Pavel Babica

EXPLORING THE NATURAL ROLE OF MICROCYSTINS—A REVIEW OF EFFECTS ON PHOTOAUTOTROPHIC ORGANISMS1

Cyanobacterial blooms and the production of cyanotoxins represent a serious global problem. Although the effects of a group of important cyanotoxins, microcystins (MCs), have been studied intensively in various organisms, little is known about the natural functions of these cyclic heptapeptides. MCs may have allelopathic effects. This paper summarizes the information from the studies that have investigated the effects of MCs on photoautotrophs in vitro and in vivo. Interactions with terrestrial plants, macrophytes, macroalgae, and planktonic microalgae are reported in detail with respect to the ecological relevancy of experimental conditions related to allelopathy. Our review shows that only a limited number of studies described harmful effects of MCs at concentrations that are typical for the environment. Consequently, the ability of MCs to act as general allelopathic compounds against photoautotrophs seems unlikely. However, further research is needed for definitive confirmation or rejection of the allelopathic hypothesis as well as, an explanation of the crucial question of MC function in the context of new information from evolutionary and molecular biology.

Single-walled carbon nanotubes dispersed in aqueous media via non-covalent functionalization: Effect of dispersant on the stability, cytotoxicity, and epigenetic toxicity of nanotube suspensions

As the range of applications for carbon nanotubes (CNTs) rapidly expands, understanding the effect of CNTs on prokaryotic and eukaryotic cell systems has become an important research priority, especially in light of recent reports of the facile dispersion of CNTs in a variety of aqueous systems including natural water. In this study, single-walled carbon nanotubes (SWCNTs) were dispersed in water using a range of natural (gum arabic, amylose, Suwannee River natural organic matter) and synthetic (polyvinyl pyrrolidone, Triton X-100) dispersing agents (dispersants) that attach to the CNT surface non-covalently via different physiosorption mechanisms. The charge and the average effective hydrodynamic diameter of suspended SWCNTs as well as the concentration of exfoliated SWCNTs in the dispersion were found to remain relatively stable over a period of 4 weeks. The cytotoxicity of suspended SWCNTs was assessed as a function of dispersant type and exposure time (up to 48 h) using general viability bioassay with Escherichia coli and using neutral red dye uptake (NDU) bioassay with WB-F344 rat liver epithelia cells. In the E. coli viability bioassays, three types of growth media with different organic loadings and salt contents were evaluated. When the dispersant itself was non-toxic, no losses of E. coli and WB-F344 viability were observed. The cell viability was affected only by SWCNTs dispersed using Triton X-100, which was cytotoxic in SWCNT-free (control) solution. The epigenetic toxicity of dispersed CNTs was evaluated using gap junction intercellular communication (GJIC) bioassay applied to WB-F344 rat liver epithelial cells. With all SWCNT suspensions except those where SWCNTs were dispersed using Triton X-100 (wherein GJIC could not be measured because the sample was cytotoxic), no inhibition of GJIC in the presence of SWCNTs was observed. These results suggest a strong dependence of the toxicity of SWCNT suspensions on the toxicity of the dispersant and point to the potential of non-covalent functionalization with non-toxic dispersants as a method for the preparation of stable aqueous suspensions of biocompatible CNTs.

Toxins produced in cyanobacterial water blooms - toxicity and risks

Toxins produced in cyanobacterial water blooms - toxicity and risks Cyanobacterial blooms in freshwaters represent a major ecological and human health problem worldwide. This paper briefly summarizes information on major cyanobacterial toxins (hepatotoxins, neurotoxins etc.) with special attention to microcystins - cyclic heptapeptides with high acute and chronic toxicities. Besides discussion of human health risks, microcystin ecotoxicology and consequent ecological risks are also highlighted. Although significant research attention has been paid to microcystins, cyanobacteria produce a wide range of currently unknown toxins, which will require research attention. Further research should also address possible additive, synergistic or antagonistic effects among different classes of cyanobacterial metabolites, as well as interactions with other toxic stressors such as metals or persistent organic pollutants.

MICROCYSTIN KINETICS (BIOACCUMULATION AND ELIMINATION) AND BIOCHEMICAL RESPONSES IN COMMON CARP (CYPRINUS CARPIO) AND SILVER CARP (HYPOPHTHALMICHTHYS MOLITRIX) EXPOSED TO TOXIC CYANOBACTERIAL BLOOMS

Two species of common edible fish, common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix), were exposed to a Microcystis spp.-dominated natural cyanobacterial water bloom for two months (concentrations of cyanobacterial toxin microcystin, 182-539 microg/g biomass dry wt). Toxins accumulated up to 1.4 to 29 ng/g fresh weight and 3.3 to 19 ng/g in the muscle of silver carp and common carp, respectively, as determined by enzyme-linked immunosorbent immunoassay. Concentrations an order of magnitude higher were detected in hepatopancreas (up to 226 ng/g in silver carp), with a peak after the initial four weeks. Calculated bioconcentration factors ranged from 0.6 to 1.7 for muscle and from 7.3 to 13.3 for hepatopancreas. Microcystins were completely eliminated within one to two weeks from both muscle and hepatopancreas after the transfer of fish with accumulated toxins to clean water. Mean estimated elimination half-lives ranged from 0.7 d in silver carp muscle to 8.4 d in common carp liver. The present study also showed significant modulations of several biochemical markers in hepatopancreas of fish exposed to cyanobacteria. Levels of glutathione and catalytic activities of glutathione S-transferase and glutathione reductase were induced in both species, indicating oxidative stress and enhanced detoxification processes. Calculation of hazard indexes using conservative U.S. Environmental Protection Agency methodology indicated rather low risks of microcystins accumulated in edible fish, but several uncertainties should be explored.

Structure-Activity–Dependent Regulation of Cell Communication by Perfluorinated Fatty Acids using in Vivo and in Vitro Model Systems

Background Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. Objectives The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. Methods We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. Results PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. Conclusions The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC.

Effects of dissolved microcystins on growth of planktonic photoautotrophs

P. Babica, K. Hilscherová, K. Bártová, L. Bláha and B. Marŝálek. 2007. Effects of dissolved microcystins on growth of planktonic photoautotrphs. Phycologia 46: 137–142. DOI: 10.2216/06-24.1 Effects of cyanobacterial toxins microcystin-LR and -RR on growth of five representatives of Chlorophyta (Chlamydomonas reinhardtii, Chlorella kesslerii, Pediastrum duplex, Pseudokirchneriella subcapitata, Scenedesmus quadricauda) and cyanobacterium Microcystis aeruginosa (Cyanophyta) were investigated in the concentration range 1–25,000 μg l−1 using microplate assays (evaluated after 4, 7 and 11 days). Our results demostrate different susceptibility of several planktonic organisms to microcystins. In some species (C. reinhardtii, C. kesslerii, P. duplex, M. aeruginosa), microcystin-RR induced more pronounced effects on growth than structural variant microcystin-LR. However, environmentally relevant concentrations of microcystins (1–10 μg l−1) did not cause significant growth alterations. Also, concentrations 100–5000 μg l−1 were ineffective in most tested species with the exception of P. subcapitata. Growth of P. subcapitata was strongly inhibited at concentrations of microcystin-LR or -RR ≥ 1000 μg l−1 after 4 days of exposure, whereas S. quadricauda was affected only at concentration 25,000 μg l−1. C. reinhardtii and C. kesslerii responded to both microcystins at the highest experimental concentration, but effects of microcystin-LR were weak and apparent only after 11 days of exposure, while microcystin-RR inhibited alga growth from day 4. Growth of P. duplex was also reduced at 25,000 μg l−1 of microcystin-RR from day 4, but only slightly inhibited by microcystin-LR (after 4 and 7 days of exposure). Growth of M. aeruginosa was only slightly affected by microcystin-LR (inhibition at 25,000 μg l−1 on day 7) and inhibited by microcystin-RR especially at the highest experimental concentration. Our results suggest that microcystin effects on phytoplankton are species specific and congener specific. However, microcystins are not likely to affect proliferation of planktonic photoautotrophs at environmentally occurring conditions. These findings do not support a hypothesis of possible direct allelopathic natural function of microcystins, at least as far as growth inhibition effects are concerned.

Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine-specific phospholipase C

Inhibition of gap junctional intercellular communication (GJIC) and the activation of intracellular mitogenic pathways are common hallmarks of epithelial derived cancer cells. We previously determined that the 1‐methyl and not the 2‐methyl isomer of anthracene, which are prominent cigarette smoke components, activated extracellular receptor kinase, and inhibited GJIC in WB‐F344 rat liver epithelial cells. Using these same cells, we show that an immediate upstream response to 1‐methylanthracene was a rapid (<1 min) release of arachidonic acid. Inhibition of phosphatidylcholine‐specific phospholipase C prevented the inhibition of GJIC by 1‐methylanthracene. In contrast, inhibition of phosphatidylinositol specific phospholipase C, phospholipase A2, diacylglycerol lipase, phospholipase D, protein kinase C, and tyrosine protein kinases had no effect on 1‐methylanthracene‐induced inhibition of GJIC. Inhibition of protein kinase A also prevented inhibition of GJIC by 1‐methylanthracene. Direct measurement of phosphatidylcholine‐specific phospholipase C and sphingomyelinase indicated that only phosphatidylcholine‐specific phospholipase C was activated in response to 1‐methylanthracene, while 2‐methylanthracene had no effect. 1‐methylanthracene also activated p38‐mitogen activated protein kinase; however, like extracellular kinase, its activation was not involved in 1‐methylanthracene‐induced regulation of GJIC, and this activation was independent of phosphatidylcholine‐specific phospholipase C. Although mitogen activated protein kinases were activated, Western blot analyzes indicated no change in connexin43 phosphorylation status. Our results indicate that phosphatidylcholine‐specific phospholipase C is an important enzyme in the induction of a tumorigenic phenotype, namely the inhibition of GJIC; whereas mitogen activated protein kinases triggered in response to 1‐methylanthracene, were not involved in the deregulation of GJIC. (Cancer Sci 2008; 99: 696–705)

