Efekti kvercetina i epikatehina na oksidativno-antioksidativni status pacova tretiranih bakar(II)-jonom

Abstract

Bakar kao esencijalni mikroelement ima važnu ulogu u biološkim sistemima međutim, povećane koncentracije mogu da izazovu toksične efekte. Kao prelazni metal, bakar može da stimuliše produkciju reaktivnih vrsta kiseonika preko Fenton-ove i Haber-Weiss-ove reakcije i dovede do oksidativnih oštećenja biomolekula u tkivima, pre svega u jetri koja učestvuje u njegovom metabolizmu. Flavonoidi su prirodna biljna polifenolna jedinjenja koja pokazuju širok spektar biološke aktivnosti. Antioksidativna svojstva mogu da ispolje različitim mehanizmima, u direktnoj reakciji sa reaktivnim vrstama ili helacijom jona prelaznih metala, koji bi u daljim reakcijama proizveli reaktivne vrste. Cilj istraživanja ove doktorske disertacije je ispitivanje efekata subhronične intoksikacije bakrom na hematološko-biohemijske parametre u krvi, oksidativni metabolizam eritrocita i parametre oksidativnog stresa u jetri pacova. Takođe, cilj je i ispitivanje protektivnog kapaciteta i sinergističkog delovanja flavonoida kvercetina (QE) i epikatehina (EC), kao čistih supstanci, u uslovima oksidativnog stresa. Upoređivani su i efekti primenjenih flavonoida i komercijalnog leka D-penicilamina (DPA), tiolnog jedinjenja koje se koristi za tretman oboljenja čiji je uzrok povećano akumuliranje bakra u tkivima (Vilsonova bolest). Studija je izvedena na mužjacima pacova soja Wistar albino, 8 nedelja starosti na početku eksperimenta, nasumično podeljenih u 5 grupa po 7 životinja: (1) kontrolna (0.3 ml fiziološkog rastvora, i.p.); (2) tretirana bakrom (CuCl2·2H2O u koncentraciji od 560 mg/L) preko vode za piće u trajanju od 5 nedelja; (3) tretirana bakrom (na način opisan prethodno) i DPA (50 mg/kg TT, u 0.3 ml redestilovane vode, i.p.) svakog drugog dana tokom poslednje 2 nedelje eksperimenta (ukupno 5 injekcija); (4) tretirana bakrom (na način opisan prethodno), a zatim flavonoidima QE i EC (40 mg QE/kg TT + 40 mg EC/kg TT, u 0.3 ml redestilovane vode, i.p.) svakog trećeg dana tokom poslednje 3 nedelje eksperimenta (ukupno 7 injekcija); (5) tretirana flavonoidima QE i EC (na način opisan prethodno). U punoj krvi određivani su hematološki parametri: broj eritrocita (RBC), koncentracija hemoglobina (Hb), hematokrit (Hct), broj leukocita (WBC), broj trombocita (Plt), hematološki indeksi, i biohemijski parametri: koncentracije elektrolita (K, Na, Ca, Fe, fosfata), koncentracije ukupnih proteina, albumina, glukoze i ukupnog holesterola, aktivnosti enzima aspartat aminotransferaze (AST), alanin aminotransferaze (ALT), alkalne fosfataze (ALP), gama-glutamil transpeptidaze (GGT) i laktat dehidrogenaze (LDH). U lizatu eritrocita, primenom standardnih analitičkih metoda, određivani su markeri oksidativnog stresa: koncentracije lipidnih peroksida (LPO), koncentracije reaktivnih vrsta kiseonika i azota (superoksid anjon radikala (O2 •-), vodonik peroksida (H2O2) i nitrita (NO2 -)), koncentracije redukovanog (GSH) i oksidovanog glutationa (GSSG) i aktivnosti enzima superoksid-dismuze (SOD), katalaze (CAT), glutation- peroksidaze (GSH-Px), glutation-reduktaze (GR), glutation-S-transferaze (GST). U izolovanom tkivu jetre, nakon homogenizacije, primenom standardnih analitičkih metoda takođe je određen redoks status (koncentracije LPO, neenzimskih antioksidanata GSH, GSSG i Vit C, i aktivnosti enzima AOS: ukupne SOD, CuZn SOD, Mn SOD, CAT, GSH-Px, GR i GST). Koncentracije bakra i ceruloplazmina određene su u plazmi i supernatantu tkiva jetre. Rezultati istraživanja pokazuju da bakar u subhroničnom tretmanu ispoljava direktan prooksidativni efekat na eritrocite i tkivo jetre pacova i pokazuje značajne toksične efekte na hematološko-biohemijske procese u krvi. Subhronična intoksikacija bakrom dovodi do povećanja LPO u eritrocitima i tkivu jetre, uzrokuje hemolizu eritrocita i anemiju, porast ukupnog holesterola, poremećaj homeostaze elektrolita. Nakon subhronične intoksikacije bakrom povišene su aktivnosti enzima AST, ALT, ALP, GGT i LDH, markera citotoksičnosti jetre u serumu. Oksidativnim oštećenjima eritrocita i tkiva jetre doprinosi povećana produkcija O2 •- i H2O2 i smanjenje neenzimskih antioksidanata, ćelijskog pula GSH i Vit C. Promene u aktivnostima enzima AOS posle subhronične intoksikacije bakrom takođe ukazuju na pojavu oksidativnog stresa u eritrocitima i tkivu jetre. Flavonoidi QE i EC pokazuju antioksidativnu sposobnost i umanjuju oksidativni stres nastao nakon subhroničnog izlaganja bakru. QE i EC stabilizuju membrane, blokiraju lipidnu peroksidaciju i time igraju ulogu u ublažavanju manifestacija toksičnosti u eritrocitima i tkivu jetre. Značajno povećavaju koncentracije neenzimskih antioksidanata GSH i Vit C u ćelijama, indukuju sintezu enzima AOS, čime doprinose povećanju antioksidativnog kapaciteta ćelija. Sinergističko delovanje flavonoida QE i EC, kao i citoprotektivna uloga koju pokazuju, može se pripisati njihovoj sposobnosti da heliraju metale i/ili uklanjaju O2 •- i N2O2. Rezultati takođe pokazuju da su QE i EC efektivniji u antioksidativnom delovanju u poređenju sa DPA, koji pored efikasnosti u heliranju metala ispoljava i prooksidativne efekte povećanjem lipidne peroksidacije u eritrocitima i tkivu jetre. Ovo istraživanje predstavlja doprinos kompletiranju naučnih saznanja o toksičnim efektima bakra, kao i kompleksnosti protektivnog delovanja flavonoida kvercetina i epikatehina na oksidativno-antioksidativni status eritrocita i ćelija tkiva jetre. Zbog širokog spektra biohemijskih i farmakoloških dejstava, posebno treba istaknuti značaj ispitivanih flavonoida u zdravoj ishrani, kao i u prevenciji i terapiji mnogih oboljenja.

