Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment.

Cyanobacteria (blue-green algae) produce toxins that may present a hazard for drinking water safety. These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are structurally diverse and their effects range from liver damage, including liver cancer, to neurotoxicity. The occurrence of cyanobacteria and their toxins in water bodies used for the production of drinking water poses a technical challenge for water utility managers. With respect to their removal in water treatment procedures, of the more than 60 microcystin congeners, microcystin-LR (L, L-leucine; R, L-arginine) is the best studied cyanobacterial toxin, whereas information for the other toxins is largely lacking. In response to the growing concern about nonlethal acute and chronic effects of microcystins, the World Health Organization has recently set a new provisional guideline value for microcystin-LR of 1.0 microg/L drinking water. This will lead to further efforts by water suppliers to develop effective treatment procedures to remove these toxins. Of the water treatment procedures discussed in this review, chlorination, possibly micro-/ultrafiltration, but especially ozonation are the most effective in destroying cyanobacteria and in removing microcystins. However, these treatments may not be sufficient during bloom situations or when a high organic load is present, and toxin levels should therefore be monitored during the water treatment process. In order to perform an adequate human risk assessment of microcystin exposure via drinking water, the issue of water treatment byproducts will have to be addressed in the future.

In vitro Interaction between Human Basophils and Polymorphonuclear Granulocytes Effect of Airborne Particulate Matter.

The effects of extracts of airborne particulate matter (APM) on morphology and mediator release from enriched basophils (HBC) and from purified neutrophils of pollen-allergic and nonallergic individuals were investigated. APM was collected on glass fiber filter sheets using high volume samplers HVS150. After preparing an aqueous extract, in which allergens are absent, followed by extraction of organic substances, the mediator-releasing activity of the APM extracts was studied under defined and physiological in vitro conditions. The results indicate that APM potentiates anti-IgE-induced histamine release (HR) and that this enhancement is significantly higher in HBC samples from pollen allergic donors than from nonallergic individuals. Electronmicroscopy of APM-exposed HBC reveals basophil degranulation in addition to strong activation of platelets and neutrophils, the latter sticking onto the surface of HBC and forming clusters with them. Incubation of purified PMN with APM prior to zymosan stimulation showed that these substances also enhance the release of LTB4, LTC4, PGE2 and IL-8. The results show that substances adsorbed to APM interfere with mediator cells of the allergic inflammation and exhibit 'priming' effects on them. Therefore, exposure of individuals to high concentrations of APM can provoke enhancement in symptoms of allergy and continuation of allergic inflammation.

Microcystin-LR toxicodynamics, induced pathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (oncorhynchus mykiss).

With this retrospective study, we investigated the temporal pattern of toxin exposure and pathology, as well as the topical relationship between hepatotoxic injury and localization of microcystin-LR, a potent hepatotoxin, tumor promoter, and inhibitor of protein phosphatases-1 and -2A (PP), in livers of MC-gavaged rainbow trout (Oncorhynchus mykiss) yearlings, using an immunohistochemical detection method and MC-specific antibodies. H&E stains of liver sections were used to determine pathological changes. Nuclear morphology of hepatocytes and ISEL analysis were employed as endpoints to detect the advent of apoptotic cell death in hepatocytes. Trout had been gavaged with lyophilized cyanobacteria (Microcystis aeruginosa, strain PCC 7806) at acutely toxic doses of 5700 microg microcystin (MC) per kg of body weight (bw), as described previously (Tencalla and Dietrich, 1997). Briefly, 3 control and 3 test animal were killed 1, 3, 12, 24, 48, and 72 h after bolus dosing, and livers were fixed and paraffin embedded for histological analysis and later retrospective histochemical analyses. The results of the immunohistochemistry reported here revealed a time dependent, discernible increase in MC-positive staining intensity throughout the liver, clearly not concurring with the kinetics of hepatic PP inhibition observed in the same fish and reported in an earlier publication by Tencalla and Dietrich (1997). After 3 h, marked and increasing MC-immunopositivity was observed in the cytoplasm, as well as the nuclei of hepatocytes. Apoptotic cell death could be detected after 48 h, at the very earliest. These data suggest that accumulation of MC and subsequent changes in cellular morphology, PP inhibition, and hepatocyte necrosis represent the primary events in microcystin induced hepatotoxicity and appear to be associated with the reversible interaction of MC with the PP. In contrast, apoptotic cell death, as demonstrated here, seems to be of only secondary nature and presumably results from the covalent interaction of MC with cellular and nuclear PP as well as other thiol containing cellular proteins.

Toxin production in cyanobacterial mats from ponds on the McMurdo ice shelf, Antarctica.

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 floating, 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 significantly 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 finding may lead to further insight into potential ecological roles of cyanobacterial phosphatase inhibiting toxins.

Airborne particulate matter modulates the production of reactive oxygen species in human polymorphonuclear granulocytes.

