Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin.

Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K(m) values amounted to 7 +/- 3 microM for OATP1B1, 9 +/- 3 microM for OATP1B3, and 20 +/- 8 microM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.

Congener-independent immunoassay for microcystins and nodularins.

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

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.

Pathological and biochemical characterization of microcystin-induced hepatopancreas and kidney damage in carp (Cyprinus carpio).

Mass occurrences of cyanobacteria, due to their inherent capacity for toxin production, specifically of microcystins (MC), have been associated with fish kills worldwide. The uptake of MC-LR and the sequence of pathological and associated biochemical changes was investigated in carp (Cyprinus carpio) in vivo over 72 h. Carp were gavaged with a single sublethal bolus dose of toxic Microcystis aeruginosa (PCC 7806) amounting to an equivalent of 400 microg MC-LR/kg body wt. Damage of renal proximal tubular cells and hepatocytes was observed as early as 1 h, followed by pathological changes in the intestinal mucosa at approximately 12 h postdosing. These alterations were characterized in hepatopancreas by a dissociation of hepatocytes, an early onset of apoptotic cell death, and delayed cell lysis. In the renal proximal tubules (P2) observations included increased vacuolation of individual tubular epithelial cells, apoptosis, cell shedding, and finally proteinaceous casts at the cortico-medullary junction. Concurrently with the pathological alterations, MC-immunopositive staining was observed in hepatocytes and the proximal tubular cells; the staining increasing in the hepatopancreas in intensity with increasing time postdosing. The presence of apoptotic cell death was determined using in situ fragment end labeling (ISEL) of the respective tissue sections and agarose gel electrophoresis for detection of DNA-laddering. The analysis of carp tissue extracts (hepatopancreas, kidney, GI tract, skeletal muscle, brain, heart, spleen, and gills) demonstrated MC-LR adducts having molecular weights of 38 kDa (putatively catalytic subunit of protein phosphatases-1 and -2A) and 28 kDa, respectively. An additional band was found to be present at 23 kDa in both hepatopancreas and kidney. The present data demonstrate that, in comparison to the pathological events in salmonids exposed to MC, where a slower development of pathology and primarily necrotic cell death prevails, the pathology in carp develops rapidly and at lower toxin concentrations. This is most likely due to a more efficient uptake of toxin, while the mechanism of cell death is primarily apoptosis.