* Environmental Toxicology, University of Konstanz, Konstanz, Germany;
Institute of Toxicology, Schwerzenbach, Switzerland; and
Turku Centre for Biotechnology, Turku, Finland
Received June 29, 1999; accepted September 27, 1999
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ABSTRACT |
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Key Words: apoptosis; fish; histopathology; immunohistochemistry; liver; microcystin; protein phosphatase-1 and -2A; serum; toxin.
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INTRODUCTION |
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The toxicity of MC in mammals is characterized by fulminant intrahepatic hemorrhage, followed by hypovolemic shock or hepatic insufficiency and death of the animals (Carmichael, 1992, 1994
). Microcystins reabsorbed from the gastrointestinal tract are believed to be taken up from the blood into the hepatocytes via a multispecific bile acid transport system (Eriksson et al., 1990
; Hooser et al., 1991b
; Runnegar et al., 1995a
, 1981
, 1991
). At acutely toxic doses, rounding of hepatocytes occurred concurrently with the loss of normal hepatic architecture. The latter pathological changes are considered to result from the interaction of MC with serine/threonine protein phosphatases-1 and -2A (PP), essential for maintaining the monomerization (phosphorylation)/polymerization (dephosphorylation) equilibrium of the cytoskeletal intermediate filaments (Eriksson et al., 1992
, 1989
; Falconer and Yeung, 1992
). Through MC-mediated inactivation of PP, this equilibrium is shifted towards monomerization and dissociation of the cytoskeleton (Eriksson et al., 1992
, 1989
; Falconer and Yeung, 1992
). Analogous, however not identical, pathological changes to those reported for mammals were also observed in fish treated with purified MC or cyanobacterial material (Phillips et al., 1985
; Råbergh et al., 1991
; Tencalla and Dietrich, 1997
).
Microcystin-LR, representative for other MC congeners, was shown to interact with the catalytic subunit of PP (PPc) in a two-step mechanism (Craig et al., 1996). This biphasic reaction involves a rapid, reversible binding (usually within minutes to a few hours) and inactivation of PPc followed by a much delayed covalent interaction (requiring several h) (Craig et al., 1996
; MacKintosh et al., 1995
; Runnegar et al., 1995b
). The reversible interaction with PP is of hydrophobic and ionic nature, involving ADDA and Masp/Glu residues (Bagu et al., 1997
; Nishiwaki-Matsushima et al., 1991
; Rinehart et al., 1988
; Stotts et al., 1993
). This reversible interaction, believed to optimally position the MC-LR molecule to PPc, is considered prerequisite for covalent binding of MC-LR to the PPc. The question, however, remains whether the pathological changes observed in the hepatocytes of mammals and fish result from the rapid but reversible interaction of MC with PP or whether covalent binding to PP, and therefore long-term PP inhibition, is necessary for the initiation of the subsequent cascade of changes. Circumstantial evidence supporting the hypothesis that the reversible binding of MC-LR/MC to the PPc is sufficient for PP inhibition and development of liver pathology is provided by the fact that mice and rats experimentally dosed with MC die within 1 to 3 h following exposure (Kaya, 1996
; Kaya and Watanabe, 1990
), i.e., a time frame in which only a small amount of MC would be expected to have undergone covalent interaction with the PP. In addition, since MC was reported to be rapidly eliminated via biliary excretion (Sahin et al., 1996
) following conjugation to glutathione and endogenous thiol containing proteins (Kondo et al., 1996
; Pflugmacher et al., 1998
) PP-MC reversible binding appears to compete with conjugation (detoxifying) and binding reactions to other thiol-containing proteins. If indeed the detoxifying reactions compete with PP for MC and the reversible reaction (MC-PP binding) is responsible for the reported PP inhibition, then the inhibition of PP, measured as total PP activity in the livers of exposed animals, should at least in part be reversible also. The latter hypothesis would also suggest that covalently bound MC should be immunohistochemically detectable in livers of exposed animals much later than the concurrent inhibition of hepatic PP.
In order to test the above hypothesis, a retrospective immunohistochemical study was conducted using yearling rainbow trout that were administered acutely toxic doses of the microcystin-producing cyanobacteria Microcystis aeruginosa PCC 7806 via gavage (Tencalla and Dietrich, 1997). Uptake of the toxin into the liver was monitored with anti-MC antibodies and compared to the onset of PP inhibition reported in an earlier publication by Tencalla and Dietrich (1997). In addition, the pathogenic time course of microcystin-induced hepatotoxicity and form of cell death (necrosis vs. apoptosis) was investigated.
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MATERIALS AND METHODS |
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In order to achieve a more complete understanding of the events involving microcystin-induced liver pathology, the results obtained in this retrospective study were combined and compared with the data on PP inhibition and blood enzyme parameters of the same fish published earlier by Tencalla and Dietrich (1997).
Anti-MC Antibodies
Aminoethyl-MC (AE-MC) was synthesized as described by Mikhailov et al. (manuscript submitted). Briefly, conjugation of AE-MC with the carrier proteins (50 µg of MCLR per mg of soybean trypsin inhibitor [SBTI, Sigma, U.S.]) was accomplished with 2% glutaraldehyde at pH 8.7, followed by multiple dialysis to ensure the absence of free toxin. Polyclonal antibodies were raised in white New Zealand rabbits (both females and males) by immunization with conjugates of the AE-MCLR with SBTI. The rabbit polyclonal antibodies were affinity purified.