Causal relationships between airborne particles (especially particulate matter < 10 microm in diameter) and increases in prevalences and symptoms of respiratory diseases have been postulated in many epidemiologic studies. Polymorphonuclear leukocytes (PMN) in the nasal or bronchial epithelium can be exposed to particulate matter (PM) and may upon exposure produce reactive oxygen species (ROS). Release of ROS can result in cellular and tissue damage and initiate or exacerbate inflammation. To elucidate the effect of PM on inflammatory reactions, we exposed human PMN to PM extracts. PM were collected with high volume samplers in two cities, Dusseldorf and Duisburg, in Germany and reflect sites with high traffic and industrial emissions respectively. The collected particles were extracted using water and then dichloromethane, resulting in an aqueous and an organic extract. The production of ROS was determined using luminol-enhanced chemiluminescence (LCL) of resting and zymosan-stimulated PMN. The present study shows that extracts of PM alone significantly stimulated the production and release of ROS in resting PMN. The effects of the PM extracts were inhibited by superoxide dismutase (SOD), catalase and sodium azide (NaN3). Zymosan-induced LCL was, however, diminished by coincubation with PM extracts. The chemical composition is important when considering the effects of particles. Our study shows that only organic substances adsorbed to particles stimulate LCL. SOZ-induced LCL is inhibited by both types of extracts, but aqueous extracts have a stronger inhibitory effect. It is at present unclear which substances are responsible for these effects.

The effect of lead on protein and DNA metabolism of normal and lead-adapted rat kidney cells in culture.

Cultures of epithelial rat kidney cells were used to investigate the effect of lead on protein and DNA metabolism. Lead-adapted cells were cultured in 5 and 10 microM Pb(NO3)2 for several months and subsequently showed the same growth characteristics but a higher resistance to increased lead doses than untreated cells. Both normal and adapted cell types were exposed to lead nitrate in concentrations from 5 to 1000 microM for incubation periods of from 1 to 24 h. 3H-leucine incorporation was increased in 10 microM lead by up to 140% in normal but not in adapted cells, pointing to a resistance forming mechanism. Not only is the overall rate of protein synthesis increased, but separations of cytosolic proteins by SDS-polyacrylamide electrophoresis indicate the novel or increased synthesis of several proteins. A difference could also be observed in the DNA metabolism, where normal cells show a very pronounced increase of up to 900% after 24 h of incubation. This was not observed in adapted cells.

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

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 1-5 micrograms/mg dry weight in Planktothrix extracts and 0.08 microgram/l in water samples, were determined via HPLC and protein-phosphatase inhibition assay. Planktothrix filaments were detected in gut contents of whitefish. In addition, microcystins could be detected in gut contents via ELISA. Similarly, immunoprobing with microcystin-antibodies demonstrated microcystin-protein adducts in liver homogenates of whitefish caught during Planktothrix blooms. Furthermore, Planktothrix extracts proved cytotoxic to trout hepatocytes as determined by MTT reduction. Whitefish eggs and larvae, obtained during blooms in winter 1998 and 2000 in a Lake Ammersee hatchery, were studied for developmental progress. Malformations typical for cyanobacterial toxin exposure were observed.

Characteristics of lead adaptation in a rat kidney cell line. I. Uptake and subcellular and subnuclear distribution of lead.

Two sublines of normal rat kidney cell (NRK 52-E) that are resistant to 5 and 10 microM lead nitrate have been developed and characterized. The cellular and nuclear uptake of 210Pb, as well as the subnuclear distribution, was unchanged by lead adaptation: it remained at values of 2.3-6.6 pmole 210Pb/microgram protein/h. Also no difference in the slope of the curve of the uptake rate of the three cell types could be detected. Studies of the nuclear uptake of 210Pb showed that in both normal and lead-adapted cells the major lead fraction was associated with the chromatin and mostly (63-67%) with the nuclear proteins. Electron microscopic studies demonstrated that particulate lead was taken up in all lead-exposed cells. It was concluded that development of resistance to lead does not arise from a reduced rate of cellular uptake, from increased excretion, or from changes in nuclear uptake or in the subnuclear distribution within the cells.

Characteristics of lead adaptation in a rat kidney cell line. II. Effect on DNA synthesis, protein synthesis, and gene expression.

The effects of adaptation of normal rat kidney cells (NRK 52-E) to growth in 5 or 10 microM lead nitrate on the rates of DNA synthesis and on the rate and pattern of protein synthesis was studied. The rate of [3H]thymidine incorporation into DNA was increased in normal cells, but remained unchanged in one lead-adapted cell line (only 5 microM NRK studied). Increased rates of [3H]leucine incorporation into nonadapted NRK cells were found only at times up to 3 h; in contrast, the lead-adapted cells showed such increases only at longer times. The most pronounced differences between normal and lead-adapted cells were found with lead concentrations of 10 or 50 microM lead nitrate. Lead-adapted control cells incorporated 170% of the [3H]leucine taken up by nonadapted cells. In both adapted and nonadapted cells the pattern of synthesis of specific proteins showed varied and dose-dependent differences between the three cell sublines examined. The changed sensitivity of both DNA and protein synthesis following lead exposure appears to be a potent parameter in the development of resistance, perhaps through the development of specific lead-binding proteins.