Copper is an essential trace element that plays an important role in biological systems, however, the increased concentration may cause toxic effects. As the transition metal, copper may stimulate the production of reactive oxygen species by Fenton's and Haber-Weiss reaction and lead to oxidative damage of biomolecules in the tissues, especially in the liver, which participates in its metabolism. Flavonoids are a natural plant polyphenol compounds which exhibit a wide spectrum of biological activity. They may exert antioxidant properties through different mechanisms, in a direct reaction with a reactive species,or by chelating transition metal ions, which in a further reaction may produce reactive species. The effects of subchronic copper intoxication on haematological and biochemical parameters in blood,oxidative metabolism of erythrocytes and oxidative stress parameters in rat liver are objectives of this dissertation research. The aim is also to examine the protective capacity and synergistic effects of flavonoids quercetin (QE) and epicatechin (EC), as a pure substances, under oxidative stress conditions. We also compared the effects of these flavonoids and a commercial drug D-penicillamine (DPA), a thiol compound used for the treatment of diseases caused by an increased accumulation of copper in the tissues (Wilson's disease). The study was performed on male albino Wistar rats, 8 weeks old at the beginning of the experiment,randomly divided into 5 groups of 7 animals: (1) control (0.3 ml of saline, i.p.); (2) copper-treated (CuCl2·2H2O at a concentration of 560 mg/L) through drinking water for 5 weeks; (3) treated with copper (as described previously), and DPA (50 mg/kg body weight in 0.3 ml of re-distilled water, i.p.) every other day during the last 2 week of the experiment (total number of injections were 5); (4) treated with copper (as described above), and flavonoids QE and EC (40 mg QE/kg body weight + 40 mg EC/kg body weight, in 0.3 ml of re-distilled water,i.p.) every third day during the last 3 weeks of the experiment (total number of injections were 7); and (5) treated with flavonoids QE and EC (as described previously). Hematological parameters: erythrocyte count (RBC),hemoglobin concentration (Hb), hematocrit (Hct), leukocyte count (WBC), platelet count (Plt), hematological indices, and biochemical parameters: concentration of electrolytes (K, Na, Ca, Fe, phosphate), total protein,albumin, glucose, and total cholesterol, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH) were measured in the whole blood. Markers of oxidative stress: lipid peroxidation (LPO),the concentration of reactive oxygen and nitrogen species (superoxide anion radicals (O2 •-), hydrogen peroxide (H2O2) and nitrite (NO2 -)), reduced (GSH) and oxidized glutathione (GSSG) concentrations, and activities of AOS enzymes: superoxide-dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione-S-transferase (GST) were determined in the erythrocyte lysate using standard analytical methods. In isolated liver tissue after homogenization, the redox status (LPO concentration, GSH, GSSG and Vit C concentration, and the activities of total SOD, CuZn SOD, Mn SOD, CAT, GSH-Px, GR, and GST) was also determined using standard analytical methods. The concentrations of copper and ceruloplasmin were determined in the plasma and in the liver tissue supernatant. The results show that copper in subchronic treatment exerts a direct pro-oxidative effect on erythrocytes and liver tissue of rats and significant toxic effects on haematological and biochemical processes in the blood. Subchronic intoxication with copper leads to an increase in LPO in liver tissue and in erythrocytes, causes haemolysis and anemia, increase in total cholesterol, electrolyte homeostasis disbalance. After subchronic copper intoxication activities of cytotoxicity liver markers AST, ALT, ALP, GGT, and LDH, in the serum were elevated. Increased production of O2 •- and H2O2 and the reduction of non-enzymatic antioxidants, GSH and Vit C, contribute to oxidative damage in red blood cells and liver tissue. Changes in the activities of the AOS enzymes after subchronic intoxication with copper also indicated the occurrence of oxidative stress in erythrocytes and liver tissue. Flavonoids QE and EC exhibit antioxidant capacity and reduce oxidative stress occurring after subchronic copper exposure. QE and EC stabilize the membrane, blocking the lipid peroxidation, and thereby play a role in mitigating the manifestation of toxicity in the erythrocytes and liver tissue. They significantly increase the concentration of non-enzymatic antioxidants GSH and Vit C in cells, induce synthesis of the AOS enzymes, thus contributing to increase the cells antioxidant capacity. The synergistic action of the flavonoids QE and EC, and a cytoprotective role which they exert, can be attributed to their ability to chelate metal ions and/or remove O2 •- and H2O2. The results also show that QE and EC were more effective in antioxidant activity when compared with the DPA, which in addition to the efficiency of chelating metal ions exerts prooxidant effects, by increasing lipid peroxidation in erythrocytes and liver tissue. This research is a contribution to the scientific knowledge of the toxic effects of copper, as well as the complexity of the protective effect of flavonoids quercetin and epicatechin in the oxidative-antioxidant status of red blood cells and liver tissue cells. Considering their wide range of biochemical and pharmacological effects, we should point out to the importance of the flavonoids in a healthy diet, as well as in the prevention and treatment of many diseases.