Glavni urednik

Research programme and results:

The overall goal of this project is to study the basic principles governing the biotransformation of complex synthetic organic compounds, especially those assigned as dangerous pollutants (xenobiotics). Specific goals are to study the catabolic activity of bacterial communities originating from pure and polluted ecosystems and to further characterize those communities which showed the activity in the transformation of the selected xenobiotics (polychlorinated biphenyls, PCBs and triazine-herbicide atrazine).

Laboratory experiments started by the enrichment of microbial communities and the selection of most active cultures in the transformation of the selected xenobiotics. In these experiments a microcosm enrichment approach was employed with the aim to isolate the bacteria which are representative of long-term biphenyl-adapted bacteria as well as atrazine-adapted bacteria. To study the structure of microbial communities and their catabolic activities modern molecular methods for indentification and characterization of bacteria (PCR, 16S rDNA) were developed.

Enrichment experiments using contaminated soils of different origin, followed by PCB biodegradation studies, resulted in the selection of three cultures (originating from the polluted soils of Trogir and Omišalj airports and of the Transformer Station in Zadar, respectively), which showed substantial PCB-degrading activity. More detailed studies of their catabolic activities suggested that the selected cultures displayed broad substrate specificity (di- to octa-chlorobiphenyls were reduced). The results of these studies were presented at the meetings organized within the EU project ICFP501A2PR02 (Assessment of the selected POPs - PCBs, PCDDs/Fs, OCPs - in the atmosphere and water ecosystems from the waste materials). These results and the more insight characterization of the selected cultures, which is still in progress, should provide the basis for the selection of appropriate strategy for PCB removal from the contaminated soil in the vicinity of the Transformer Station, Zadar (TS 110/35 kV), damaged during recent warfare operations in Croatia, and from other sites with proved PCB contamination. Although the extent of the pollution as a consequence of warfare operations has not as yet been assessed, the existence of contaminated areas in Croatia that would necessitate remediation is very probable.

Similar microcosm enrichment experiments using effluents and polluted soil of the herbicide factory resulted in the selection of three mixed cultures which showed substantial atrazine-mineralizing activity (determined by HPLC analysis and carbon dioxide evolution test). These results were presented at three international scientific conferences. Selected mixed cultures and isolated pure strains were additionally examined to determine the factors that govern their atrazine-transformation activities. Part of this research work was published in a SCI journal and one paper is in preparation. The results of this fundamental research will contribute to the better understanding of the fate and behaviour of triazine compounds in the biosphere. The results obtained in the scientific-research project will also find application in the research and development project TP-01/0098-19 (Conceptual model for biotechnological treatment of atrazine production wastewater) which has been approved within the Croatian Innovation-Technological Development Programme. The objective of this project is to provide the basis for designing the biotechnological treatment plant for atrazine production wastewater.

Oznaka: 0098135

MEHANIZAM MULTIKSENOBIOTIČKE OTPORNOSTI KAO POKAZATELJ KVALITETE VODENOG OKOLIŠA

MULTIXENOBIOTIC RESISTANCE MECHANISM AS A BIOMARKER OF ENVIRONMENTAL QUALITY

Voditelj/ica projekta: dr. sc. Tvrtko Smital

Tel. ++385 1 4561 088   e-mail: [e-mail]

Suradnici na projektu:
Marijan Ahel, doktor kem. znanosti, znanstveni savjetnik, Institut “Ruđer Bošković” (konzultant)
Smiljana Britvić, doktorica biol. znanosti, viša znanstvena suradnica, Institut “Ruđer Bošković”
Sanja Krča, doktorica biol. znanosti, znanstvena suradnica, Institut “Ruđer Bošković”
Tarzan Legović, doktor fiz. znanosti, znanstveni savjetnik, Institut “Ruđer Bošković” (konzultant)
Maja Osmak, doktorica biol. znanosti, znanstvena savjetnica, Institut “Ruđer Bošković” (konzultantica)
Roberta Sauerborn Klobučar, magistrica biol. znanosti, znanstvena novakinja, Institut “Ruđer Bošković”
Tvrtko Smital, doktor biol. znanosti, znanstveni suradnik, Institut “Ruđer Bošković” - voditelj projekta

Tehnički suradnici:
Dalibor Jelaska, Institut “Ruđer Bošković”

