Distribution of selected metals among cytosolic biomolecules in gills and liver of chub (Squalius cephalus (Linnaeus) and Squalius vardarensis Karaman; Actinopterygii, Cyprinidae) from metal contaminated natural waters

Citosolske raspodjele nekoliko odabranih elemenata (Cd, Co, Cu, Fe, Mn, Mo, Pb, Se i Zn) u jetrima i škrgama dvaju slatkovodnih bioindikatorskih organizama, klena (Squalius cephalus) iz rijeke Sutle u Hrvatskoj i vardarskog klena (Squalius vardarensis) iz triju rijeka sjeveroistočne Sjeverne Makedonije, izučavane su primjenom tekućinske kromatografije visoke djelotvornosti s isključenjem po veličini (SEC-HPLC) i spektrometrije masa visoke rezolucije s induktivno spregnutom plazmom (HR ICP-MS). Određene su raspodjele metala/nemetala među citosolskim biomolekulama različitih molekulskih masa svojstvene uvjetima niske izloženosti metalima u riječnoj vodi, kao i promjene u raspodjelama koje su javljaju kao posljedica povišenih razina izloženosti. Primjena anionsko izmjenjivačke kromatografije (AEC-HPLC) kao drugog stupnja razdvajanja i pročišćavanja citosolskih biomolekula te dviju tehnika spektrometrije masa (MALDI-TOF-MS i LC-MS/MS) omogućila je identifikaciju ili precizno određivanje molekulskih masa nekoliko citosolskih biomolekula koje vežu metale u jetrima i škrgama vardarskog klena, poput izoformi metalotioneina koje vežu Cd, Cu i Zn, hemoglobinskih podjedinica koje vežu Fe te toplinski stabilnih biomolekula koje vežu Mo. Primijenjeni analitički pristup pokazao se korisnim u istraživanju unutarstanične sudbine i ponašanja metala u organima riba te otkrivanju potencijalnih novih biomarkera izloženosti i učinaka metala.The cytosolic distributions of several selected elements (Cd, Co, Cu, Fe, Mn, Mo, Pb, Se, and Zn) in the liver and gills of two freshwater bioindicators, European chub (Squalius cephalus) from the Sutla River in Croatia and Vardar chub (Squalius vardarensis) from three rivers of north-eastern Macedonia, were studied using size-exclusion high performance liquid chromatography (SEC-HPLC) and high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). Distributions of metals/non-metal among the biomolecules of different molecular masses which are characteristic for low metal exposure in the river water were determined, as well as the changes in distribution which occur as a consequence of increased exposure to metals. The use of anion-exchange high performance liquid chromatography (AEC-HPLC) as the second step of separation and purification of cytosolic biomolecules, as well as the use of two mass spectrometry techniques (MALDI-TOF-MS and LC-MS/MS) have enabled the identification or exact determination of molecular masses of several cytosolic biomolecules that bind metals in the liver and gills of Vardar chub, such as the metallothionein isoforms that bind Cd, Cu and Zn, the hemoglobin subunits that bind Fe and heat stable biomolecules that bind Mo. Applied analytical approach has proven to be useful in the study of intracellular fate and behavior of metals in organs of fish, as well as in the process of discovery of potential new biomarkers of metal exposure and effects