Detoxification and oxidative stress responses along with microcystins accumulation in Japanese quail exposed to cyanobacterial biomass

The cyanobacterial exposure has been implicated in mass mortalities of wild birds, but information on the actual effects of cyanobacteria on birds in controlled studies is missing. Effects on detoxification and antioxidant parameters as well as bioaccumulation of microcystins (MCs) were studied in birds after sub-lethal exposure to natural cyanobacterial biomass. Four treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to controlled doses of cyanobacterial bloom during acute (10 days) and sub-chronic (30 days) experiment. The daily doses of cyanobacterial biomass corresponded to 0.2-224.6 ng MCs/g body weight. Significant accumulation of MCs was observed in the liver for both test durations and slight accumulation also in the muscles of the highest treatment group from acute test. The greatest accumulation was observed in the liver of the highest treatment group in the acute test reaching average concentration of 43.7 ng MCs/g fresh weight. The parameters of detoxification metabolism and oxidative stress were studied in the liver, heart and brain. The cyanobacterial exposure caused an increase of activity of cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase representing the activation phase of detoxification metabolism. Also the conjugation phase of detoxification, namely the activity of glutathione-S-transferase, was altered. Cyanobacterial exposure also modulated oxidative stress responses including the level of glutathione and activities of glutathione-related enzymes and caused increase in lipid peroxidation. The overall pattern of detoxification parameters and oxidative stress responses clearly separated the control and the lowest exposure group from all the higher exposed groups. This is the first controlled study documenting the induction of oxidative stress along with MCs accumulation in birds exposed to natural cyanobacterial biomass. The data also suggest that increased activities of detoxification enzymes could lead to greater biotransformation and elimination of the MCs at the longer exposure time.

Immunomodulatory Potency of Microcystin, an Important Water-Polluting Cyanobacterial Toxin

Microcystins (MCs) are primarily hepatotoxins produced by cyanobacteria and are responsible for intoxication in humans and animals. There are many incidents of chronic exposure to MCs, which have been attributed to the inappropriate treatment of water supplies or contaminated food. Using RAW 264.7 macrophages, we showed the potency of microcystin-LR (MC-LR) to stimulate production of pro-inflammatory cytokines (tumor necrosis factor α and interleukin-6) as a consequence of fast nuclear factor κB and nitrogen-activated protein kinase activation. In contrast to other studies, the observed effects were not attributed to the intracellular inhibition of protein phosphatases 1/2A due to lack of specific transmembrane transporters for MCs. However, the MC-LR-induced activation of macrophages was effectively inhibited by a specific peptide that blocks signaling of receptors, which play a pivotal role in the innate immune responses. Taken together, we showed for the first time that MC-LR could interfere with macrophage receptors that are responsible for triggering the above-mentioned signaling pathways. These findings provide an interesting mechanistic explanation of some adverse health outcomes associated with toxic cyanobacteria and MCs.

Effects of microcystin and complex cyanobacterial samples on the growth and oxidative stress parameters in green alga Pseudokirchneriella subcapitata and comparison with the model oxidative stressor—herbicide paraquat

Oxidative stress is one of the biochemical mechanisms involved in toxicity of cyanobacterial toxins microcystins (MC), but its role in the effects of complex water blooms is elusive. The aim of this study was to investigate effects of pure MCs and different complex mixtures of cyanobacterial metabolites on the growth and biochemical markers of oxidative stress and detoxification in green alga Pseudokirchneriella subcapitata. Pure MCs at high concentrations (300 μg/L) had no effects on the growth of P. subcapitata (up to 10 day exposures) but stimulated activity of glutathione reductase (GR) after short 3 and 24 h exposures. Other biomarkers (levels of glutathione, GSH, and activities of glutathione‐S‐transferase, GST, and glutathione peroxidase, GPx) were not affected by pure MCs). Crude extract of the laboratory culture of cyanobacteria Microcystis aeruginosa (containing 300 μg/L of MCs) had no effects on algal growth or any of the biomarkers. Weak growth stimulations after 4–7 days were observed after exposures to the growth‐spent medium of the M. aeruginosa culture, which also inhibited activities of GST after prolonged exposures. Other investigated parameters (reduced GSH and activity of GPx) were not affected by any of the cyanobacterial samples. The results were compared with effects of model oxidative stressor herbicide paraquat, which exhibited variable effects on both algal growth and biomarkers (decrease in reduced GSH, stimulations of GR). Taken together, although pure MCs induce oxidative stress in green alga, the effects of cyanobacterial mixtures, which are more relevant to the natural situation, are more complex and they differ from the pure toxin. High variability in the biochemical responses to the oxidative stress makes the interpretation of results complicated, which limits the use of these biomarkers as early warnings of toxicity under natural conditions.