f) and purified NODLN on the infaunal clam Macoma balthica from the Baltic Sea were examined. N. sphaerocarpa (2.4 and 12.5 mg dw l(-1)), N. spumigena (4 and 20 mg dw l(-1), intracellular NODLN content ca. 4 and 20 microg l(-1)) and purified NODLN (10 and 50 microg l(-1)) were applied in experimental tanks at 24 h intervals for 96 h. Water samples were taken during the experiment for the measurement of soluble NODLN concentrations. The concentrations of total hepatotoxins in the soft tissues were analysed with enzyme-linked immunosorbent assay (ELISA) and NODLN with high-performance liquid chromatography/diode array detection (HPLC/DAD). Acetylcholinesterase (AChE) activity was measured from the foot tissue samples taken at 0, 24 and 96 h. In the water phase, 60-100% of NODLN in the pure-toxin treatments and all the NODLN in N. spumigena treatments appeared as a yet unidentified form with NODLN-like spectral characteristics. The compound was present in similar quantities also in the non-toxic N. sphaerocarpa treatments. In the toxic N. spumigena treatments the tissue concentration of hepatotoxic NODLN-like compounds (measured with ELISA) increased from the control levels of 0.16 to 16.6 microg g(-1) dw (24 h), reaching 30.3 microg g(-1) dw at 96 h. However, <5% of the toxin detected by ELISA could be shown to be NODLN in HPLC/DAD analysis. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/MS) analyses revealed no NODLN-glutathione (GSH) conjugates in the tissues of M. balthica. Combining the responses in the AChE activity recorded after 24 and 96 h exposure, increases by 46% (N. spumigena) and 36% (soluble NODLN) compared with the control activity were observed in the low-level toxic exposures. Oppositely, decreases by 19% (N. spumigena) and 27% (soluble NODLN) of control activity were observed in the high-level exposures. Only the differences between the increased and decreased AChE activities were statistically significant, with individuals exposed to high levels expressing approximately 55% of the activity of those exposed to low concentrations. The results show that M. balthica readily ingests toxic N. spumigena and that accumulation of peptides takes place rapidly, which has potential food chain effects through toxin enrichment. However, it appears that M. balthica is at least partly able to metabolise NODLN. In addition to hepatotoxicity, NODLN seems to induce concentration-dependent neurotoxic effects; this must be taken into consideration when applying AChE activity as a biomarker of specific anthropogenic contamination (e.g. organophosphate and carbamate pesticides).

Accumulation of microcystin congeners in different aquatic plants and crops - A case study from lake Amatitlán, Guatemala

Microcystins (MCs) fate in natural environments can lead to its transfer into aquatic organisms, e.g. aquatic plants. Moreover, lakes in several countries sustain agriculture activities posing a serious health threat for the public. The case of Lake Amatitlán in Guatemala, was addressed to better understand MCs accumulation of four aquatic plants (Polygonum portoricensis, Eichhornia crassipes, Typha sp. and Hydrilla verticillata) coexisting with Microcystis aeruginosa blooms. These findings were further corroborated with an uptake/accumulation laboratory study. Finally crop products (Solanum lycopersicum and Capsicum annuum) irrigated with lake water were also evaluated for MCs. The obtained results suggest that Lake Amatitlán is highly contaminated with MCs (intra- and extracellular 1931 and 90 µg/L, respectively). In fruits of S. lycopersicum and C. annuum a concentration of 1.16 and 1.03 µg/kg dry weight (DW), respectively could be detected. All four aquatic plants showed a high MCs uptake capacity based on obtained bioconcentration factors (BCF) 165, 18, 16 and 11, respectively. These results were further corroborated in a laboratory study with 30 percent of total MCs taken up by H. verticillata within just 7 days. Additionally it was evidenced that all plants accumulated more MC-LR than other MCs congeners. Monitoring of crop products irrigated with lake water needs further consideration.

β-N-Methylamino-l-alanine (BMAA) uptake by the aquatic macrophyte Ceratophyllum demersum

Free-living freshwater cyanobacteria contain BMAA in both free cellular and protein-associated forms. Free BMAA released on bloom collapse or during cellular turnover creates a potential source of the non-proteinogenic amino acid for bioaccumulation and biomagnification in aquatic ecosystems. Uptake of free amino acids is well documented in macrophytes and the potential for aquatic macrophytes to bioaccumulate BMAA therefore poses a potential threat where such macrophytes constitute a food source in an ecosystem. BMAA uptake and accumulation by the aquatic macrophyte Ceratophyllum demersum was therefore investigated. Rapid uptake of significant amounts of BMAA was observed in C. demersum. Both free and protein-associated BMAA were observed with protein association following accumulation of free BMAA. The protein association suggests potential biomaccumulation by aquatic macrophytes and offers a possibility of phytoremediation for BMAA removal.

The fate of the cyanobacterial toxin β-N-methylamino-l-alanine in freshwater mussels

The cyanobacterial neurotoxin, β-N-methylamino-l-alanine (BMAA) has been suggested as a causative agent for certain neurodegenerative diseases. This cyanotoxin bioaccumulates in an array of aquatic organisms, in which it occurs as both a free amino acid and in a protein-associated form. This study was intended to investigate the environmental fate of BMAA by examining the metabolism of isotopically labeled BMAA in four freshwater mussel species. All species showed substantial uptake of BMAA from the culture media. Data showed no significant evidence for BMAA catabolism in any of the animals but did suggest metabolism via the reversible covalent modification of BMAA in freshwater mussels, a process that appears to be variable in different species.

The effect of β-N-methylamino-l-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum

Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes.

Dissolved humic substances can directly affect freshwater organisms

Direct effects of pure humic substance (BS1 FA) on three different aquatic organisms (Ceratophyllum demersum, Dreissena polymorpha, and Chaetogammarus ischnus) were demonstrated in this study. Exposure to environmentally relevant concentrations (0.5 mg/L) of this humic substance led to the activation of the microsomal and soluble glutathione S-transferase (GST) in Ceratophyllum demersum. Exposure to 3-chlorobiphenyl showed also an elevation of GST activity, which is due to the proposed detoxication metabolism of these chlorinated biphenyl. Coexposure to a mixture of 3-chlorobiphenyl and BS1 FA showed a reduction of enzyme elevation, but still significant over an untreated control. The direct impact of humic substances seems not to be restricted to a specific class of organisms.