18 research outputs found
OdreÄivanj prisutnosti ekotoksikoloÅ”ki važnih Pgp i MRP-proteina u Å”arankama na genskoj i proteinskoj razini
One of the most intriguing defence strategies which aquatic organisms developed through evolution is multixenobiotic resistance (MXR). The key mediators of MXR activity are ATP-binding cassette (ABC) transport proteins. They provide resistance of aquatic organisms by binding xenobiotics and extruding them from cells in an energy-dependent manner. Since Cyprinid fi sh species are common target in freshwater biomonitoring programs, we have studied the presence of two main MDR/MXR effl ux transporters Pglycoprotein
(Pgp, Abcb1) and MRP-like protein(s) (Abcc) in the liver of fi ve Cyprinid species: common carp, European chub, sneep, barbel, and silver prussian carp. Their presence was evaluated on the mRNA
and protein level. Various pairs of primers were designed to clone homologous fragments of MXR-related genes. At the protein level, we used Western blotting with specifi c monoclonal antibodies against human Pgp (Abcb1, Ab C219), MRP1 (Abcc1; Ab MRPm6) or MRP2 (Abcc2; Ab M2I-4). Transcripts of both key types of MXR transporters were identifi ed in all species examined and here we provide the phylogenetic analysis of new partial sequences. Immunochemical determinations with mammalian antibodies failed to identify the presence of MRP(s), but Pgp expression was found in all fi ve Cyprinid species. These results support that MXR is a defence system mediated by both Pgp and MRP types of ABC transport proteins.Mehanizam multiksenobiotiÄke otpornosti (MXR) staniÄni je obrambeni sustav odgovoran za svojstvo istovremene otpornosti na razliÄite ksenobiotike, koje se oÄituje smanjenjem akumulacije, odnosno poveÄanjem izbacivanja potencijalno toksiÄnih tvari iz stanica vodenih organizama. MXR-mehanizam pokazuje odlike analogne fenomenu istovremene otpornosti na razliÄite lijekove (engl. multidrug resistance, MDR) prvi put dokazanom u tumorskim stanicama. Posredovan je istim ABC transportnim proteinima kao
i MDR. Istraživanja vezana uz MXR vodenih životinja uglavnom su imala težiÅ”te na odreÄivanju prisutnosti i funkcije P-glikoproteina (Pgp). BuduÄi da se ribe iz reda Å”aranki Äesto rabe u biomonitoringu slatkovodnih voda, cilj ovog istraživanja bilo je odreÄivanje prisutnosti dvaju glavnih MDR/MXR-tipova proteina - Pgp i MRP-tip proteina - u jetri iz pet vrsta Å”aranki; Å”arana (Cyprinus carpio), klena (Sqalius cephalus), mrene (Barbus barbus), babuÅ”ke (Carassius auratus gibelio) i podusta (Chondrostoma nasus). Njihova prisutnost utvrÄena je na razini mRNA te na proteinskoj razini. RazliÄiti parovi poÄetnica dizajnirani su kako bi se identifi cirali homologni fragmenti gena sliÄnih MXR-u. Detekcija na razini proteina napravljena
je putem Western blot analize s pomoÄu specifi Änih monoklonskih protutijela proizvedenih da prepoznaju konzervirane epitope; Pgp (C219), MRP1 (MRPm6) ili MRP2 (M2I-4) u sisavaca. Transkripti obaju kljuÄnih MXR-transportera identifi cirani su u svim jedinkama, a napravljena je i fi logenetska analiza dobivenih sekvenci. Imunokemijskom detekcijom s protutijelima sisavaca nismo uspjeli detektirati prisutnost MRP-proteina, dok je Pgp-ekspresija potvrÄena u svih pet vrsta Å”aranki. Nove spoznaje da je za MXRmehanizam
zaslužno viÅ”e transportnih proteina zasigurno Äe pridonijeti potpunijoj karakterizaciji MXR-a kao integralnog dijela detoksikacijskog, odnosno temeljnog obrambenog sustava vodenih organizama te
njegovoj znanstvenoj afi rmaciji kao vjerodostojnog pokazatelja kvalitete okoliŔa
First evidence of the P-glycoprotein gene expression and multixenobiotic resistance modulation in earthworm
Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators
(verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthwormās MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity