Parametri antioksidacione zaštite u tkivima nekih vrsta slatkovodnih riba iz jezera Gruža

Abstract

Ribe mogu direktno da budu izložene toksinima cijanobakterija i zato su veoma dobri modeli za proučavanje uticaja cijanotoksina na ćelijski antioksidacioni sistem zaštite. Postoji mnogo informacija o biohemijskim i ultrastrukturnim promenama u različitim tkivima riba nakon izlaganja cijanotoksinima u laboratorijskim uslovima, ali su podaci o dejstvu cvetanja cijanobakterija u prirodnim uslovima veoma retki. Cilj ove doktorske disertacije bio je da se istraži uticaj cvetanja cijanobakterija u akumulacionom jezeru Gruža na parametre antioksidacione zaštite u jetri, škrgama i mišiću kod slatkovodnih vrsta riba Rutilus rutilus (bodorka), Blicca bjoerkna (blika), Carassius gibelio (babuška) i Perca fluviatilis (grgeč). Određivani su aktivnost enzima antioksidacione zaštite: ukupne, mangan i bakar cink sadržavajuće superoksiddismutaze (Uk SOD, Mn SOD, CuZn SOD, EC 1.15.1.1), katalaze (CAT, EC 1.11.1.6), glutation-peroksidaze (GSH-Px, EC 1.11.1.9), glutation-reduktaze (GR, EC 1.6.4.2) i enzima faze II biotransformacije glutation-S-transferaze (GST, EC 2.5.1.18), kao i koncentracija ukupnog glutationa (GSH) i sulfhidrilnih (SH) grupa. Histopatološka analiza jetre riba proučavana je na nivou svetlosne i elektronske mikroskopije. Svi ispitivani parametri mereni su pre i tokom cvetanja cijanobakterija. Aktivnost Uk SOD i CuZn SOD u jetri B. bjoerkna, kao i aktivnost Mn SOD u jetri kod svih ispitivanih vrsta riba bila je povećana tokom cvetanja cijanobakterija. Elektroforetskom analizom enzima SOD konstatovane su inducibilne izoforme SOD u jetri B. bjoerkna tokom perioda cvetanja. Aktivnost Uk SOD, Mn SOD i CuZn SOD u škrgama i mišiću ispitivanih vrsta imala je različit stepen smanjenja tokom cvetanja cijanobakterija. Izmerena aktivnost CAT u jetri svih ispitivanih vrsta riba bila je značajno manja tokom cvetanja u odnosu na period pre cvetanja. Suprotan trend detektovan je u škrgama R. rutilus i P. fluviatilis, kao i u mišiću sve četiri vrste riba. Smanjenje enzimske aktivnosti GSH-Px u jetri i mišiću svih ispitivanih vrsta i u škrgama B. bjoerkna i C. gibelio tokom cvetanja cijanobakterija, ukazalo je da cijanotoksini narušavaju integritet ćelijskih membrana u svim ispitivanim tkivima. Aktivnost GR bila je značajno smanjena u jetri C. gibelio, škrgama sve četiri vrste riba i u mišiću R. rutilus i C. gibelio. Povećanje aktivnosti enzima faze II biotransformacije GST u jetri B. bjoerkna tokom perioda cvetanja ukazuje na moguću aktivaciju procesa detoksifikacije toksina cijanobakterija. Smanjenje aktivnosti GST u škrgama i mišiću svih ispitivanih vrsta ukazuje na narušenu efikasnost ovih tkiva da konjuguju cijanotoksine i to može da dovede do njihove izražene unutarćelijske akumulacije tokom cvetanja cijanobakterija. Promena koncentracije ukupnog GSH i SH grupa pokazala je različit odgovor u zavisnosti od ispitivanog tkiva i vrste. Ultrastrukturna istraživanja su ukazala da su hepatociti ispitivanih vrsta riba imali izražene promene gotovo svih organela tokom cvetanja cijanobakterija. Neki hepatociti su imali znakove nekroze, dok je bojenje propidijum-jodidom ukazalo da period cvetanja dovodi i do apoptotskih promena u jetri svih ispitivanih vrsta riba. Dobijeni rezultati su pokazali da cvetanje cijanobakterija indukuje odgovor na oksidacioni stres u ispitivanim tkivima riba i dovodi do ultrastrukturnih promena u jetri. Rezultati ove doktorske disertacije predstavljaju prvi izveštaj o uticaju cvetanja cijanobakterija u akumulacionom jezeru Gruža na parametre antioksidacione zaštite (aktivnost SOD, CAT, GSH-Px, GR i enzima faze II biotransformacije GST, kao i koncentraciju GSH i SH grupa) u jetri, škrgama i mišiću slatkovodnih riba Rutilus rutilus, Blicca bjoerkna, Carassius gibelio i Perca fluviatilis. Biomonitoring antioksidacionih biomarkera u tkivima riba može da posluži kao rani upozoravajući signal koji ukazuje na oštećenja ćelija usled izloženosti cijanotoksinima u slatkovodnim ekosistemima. Ova disertacija pruža veliki doprinos na polju ekofiziologije akvatičnih organizama.