Histopathology
Tissues were processed in a standard fashion. Briefly, liver samples were fixed in 4% neutral buffered formalin for several h, dehydrated, paraffin embedded, and archived. Sections of 35 µm were mounted on aminopropyltriethoxysilane-coated slides (APTS, A-3648, Sigma, USA). Following deparaffinization in 3 xylene baths, sections were rehydrated, stained with hematoxylin and eosin (H&E), and mounted with Crystal/MountTM (Biømeda, USA) for later pathological assessment. Liver samples were also fixed in osmium tetraoxide for electron microscopic analyses.
Immunohistochemistry
Paraffin-embedded sections were deparaffinized, rehydrated, and incubated with type XIV bacterial protease (P-5147, Sigma, USA) in PBS for antigen retrieval at 37°C for 15 min. Endogenous peroxidase was blocked with 3% H2O2 for 15 min. Endogenous biotin was blocked with a specific blocking kit (Avidin/Biotin blocking kit, Vector Inc., USA). Microcystin-LR antiserum was applied in a humidified atmosphere for 1620 h at 4°C. Antigen-primary antibody complex visualization was achieved using biotin-conjugated secondary antibodies, HRP-labeled streptavidin and AEC-chromogen (3-amino-9-ethylcarbazole) (Super SensitiveTM, BioGenex, USA). Sections were counterstained for 15 sec with hematoxylin, rinsed in tap water and mounted with Crystal/MountTM (Biømeda, USA).
Detection of Apoptotic Cells
Recognition of apoptotic nuclei/bodies in the paraffin-embedded tissue sections was achieved either by morphological evaluation of H&E-stained slides or by histochemical fragment end labeling of DNA (ISEL). The latter was performed using an in situ DNA-fragment end labeling kit (FragELTM; Oncogene, USA) according to the manufacturer`s instructions. A semiquantitative apoptotic index (apoptotic cells/field) was determined by counting ISEL-positive cells of 7 randomly chosen fields. Fields were defined by a 10 x 10 grid (10 x ocular) and a 20 x objective (= 200 x magnification).
Statistics
The Shapiro-Wilks and Bartlett`s tests were used to assess the normality of the data distribution and the homogeneity of variance, respectively, and the semiquantitative apoptotic index was analyzed using ANOVA followed by Tukey's multiple comparison test (Toxstat, 1991) via log base-10 transformation of the original data.
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RESULTS |
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Mortality and Gross Morphology
As reported by Tencalla and Dietrich (1997), no mortality occurred either during gavage or throughout the test duration (72 h). Visual inspection of the gastrointestinal tract at the respective time points during the experiment had demonstrated a progressive development of yellowish discoloration in livers of treated fish. However, no increase in either liver size or liver weight had been observed, when compared to the respective controls.
Retrospective Histopathology
Changes in the cord-like organization of hepatocytes could already be seen 1 h post application of the bolus dose. These changes appeared primarily in the pericentral region of the liver (Fig. 1:II) and were characterized by the appearance of hepatocytes with condensed cytoplasm (Figs. 1:II1:VII
). Between 3 and 12 h, the latter changes became progressively more pronounced involving larger areas of the liver. In addition, small hemorrhages could be detected where sinusoids appeared to be ruptured (Fig. 1:VI
). An involvement of the entire liver was observed as of 24 h post-dosing. Overt lysis of hepatocyte membranes (necrotic cells) was observed, as of 48 h post-dosing, in many cases associated with pyknotic nuclei (Fig. 1:VI and 1:VII
).
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DISCUSSION |
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The comparison of the retrospective study presented here with the biochemical data of the original study (Tencalla and Dietrich, 1997) (Fig. 5
), suggests some incoherence with regard to the mechanism underlying the development of microcystin-induced liver pathology. Indeed, a rapid uptake of microcystin from the gastrointestinal tract into the blood, concurrent with high levels of extractable microcystin in the liver and almost complete PP inhibition within 3 h following gavage, was reported in the original study (Tencalla and Dietrich, 1997
), whereas the concentrations of microcystin in the blood and liver decrease and PP activity recuperates to almost 50% the original value by end of the study (Fig. 5
). These observations contrast the early onset of necrosis which progressively increased in severity toward the end of our study period (72 h, Fig. 1
), as well as to a rather late appearance of immunohistochemically detectable microcystin in the tissue sections (Fig. 4
), as reported in the retrospective study here. These seemingly contradictory findings, however, indicate that rapid MC uptake is responsible for the early onset of PP inhibition and necrosis. Methanol-extractable (free or non-covalently bound) hepatic MC, also reaches peak levels simultaneously with the almost complete inhibition of endogenous hepatic PP activity (3 h post gavage). The subsequent decrease in free or non-covalently-bound hepatic MC and concurrent recuperation of endogenous PP activity, suggests that rapid PP inhibition is (1) directly dependent on the concentration of available unbound MC; and (2) a result of this reversible interaction of MC with the catalytic subunit of the protein phosphatases-1 and -2A (PPc). The latter interpretation is supported by several observations:
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ACKNOWLEDGMENTS |
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NOTES |
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