Ondřej Adamovský

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.

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.

Phytoestrogens and mycoestrogens in surface waters--Their sources, occurrence, and potential contribution to estrogenic activity.

This review discusses the potential contribution of phytoestrogens and mycoestrogens to in vitro estrogenic activities occurring in surface waters and in vivo estrogenic effects in fish. Main types, sources, and pathways of entry into aquatic environment of these detected compounds were summarized. Reviewed concentrations of phyto/mycoestrogens in surface waters were mostly undetectable or in low ng/L ranges, but exceeded tens of μg/L for the flavonoids biochanin A, daidzein and genistein at some sites. While a few phytosterols were reported to occur at relatively high concentrations in surface waters, information about their potencies in in vitro systems is very limited, and contradictory in some cases. The relative estrogenic activities of compounds (compared to standard estrogen 17β-estradiol) by various in vitro assays were included, and found to differ by orders of magnitude. These potencies were used to estimate total potential estrogenic activities based on chemical analyses of phyto/mycoestrogens. In vivo effective concentrations of waterborne phyto/mycoestrogens were available only for biochanin A, daidzein, formononetin, genistein, equol, sitosterol, and zearalenone. The lowest observable effect concentrations in vivo were reported for the mycoestrogen zearalenone. This compound and especially its metabolites also elicited the highest in vitro estrogenic potencies. Despite the limited information available, the review documents low contribution of phyto/mycoestrogens to estrogenic activity in vast majority of surface waters, but significant contribution to in vitro responses and potentially also to in vivo effects in areas with high concentrations.

Microcystin kinetics (bioaccumulation, elimination) and biochemical responses in common carp and silver carp exposed to toxic cyanobacterial blooms

The study investigating microcystin kinetics (bioaccumulation, elimination) and biochemical responses in common carp and silver carp exposed to toxic cyanobacterial blooms

Novel metabolites in cyanobacterium Cylindrospermopsis raciborskii with potencies to inhibit gap junctional intercellular communication

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H](+) ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.