Suradnici iz druge ustanove:
David Epel, doktor biol. znanosti, redovni profesor, Stanford University (konzultant)
Branimir Hackenberger-Kutuzović, doktor biol. znanosti, viši asistent, Biološki odjel, Sveučilište J.J. Strossmayera u Osjieku

Program rada i rezultati na projektu:

Novija saznanja ukazuju da fenomen multiksenobiotičke otpornosti (engl., MXR) vodenih organizama po svemu sudeći nije posredovan aktivnošću samo jednog proteina (Pgp), prema čijoj su kakakterizaciji bila usmjerena naša dosadašnja istraživanja. Među nekoliko novih transportnih proteina nedavno identificiranih u različitim humanim i životinjskim tkivima najviše se zna o tzv. multidrug resistance-associated proteinu (engl., MRP). Pretpostavljamo da bi MRP mogao biti prisutan i u stanicama vodenih organizama, odnosno da bi MXR kao fenomen u cjelini mogao biti posredovan aktivnošću bar dva različita proteina. U ekotoksikološkom smislu ključne uloge tih proteina bile bi izbacivanje ksenobiotika iz stanica vodenih organizama i/ili transport detoksikacijskih produkata (metabolita faze I. i II.) izvan stanice. Stoga ćemo u okviru ovog Projekta nastojati: (1) imunokemijski identificirati transportne proteine uključene u MXR sustav vodenih organizama i razviti odgovarajuće in vitro (kultura MXR-stanica) i in vivo (beskralješnjaci i kralješnjaci) metode za funkcionalno mjerenje transportne aktivnosti ovih proteina, odnosno mjerenje koncentracije MXR-inhibitora; (2) objasniti ulogu MXR-transportnih proteina kao integralnog dijela staničnog detoksikacijskog sustava; i (3) pokazati da će inhibicija MXR mehanizma dovesti do povećanja akumulacije i učinka genotoksičnih, ksenoestrogenih i drugih toksičnih ksenobiotika i njihovih metabolita prisutnih u vodenom okolišu. Primarno značenje predloženog istraživanja jest pružanje znanstvene podloge za afirmaciju mjerenja aktivnosti MXR mehanizma kao relevantnog biomarkera kvalitete okoliša. Prvi rezultati naših istraživanja u okviru ovog Projekta jasno ukazuju na prisustvo gena MRP porodice transportnih proteina u jetri i tankom crijevu slatkovodnih (Cyprinus carpio, Salmo trutta) i morskih riba (Mullus barbatus).

Research programme and results:

Recent evidences suggest that multixenobiotic resistance (MXR) phenomenon in aquatic organisms could also be mediated by the activity of other, non-Pgp transport proteins. Based on the main characteristics and on the tissue distribution of recently discovered transport proteins we presume that the members of the so-called multidrug resistance-associated protein (MRP) family, may also be present in aquatic organisms. Consequently, the MXR phenomenon in general could be mediated by the activity of at least two different transport proteins. From the ecotoxicological perspective the key role of those proteins would be the transport of xenobiotics and/or detoxification products (phase I. and II. metabolites) out of cell. In order to prove these hypothesis our work would be directed to: (1) immunochemical identification of transport proteins involved in MXR of aquatic organisms, and development of both in vitro and in vivo methods for the functional determination of their transport activity; (2) explanation of the role of MXR transport proteins as an integral part of cell detoxification system; (3) we will try to demonstrate that the inhibition of MXR mechanism may result in the increase in accumulation and effect of genotoxic, xenoestrogenic or other toxic xenobiotics or their metabolites present in aquatic environment. General aim of the proposed investigation is to offer a scientific argumentation for the validation of MXR as a relevant biomarker of environmental quality. First results of investigations performed within this Project clearly showed that the gene encoding for MRP related proteins are present in liver and intestine tissue of several freshwater (Cyprinus carpio, Salmo trutta) and marine (Mullus barbatus) fish species.

PRILOZI

Znanstveni radovi objavljeni u časopisima koji su indeksirani u Current Contents-u:

1.	Ahel, Marijan; Terzić, Senka. Biogeochemistry of aromatic surfactants in microtidal estuaries. // Chimia. 57 (2003) ; 550-555.
2.	Bayraktaroglu, Emine; Legović, Tarzan; Velasquez, Zoila R.; Cruzado, Antonio. Diatom Thalassiosira weissflogii in oligotrophic versus eutrophic culture: models and ultrastructure. // Ecological Modelling. 170 (2003) ; 237-243.
3.	Bermanec, Vladimir; Furić, Krešimir; Rajić, Maša; Kniewald, Goran. Thermal stability and vibrational spectra of the sheet borate tuzlaite, NaCa(B5O8(OH)2)x3H2O. // American Mineralogist. 88 (2003) , 2; 271-276.
4.	Ciglenečki, Irena; Helz, George R. Voltammetric behavior of MoS42- and SbS43-, possible components of "dissolved sulfide" in oxic natural waters. // Electroanalysis. 15 (2003) , 2; 145-150.
5.	Ciglenečki, Irena; Plavšić, Marta; Vojvodić, Vjeročka; Ćosović, Božena; Pepi, Milva; Baldi, Franco. Mucopolysaccharide transformation by sulfide in diatom culture and natural mucilage. // Marine Ecology Progress Series. 263 (2003) , 28; 17-27.
6.	Djogić, Renata; Krznarić, Damir. Adsorption of Tri-n-butyl Phosphate (TBP) from NaClO4 Aqueous Solution on Hg Electrode. // Electroanalysis. 15 (2003) , 4; 312-318.
7.	Filipović, Vlatka; Raspor, Biserka. Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea. // Water Research. 37 (2003) , 13; 3253-3262.
8.	Frančišković-Bilinski, Stanislav; Bilinski, Halka; Barišić, Delko; Horvatinčić, Nada; Yuan, Daoxian. Analysis of tufa from Guangxi (China). // Acta Geologica Sinica (English Edition). 77 (2003) , 2; 267-275.
9.	Frančišković-Bilinski, Stanislav; Bilinski, Halka; Vdović, Neda; Balagurunathan, Yoganand; Dougherty, Edward Russel. Application of image-based granulometry to siliceous and calcareous estuarine and marine sediments. // Estuarine Coastal and Shelf Science. 58 (2003) , 2; 227-239.
10.	Gašparović, Blaženka; Ćosović, Božena. Surface-active properties of organic matter in the North Adriatic Sea. // Estuarine, Coastal and Shelf Science. 58 (2003) ; 555-566.
11.	Horvat-Radošević, Višnja; Kvastek, Krešimir. Impedance spectra of the hydrogen evolution reaction at low cathodic overpotentials. // Croatica Chemica Acta. 76 (2003) , 4; 313-322.
12.	Jeličić, Ivana; Ahel, Marijan. Occurrence of phenazone analgesics and caffeine in Croatian municipal wastewaters. // Fresenius Environmental Bulletin. 12 (2003) , 1; 46-50.
13.	Komorsky-Lovrić, Šebojka; Vukašinović, Nevenka; Penovski, Rahela. Voltammetric determination of micro particles of some local anaesthetics and antithusics immobilised on the graphite electrode. // Electroanalysis. 15 (2003) , 5-6; 544-547.
14.	Kozarac, Zlatica; Ćosović, Božena; Frka, Sanja; Moebius, Dietmar; Hacke, Sussane. Complex methodological approach to the studies of natural microlayers at the air/water interface. // Colloids and Surfaces A: Physicochemical and Engineering Aspects. 219 (2003) ; 173-186.
15.	Legović, Tarzan. Prediction of seawater quality around Island Rab (Adriatic Sea). // Ecological Modelling. 160 (2003) ; 131-143.
16.	Legović, Tarzan; Janeković, Ivica; Viličić, Damir; Petricioli, Donat; Smoljan, Zorica. Effects of freshwater release to a marine bay. // Journal of Environmental Science and Health Part A - Toxic/Hazardous Substances and Environmental Engineering. A38 (2003) , 8; 1411-1420.
17.	Lovrić, Milivoj; Komorsky-Lovrić, Šebojka. Distribution of three ions in the thin film experiment. // Electrochemistry Communications. 5 (2003.) , 8; 637-643.
18.	Lovrić, Milivoj; Scholz, Fritz. Modelling cyclic voltammograms of simultaneous electron and ion transfer reactions at a conic film three-phase electrode. // Journal of Electroanalytical Chemistry. 540 (2003) ; 89-96.
19.	Lovrić, Milivoj; Turyan, Yakov. A model of CE mechanism on spherical electrodes. // Croatica Chemica Acta. 76 (2003) , 3; 189-197.