Since fish can be directly exposed to cyanobacterial toxins, they are very good models to study the influence of cyanotoxins on the cellular antioxidant defence system. There is much information on biochemical and ultrastructural alterations in different fish tissues after exposure to cyanotoxins under laboratory conditions, however, data on the effects of cyanobacterial blooms in natural conditions are very scarce. The aim of this doctoral dissertation was to investigate the influence of the cyanobacterial bloom in the Gruža Reservoir on the antioxidant defence parameters in the liver, gills and muscle of freshwater fish Rutilus rutilus (roach), Blicca bjoerkna (white bream), Carassius gibelio (prussian carp) and Perca fluviatilis (European perch). The activity of antioxidant defence enzymes: total, manganese and copper zinc containing superoxide dismutases (Tot SOD, Mn SOD, CuZn SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and the biotransformation phase II enzyme glutathione-S-transferase (GST, EC 2.5.1.18), as well as the concentrations of total glutathione (GSH) and sulphydryl (SH) groups were determined. Histopathological examination of fish liver was performed by light and electron microscopy. All investigated parameters were measured before and during cyanobacterial bloom. The activities of Tot SOD and CuZn SOD in the liver of B. bjoerkna, as well as Mn SOD activity in the liver of all investigated fish species increased during the cyanobacterial bloom. Electrophoretic analysis of SOD revealed the presence of inducible SOD isoforms in the liver of B. bjoerkna during the bloom period. The activities of Tot SOD, CuZn SOD and Mn SOD in the gills and muscle of the investigated species exhibited different degrees of reduction during the cyanobacterial bloom. The activity of CAT in the liver of all investigated fish species was significantly lower during the bloom period than in the pre-bloom period. The opposite trend was detected in the gills of R. rutilus and P. fluviatilis, as well as in the muscle of all four fish species. Reduction in the enzymatic activity of GSH-Px in the liver and muscle of all investigated species and in the gills of B. bjoerkna and C. gibelio during the cyanobacterial bloom indicates that cyanotoxins impair the integrity of the cell membranes in all examined tissues. The activity of GR diminished significantly in the liver of C. gibelio, the gills of all four fish species, and in the muscle of R. rutilus and C. gibelio. The increase in the activity of the biotransformation phase II enzyme GST in the liver of B. bjoerkna during the bloom period points to a potential activation of the detoxification process of cyanobacterial toxins. The decrease in GST activity in the gills and muscle of all tested species indicates a perturbed efficiency of these tissues to conjugate cyanotoxins, which could lead to their intracellular accumulation during the cyanobacterial bloom. The concentrations of total GSH and SH groups exhibited a tissue- and species-dependent response. Ultrastructural examinations showed that hepatocytes displayed profound changes in almost all organelles during the cyanobacterial bloom. Some hepatocytes showed signs of necrosis, while propidiumiodide staining revealed that the bloom period also induced apoptotic changes in the liver of all investigated fish species. The presented results show that the cyanobacterial bloom-induced response to oxidative stress caused ultrastructural alterations in the liver. The results of this doctoral dissertation are a first report on the influence of the cyanobacterial bloom in the Gruža Reservoir on antioxidant defence parameters (SOD, CAT, GSH-Px, GR and biotransformation phase II enzyme GST activities, as well as concentrations of GSH and SH groups) in the liver, gills and muscle of freshwater fish Rutilus rutilus, Blicca bjoerkna, Carassius gibelio and Perca fluviatilis. Biomonitoring of antioxidant biomarkers in fish tissue could serve as an early warning signal of cellular damage resulting from exposure to cyanotoxins in freshwater ecosystems. This dissertation is an important contribution in the field of ecophysiology of aquatic organisms.