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.

Biochemical characterization of microcystin toxicity in rainbow trout (Oncorhynchus mykiss).

The kinetics and biochemical effects of microcystins in rainbow trout were studied with freeze-dried toxic cells of Microcystis aeruginosa, strain PCC 7806. Following in vivo exposure the changes in liver histology were observed over a 72 hr period and the absorption of microcystins from the gastrointestinal tract into the blood and liver, as well as the inhibition of hepatic protein phosphatase 1 and 2A activities, were recorded using the protein phosphatase inhibition assay. The interaction between microcystins and trout liver phosphatases was further tested in vitro using the protein phosphatase inhibition assay. The in vivo experiments demonstrated a high organotropy of microcystins for the liver, where rapid and total inhibition of protein phosphatase 1 and 2A activity was observed. Maximal inhibition of phosphatases was observed 3 hr after gavage. At that time-point, approximately 63% of the toxin present in the liver was refractive to detection via the phosphatase inhibition assay and therefore most likely covalently bound to cellular proteins. The inhibition of hepatic protein phosphatases 1 and 2A proved to be transient only, as a progressive increase in phosphatase activity was observed beginning 12 hr after gavage of the fish, reaching approximately 50% of the control activity at 72 hr. In contrast, liver damage continued to progress despite this renewed protein phosphatase activity.

Biliary excretion of biochemically active cyanobacteria (blue-green algae) hepatotoxins in fish.

Previous reports demonstrated that microcystin and related cyanobacteria polypeptides are rapidly cleared from plasma and accumulate in liver tissue. In the present study, we have used their ability to inhibit protein phosphatases to show that these cyanobacteria hepatotoxins are excreted into the bile of experimentally poisoned rainbow trout. At various times after oral administration of hepatotoxic Microcystis aeruginosa, bile samples were analysed for microcystin content by methanol extraction and protein phosphatase assays. An inhibitory principle that specifically suppressed protein phosphatase activity was detected in all bile samples removed between 1 and 72 h after oral exposure to toxic algae. These results indicate that biochemically active microcystin molecules are excreted into the biliary tract of poisoned fish.

Intra- and interindividual variation in flank gland secretions of free-ranging shrewsCrocidura russula.

Individual differences in flank gland secretions were examined among males of the monogamous shrewCrocidura russula during the breeding and nonbreeding seasons. Gas chromatography was used to measure intra- and interindividual variation of flank gland secretions of free-ranging shrews from different populations. The number of compounds detected by gas Chromatographic analyses was correlated with body mass, flank gland size, and the presence of blood parasites in individual shrews. Very few compounds were detected from the flank gland area of juvenile males. After they reached sexual maturity, however, the number of compounds detected from the flank gland secretions increased significantly. At the beginning of the reproductive season 48 different compounds were detected from male flank gland secretions. In the middle of the breeding season 70 compounds were detected, while only 11 compounds were detected during the nonbreeding season. Few compounds were common to all males. There were more volatile compounds in the flank gland secretions of males in the beginning of the breeding season than later in the breeding season. Males from the same population had fewer differences in the elution profile of compounds than males from different populations indicating that individuals from a distinct population have similar elution profiles of compounds and that each population has its own type of elution profile. No correlations were found between the number of compounds detected by gas chromatography for each male and the male's body mass or flank gland size. Blood parasites (trypanosomes,Trypanosoma crocidurae) were found in only three of 30 males investigated.

Enzymatic analysis of liver samples from rainbow trout for diagnosis of blue-green algae-induced toxicosis.

Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent and selective inhibitors of protein phosphatases 1 and 2A. We adapted existing enzymatic techniques to analyze the liver of rainbow trout after oral administration of hepatotoxic cyanobacteria. Liver tissue was removed 3 and 12 hours after treatment, and phosphatase activity was determined in liver extracts, using a specific phosphoprotein substrate. In all samples from fish exposed to toxic cyanobacteria, phosphatase activity was suppressed, whereas the control enzyme, lactate dehydrogenase, present in the same liver extract, was not affected by cyanobacteria. Thus, experimental poisoning by hepatotoxic cyanobacteria resulted in an abnormally low ratio of phosphatase to lactate dehydrogenase activity in the liver extracts. These results indicate that specific inhibition of phosphatases 1 and 2A may provide a useful diagnostic tool to determine the early effects of cyanobacteria toxic peptides directly in liver samples from poisoned animals. Although this test was developed with rainbow trout, it should be possible to extend the analysis of liver phosphatase activity to other species, including sheep and cattle, which are frequently affected by hepatotoxic cyanobacteria.