20.	Malej, Alenka; Mozetič, Patricija; Turk, Valentina; Terzić, Senka; Ahel, Marijan; Cauwet, Gustave. Changes in particulate and dissolved organic matter in nutrient-enriched enclosures from an area influenced by mucilage: the northern Adriatic Sea. // Journal of Plankton Research. 25 (2003) ; 949-966.
21.	Mikac, Nevenka; Foucher, Delphine; Niessen, Sylvie; Lojen, Sonja; Fisher, Jean-Claude. Influence of chloride and sediment matrix on the extractability of HgS (cinnabar and metacinnabar) by nitric acid.. // Analytical and Bioanalytical Chemistry. 377 (2003) ; 1196-1201.
22.	Mlakar, Marina. Determination of trace ytterbium by cathodic stripping voltammetry. // Electroanalysis. 15 (2003) ; 27-35.
23.	Mlakar, Marina; Boulmaaz, Souad; Schönberg, Hartmut; Grützmacher, Hansjörg. Electrochemical investigations give some insight into the coordination chemistry of new stable iridium(+1), iridium(0) and iridium(-1) complexes. // Electroanalysis. 15 (2003) , 12; 1043-1053.
24.	Niessen, Sylvie; Foucher, Delphine; Clarisse, O; Fischer, Jean-Claude; Mikac, Nevenka; Kwokal, Željko; Fajon, V; Horvat, Milena. Influence of sulphur cycle on mercury methylation in estuarine sediment (Seine estuary, France). // Journal de Physique IV. 107 (2003) ; 953-956.
25.	Omanović, Dario; Branica, Marko. Pseudopolarography of trace metals. Part 1. The automatic ASV measurements of reversible electrode reactions. // Journal of Electroanalytical Chemistry. 543 (2003) , 1; 83-92.
26.	Picer, Mladen; Holoubek, Ivan. PCBs in the karstic environment of Slovenia and Croatia as consequence of their accidental release. // Fresenius Environmental Bulletin. 12 (2003) ; 86-93.
27.	Pižeta, Ivanka; Billon, Gabriel; Fischer, Jean-Claude; Wartel, Michel. Solid microelectrodes for in situ voltammetric measurements. // Electroanalysis. 15 (2003) , 17; 1389-1396.
28.	Plavšić, Marta. Electroanalytical techniques applied for studying the interaction of organic matter and particles with metal ions in natural waters. // Analytical Letters. 36 (2003) , 1; 143-157.
29.	Risović, Dubravko; Gašparović, Blaženka; Ćosović Božena. Hydrodynamic influence on the fractal morphology of the linoleic acid adsorbed layer at the mercury/electrolyte interface. // Colloids and Surfaces A: Physicochemical and Engeneering Aspects. 223 (2003) ; 145-156.
30.	Smital, Tvrtko; Sauerborn, Roberta; Hackenberger, Branimir. Inducibility of the multixenobiotic resistance mechanism (MXR) transport activity in the marine mussel Mytilus galloprovincialis and the freshwater mussel Dreissena polymorpha. // Aquatic Toxicology. 65 (2003) ; 443-465
31.	Sondi, Ivan; Goia, V.Dan; Matijević, Egon. Preparation of highly concentrated stable dispersions of uniform silver nanoparticles. // Journal of Colloid and Interface Science. 260 (2003); 75-81.
32.	Sondi, Ivan; Matijević, Egon. Homogeneous precipitation by enzyme catalyzed reactions II. Strontium and barium carbonates. // Chemistry of Materials. 15 (2003), 6; 1322-1326.
33.	Sondi, Ivan; Slovenec, Dragutin. The mineralogical characteristics of the Lamboglia 2 Roman-age amphorae from the central Adriatic (Croatia). // Archaeometry. 45 (2003) , 2; 251-262.
34.	Tomec, Marija; Teskeredžić, Zlatica; Teskeredžić, Emin. Food and nutritive value of gut contents of rudd (Scardinius erythrophtalmus L.) from Lake Vrana, Cres Island, Croatia. // Czech Journal of Animal Science. 48 (2003) , 1; 28-34.
35.	Udiković, Nikolina; Hršak, Dubravka; Mendaš, Gordana; Filipčić, Daša. Enrichment and characterization of atrazine degrading bacterial communities. // Food Technology and Biotechnology. 41 (2003) , 3; 211-217.
36.	Zelić, Marina. Electrochemical reduction of europium(3+) at increasing concentrations of different salts. Part I. Voltammetric measurements. // Croatica Chemica Acta. 76 (2003) , 3; 241-248.
37.	Zelić, Marina; Lovrić, Milivoj. The influence of electrolyte concentration on the parameters of the Frumkin isotherm in the Cd2+ - I- system. // Journal of Electroanalytical Chemistry. 541 (2003) ; 67-76.

