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Vloga mikrocistinov pri razvoju degenerativnih obolenj parenhimskih organov
Avtor: Valentina O. Giacomelli, Andrej Repež
Mentor: prof. dr. Dušan Šuput
IZHODIŠCA. Mikrocistini v jetrnih celicah prizadenejo jetrno strukturo tako, da okvarijo citoskelet in kri uhaja v jetrno tkivo. Notranja krvavitev lahko vodi v hipovolemicni šok. Onesnaženost voda s cvetenji toksicnih cianobakterij sta resen problem v severovzhodnem delu Slovenije, kjer intenzivno kmetijstvo povzroca visoko stopnjo evtrofikacije voda.
NAMEN najine raziskave je bil vpeljati metodo izolacije hepatocitov in ugotoviti, ali v Sloveniji izoliran mikrocistin RR povzroci že opisane spremembe na jetrnih celicah. Želela sva ugotoviti, ali se bodo v akutnem postopku in vitro pokazale spremembe v krvnih parametrih kot posledica krvavitve in kakšne so spremembe na jetrnih izvidih in jetrni morfologiji po kronicni izpostavljenosti zmesi mikrocistinov LR in RR. Preverjala sva, ali je slikanje na osnovi magnetne resonance ustrezna metoda za zasledovanje akutnih in kronicnih sprememb jetrnega parenhima po delovanju zmesi mikrocistinov.
METODE. Poskuse sva opravila na 37 podganjih samcih soja Wistar. V akutnih poskusih sva žrtvovala 20 poskusnih živali. S polovico sva preverila delovanje izbranega toksina z metodo izolacije hepatocitov s perfuzijo jeter in situ, z drugo polovico pa sva ugotavljala ucinke zmesi mikrocistinov v poskusih in vivo z metodo magnetne resonance. V kronicnem poskusu sva 12 samcev dva meseca izpostavljala subletalnim odmerkom zmesi mikrocistinov v pitni vodi, 5 živali pa je služilo kontroli. Na koncu poskusov sva opazovala spremembe rdece krvne slike, sestavin krvnega seruma, izvidov jetrnih testov in pregledala jetra s histološkimi metodami. Meritve in vivo sva dopolnila s slikanjem z magnetno resonanco in z merjenjem sprememb teže jeter.
REZULTATI. Pri metodi izolacije hepatocitov s perfuzijo smo dosegli 83% preživetje celic. Izbrani mikrocistin RR povzroci na izoliranih hepatocitih spremembe, ki ustrezajo opisom v literaturi. Zmes mikrocistinov je v akutnih poskusih povzrocila povecanje teže jeter za 35%, površine prereza jeter na magnetnoresonancnih slikah pa za 19,3%. Hematokrit je upadel za 13% (p<0,01). Histološki pregled jeter je pokazal žarišcne krvavitve. V kronicnih poskusih smo ugotovili neznacilne spremembe v patohistoloških in magnetnoresonancnih slikah. Jetrni testi so pokazali patološke spremembe: povecanje koncentracije alkalne fosfataze za 27%, gama-glutamiltransferaze za 25%, aspartatne aminotransferaze za 19,2% in alaninske aminotransferaze za 12%. V rdeci krvni sliki sva našla upad hematokrita za 10%, koncentracije prostega železa v serumu za 39,5%. Povprecna vsebnost in koncentracija hemoglobina ter prostornine eritrocita se zmanjša.
ZAKLJUCKI. Iz navedenega lahko zakljucimo, da je hepatotoksicnost uporabljenih mikrocistinov primerljiva z opisi v literaturi. Slikanje z magnetno resonanco je ustrezna metoda za ugotavljanje akutnih zastrupitev z mikrocistini, krvavitev pa se odraža v znacilnem padcu hematokritske vrednosti. V kronicnih poskusih so spremembe izvidov jetrnih testov in mikrocitna anemija znacilni spremljevalci morfoloških sprememb jetrnega parenhima, ki ga lahko potrdimo predvsem s histološkimi metodami, medtem ko je slikanje z magnetno resonanco nezanesljiv kazalec patoloških sprememb jetrnega parenhima.
[Abstract / English version]
A role of microcystins in evolution of parenchymatic organs diseases
Author: Valentina O. Giacomelli, Andrej Repež
Mentor: prof. dr. Dušan Šuput
BACKGROUND. Microcystins damage liver tissue with disorganization of cytoskeleton. Consequently, blood penetrates liver tissue. Internal bleeding mey lead to a hemoragic shock. Water pollution with blooming of toxic strains of cianobacteria as a result of intensive agriculture is documented in north-eastern Slovenia.
INTENTION. To develop the method of isolation of the hepatocytes and to assess the potency of microcystin RR, isolated from poluted water in Slovenia. With acute exposure to mixture of microcystins LR and RR we wanted to ascertain if the internal bleeding, produced by the toxin, would reflect in changes in hematokrit. We tried to determine the morphological changes after chronic exposure of animals to microcystin mixture. Simultaneusly we have tested the hypotesis that magnetic resonance imaging was suitable method for assesment of acute and chronic toxicity of the microcystins.
METHODS. Experiments were performed on 37 male rats (Wistar). In the acute experiments 20 animals were sacrificed. With one half we tested the potency of the chosen toxin on isolated hepatocytes, optaind by in situ perfusion tehnique with collagenase. With the other half in vivo experiments using the magnetic resonance imaging for assessment of the toxin mixture efects. In the chronic experiments the rats were fed with microcystins for two months. Measurments in vivo were performed with the magnetic resonance imaging. At the end of experiments the animales were observed with the magnetic resonance imaging, then sarificed. The hematokrit and liver tests were performed on samples of blood taken from inferior cava vein. Samples of liver were weighted and investigated with hystological methods.
RESULTS.With the hepatocytes isolation method we have reached 83% survival rate. The chosen microcystin RR was as hepatotoxic as other microcystins used in other studies. In acute experiments with the mixtureof toxins we have found an increase of 36% in the live weight and increase of 19.3% in the cross-section area of the liver with magnetic resonance imaging. Histologicaly focal intrahepatic bleeding have been fund. Hematokrit decreased by 13% (p < 0.01). Chronic application of the same mixture of toxins resulted in insignificant changes in the morphology of the liver and magnetic resonance images. Hepatic function tests showed following abnormalities: concentracions of alkaline phosphatase increased by 27%, gamaglutamyl-transferase by 25%, aspartat aminotransferase by 19.2% and alanin aminotransferaze by 12%. We have found a decrease of hematokrit by 10% and a fall in concentrations of mean corpuscular hemoglobin, hemoglobin concentration and red cell volume.
DISCUSSION. The potency of chosen microcystins can be compared with the other that have been already used. Magnetic resonance imaging is a suitable method for the assessment of acute intoxication with microcystins. Bleeding can also be monitored by the measurment of hematokrit. Pathological liver tests and microcistyc hypochromic anemia are characteristic changes which accompany chronic intoxication. Hystological examination of liver tissue confirms nonspecific changes.