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PATHOLOGICAL EFFECTS AND TISSUE DISTRIBUTION OF
MICROCYSTIN‐LR (MC‐LR) AFTER 48 HOURS NON INVASIVE
EXPOSITION OF NEWLY HATCHED MEDAKA
(ORYZIAS LATIPES) ELEUTHERO‐EMBRYOS
Hélène Huet 1 , Delphine Franko 2 , Chakib Djediat 2 ,
Eva Perez 2 , François Crespeau 1 , Amaury de Luze 2
1 Ecole Nationale Vétérinaire d’Alfort (France)
2 Muséum National d’Histoire Naturelle de Paris (France)
Abstract
Medaka fishes eulethero‐embryos were submitted at 48 hours period of immersion in MC‐LR
containing media at 1, 5, 10, 15 and 20 µg/ml concentration. Significant mortality (>10%) is only
observed for higher MCLR concentration (20µg/ml) in medium.
Microscopic pathological effects after 48 hours immersion in MC‐LR solution were searched on
transverse sections of paraffin embedded embryos fixed in formaldehyde. Histopathologic
studies of paraffin embedded embryos shows, from 10 µg/ml MC‐LR concentration, with variable
intensity, vacuolization of enterocytes and hepatocytes, degenerative and necrotic changes.
On same material, immuno‐labeling with specific monoclonal antibodies against MC‐LR was also
performed and appeared constantly and strongly positive in intestine (enterocytes and lamina
propria), liver (hepatocytes and probably macrophagic cells along sinusoids border). A faint
labeling was also characterized in kidneys epithelial cells, pancreas and yolk vesicle epithelial
border. No labeling was observed in skin, gills, oral epithelium, esophagus and stomach, heart
and vascular system, muscles, nervous central system…
Ultramicroscopic pathological effects after 48 hours immersion in MCLR solution were also
searched on ultrathin sections of embryos fixed in paraformaldehyde and embedded in resin.
From 10 µg/ml MC‐LR concentration, transmission electronic microscopy clearly shows disruption
of intercellular junctions in enterocytes and hepatocytes, some cytoplasmic vacuolation of
enterocytes, alteration of hepatocytes membrane with regression of microvillous cell border with
reduction of Disse space length and abnormal biliary canaliculi border.
INTRODUCTION
Microcystins (MCs) are a family of hepatotoxic cyclic heptapeptides comprising at least 80
variants and congeners. All toxic microcystin variants contain a unique hydrophobic amino acid
3‐amino‐9‐methoxy‐10‐phenyl‐2,6,8‐trimethyl‐deca‐4(E)‐dienoic acid (ADDA) (kongsuwan et
al., 1988). The prototype compound is MC‐LR, which have Leucine (L) and arginine (R) at the
two hypervariable positions in the ring structure (Rinehart et al., 1988; Carmichael, 1992).
These toxins are produced by a wide variety of planktonic cyanobacteria, which are one of the
most primitive and worldwide distributed families of photosynthetic organisms (Brock, 1973,
Ueno et al., 1998).
MCs represent potential environmental fresh water toxins, mainly when climatic conditions
promote blooms of cyanobacteria in pools, pond or lakes.
The primary toxic effect of microcystins is inhibitory reversible binding at the catalytic site of
protein phosphatases 1 and 2A. This interaction involved principally the ADDA group. The
presence of the methyl‐dehydro‐alanine (Mdha) inducted an irreversible inhibition by covalent
linkage to the cystein sulphur on the phosphatases PP1 and PP2A (MacKintosh et al., 1995;
Runnegar et al., 1995). Direct injection of MCLR in mammals induced protein phosphatases
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