Knjige i poglavlja u knjigama:

1.	3. znanstveni skup hrvatskih biofizičara / Svetličić, Vesna; Lučić, Bono (ur.). Zagreb : Institut “Ruđer Bošković”, Zagreb, 2003.
2.	Legović, Tarzan; Geček, Sunčana. Jablanac: Hidrografija, kretanje vodenih masa, predviđanje utjecaja akvakulture na morski stupac i dotok čestica na dno // Studija utjecaja na okoliš uzgajališta pastrva u Velebitskom Kanalu - lokacija Jablanac / Koren, Željko (ur.). Zagreb : Oikon, 2003. 30-40, 98-108.
3.	Legović, Tarzan; Geček, Sunčana. Lukovo Šugarje: Hidrografija, kretanje vodenih masa te predviđanje utjecaja akvakulture na vodeni stupac i donosa na dno. // Studija utjecaja na okoliš uzgajališta pastrva u Velebitskom Kanalu - lokacija Lukovo Šugarje / Koren Željko (ur.). Zagreb : Oikon, 2003. 30-41, 62-64, 96-106.
4.	Picer, Mladen; Picer, Nena; Hodak Kobasić, Vedranka. PCB levels in soil inside electrotransformer station 110/35 kv in Zadar // Organohalogen compounds, Dioxin 2003 ; environmental levels emerging POPs site assessment / Hunt, Gary ; Clement, Ray (ur.). Boston, Massachusetts, USA : RPJ Associates, Massachusetts, USA, 2003. 472-475.
5.	Rovinj 2003 experiment: Northern Adriatic mesocosm. Experimental data / Svetličić, Vesna; Hozić, Amela (ur.). Zagreb : Institut “Ruđer Bošković”, Zagreb, 2003.
6.	The Third European Ecological Modelling Conference. / Legović, Tarzan (gostujući ur.). Amsterdam : Elsevier, 2003.

Radovi objavljeni u ostalim časopisima:

1.	Ćosović, Božena; Kozarac, Zlatica; Raspor Biserka. 8. Stručni sastanak laboratorija ovlaštenih za ispitivanje voda. // Hrvatska vodoprivreda. 7/8 (2003), 127-128; 81-83.
2.	Horvat-Radošević, Višnja; Kvastek, Krešimir. Electrochemical impedance spectroscopy of some noble metal electrodes. // Impedance Contributions Online. 1 (2003.); L1-1 - L1-11.
3.	Kapetanović, Damir; Teskeredžić, Emin. Bakterijska flora ličinki i mlađi kalifornijske pastrve (Oncorhynchus mykiss). // Ribarstvo. 61 (2003), 3; 103-119.
4.	Legović, Tarzan. The exclusive economic zone - a first step toward sustainable management of marine ecosystems. // Croatian International Relations Review. 9 (2003), 32 Dossier; 35-39.
5.	Marijanović Rajčić, Marija; Raspor, Biserka. Pregled hrvatskih norma iz područja kakvoće vode (II dio). // Hrvatska vodoprivreda. XII (2003), 127-128; 68-76.
6.	Picer, Mladen; Picer, Nena. Hazards posed by petroleum oil and some chlorinated hydrocarbons to aquatic ecosystem in croatian karst during 1991-1995 war in Croatia. // Periodicum Biologorum. 105 (2003), 3; 345-354.
7.	Tomec, Marija. Mikrofitobentos Vranskog jezera (otok Cres). // Ribarstvo. 61 (2003), 2; 55-73.