Primjena kulture stanica prednjeg režnja hipofize u toksikološkim istraživanjima

High density plating procedure was used to evaluate the effect of atrazine on anterior pituitary cells of rats in monolayer culture. Collagenase-dispersed pituitary cells plated in suspension with medium-199 and 10% foetal calf serum attached quantitatively to plastic surfaces within 24 hours. Electron microscopy showed subpopulations of different cell types. After prolonged cultivation, most cells established small colonies with extensive contacts among them. Cell-to-cell formation of aggregates was significant and the colonies manifested morphological changes. The cells retained their enzymatic activity, converting testosterone into 5alfa-dihydrotestosterone by enzyme 5alfa-reductase. Immunohistochemical techniques facilitated differentiation of gonadotrophs producing follicle stimulating (FSH) and luteinising hormones (LH). Atrazine in concentrations of 5 to 50 µg/ml of medium was associated with a significant reduction in the number of viable cells within 72 hours. The results suggest that the pituitary cell culture may prove useful in toxicological testing of various toxic compounds and reduce or replace in vivo animal experiments.Atrazin, selektivni s-triazinski herbicid, danas se rabi u velikim količinama za zaštitu kukuruznih polja, a zbog svoje liposolubilnosti i perzistentnosti u okolišu ulazi u hranidbeni lanac. In vitro sustavi tkiva i organa sisavaca uz in vivo pokuse još i danas su pretežite tehnike u toksikologiji koje se pouzdano primjenjuju. Potrebe za smanjenjem broja pokusa na životinjama dovele su do intenzivna razvoja tehnika sa životinjskim stanicama, bilo da se radi o primarnim kulturama ili o staničnim linijama. Primarne kulture stanica središnjega živčanog sustava nisu do danas doživjele punu primjenu. Primjena životinjskih stanica mnogo je jeftinija tehnika koja omogućuje provođenje istodobnog ispitivanja na velikom broju uzoraka. Cilj istraživanja bio je ispitati toksičnost atrazina u in vitro uvjetima na modelu kulture stanica hipofize, koje su odgovorne za normalno funkcioniranje reprodukcijskog procesa. U radu se dobrom izolacijskom metodom i visokim stupnjem uspješnog uspostavljanja kulture stanica, mogao pratiti utjecaj atrazina na razini staničnih promjena. Stanice raspršene kolagenazom u suspenziji s medijem 199 i 10%-tnim telećim serumom prihvatile su se za plastičnu podlogu unutar 24 sata. Elektronskom mikroskopijom nađeni su različiti tipovi stanica. Nakon produljene kultivacije, najveći broj stanica stvorio je male kolonije s uočljivim vezama između stanica. Stanice su pokazale dobru enzimsku aktivnost pri pretvorbi testosterona u aktivni metabolit 5alfa-dihidrotestosteron. Imunohistokemijskim metodama utvrđene su stanice koje sintetiziraju hormone što stimuliraju razvoj folikula (FSH) i hormone luteinizacije (LH). Atrazin je dodan kulturi stanica hipofize u koncentracijama 5–50 µg/ml medija koji je sadržavao 3x105 stanica. Tijekom 24, 48 i 72 sata nađena su značajna smanjenja broja živih stanica, od 36 do 46%. Učinci su ovisili o dozi atrazina. Rezultati upućuju na to da se kulture stanica adenohipofize mogu uspješno rabiti u budućim istraživanjima toksičnosti različitih tvari radi smanjenja ili zamjene in vivo pokusa na životinjama

Alternativni modeli ispitivanja toksičnosti ksenobiotika

The alternatives to whole-animal testing include endpoint assays, cell and tissue cultures, use of tissue slices, toxicokinetic modelling, and structure-activity relationships and databases. The use of in vitro systems (subcellular fractions, cell lines, primary cell cultures, tissue slices, organ cultures, etc.) as research tools in toxicology is widespread. In the past few years, the apoptosis phenomena were followed by very precise intracellular changes where, through programmed cell death, a cell can be removed from a population. The in vitro systems are ideally suited for investigations of the molecular, cellular and physiological mechanisms of chemically induced toxicity, which cannot readily be studied in vivo for known target organ and target species toxicity studies and for answering specific questions about toxic effects. The main justification for developing in vitro toxicity tests is that they will make toxicology a more scientifically based practice. It is increasingly apparent that the development and incorporation of stepwise testing strategies, combining experimental data from a range of alternative methods (physicochemical techniques, quantitative structure-activity relationships - QSAR, metabolic and kinetic modelling and in vitro tests), provide the most advanced way to predict toxicity, reducing at the same time the number of laboratory animals used for testing.Alternativni sustavi ispitivanja toksičnosti na životinjama obuhvaćaju postupke koji uključuju stanice i kulture, uporabu dijelova tkiva, toksikokinetičko modeliranje, kao i odnose aktivnosti prema strukturi ksenobiotika. Uporaba in vitro sustava (staničnih frakcija, staničnih linija, primarnih staničnih kultura, dijelova tkiva, kulture organa itd.) ima široku primjenu u toksikološkim istraživanjima. U posljednjih nekoliko godina fenomen apoptoze može se pratiti prema vrlo preciznim unutarstaničnim promjenama te utvrđena programirana smrt stanice iskoristiti u uklanjanju takvih stanica iz populacije. In vitro sustavi idealno se rabe u istraživanjima molekularnih, staničnih i fizioloških mehanizama toksičnosti izazvanih kemikalijama, dok in vivo studije toksičnosti ne mogu dati te odgovore. Glavna potvrda razvoja i praćenja toksičnosti spojeva u in vitro testovima jest znanstvena utemeljenost toksikologije. Razvoj i uvođenje strategija testiranja koje primjenjuju kombinaciju eksperimentalnih podataka iz niza alternativnih metoda (fizikalno-kemijske tehnike, QSAR tehnike, metaboličko i kinetičko modeliranje) omogućuje najefikasniju procjenu toksičnosti, kao i istodobno smanjenje broja laboratorijskih životinja potrebnih u postupcima testiranja toksičnosti spojeva

The Zrmanja River Estuary (Adriatic Coast, Croatia) – the Need for Interdisciplinary Approach to Protection of Coastal Areas

The Zrmanja River estuary is a semi-enclosed bay immersed in a karstic landscape of the eastern Adriatic coast. It represents a highly indented system which includes part of the Zrmanja River, downstream from the Jankovića Buk, the Novigrad and the Karin Seas, and the strait connecting the Novigrad Sea with the Velebit Channel. The very fact that the estuary of the Zrmanja River is located in the karst area makes this system highly vulnerable and susceptible to possible anthropogenic pressures. Preservation of such and similar water systems relies on the knowledge of the main factors that govern its physical, chemical, biological and geochemical features. Due to the lack of geochemical data for the Zrmanja River estuary, a comprehensive study was carried out to explain sedimentation processes and the origin of the material within this estuary and provide additional knowledge necessary for its sustainable use and preservation. This paper, therefore, attempts to summarize the obtained data, focusing on the main natural and anthropogenic factors that define the geochemistry of the Zrmanja River estuary

Marked improvement in survival among adult Croatian AIDS patients after the introduction of highly active antiretroviral treatment

We compared the survival of patients following the first AIDS event in Croatia from the period 1986-1996 to the period 1997-2000. A total of 72 (81.8%) out of 88 patients from 1986-1996 and 18 (32.1%) out of 56 from 1997-2000 died. Survival following the first AIDS-defining illness markedly improved in the period 1997-2000 compared to the period 1986-1996 (adjusted Hazard Ratio (HR) for patients surviving more than 6 months: 0.11, 95% confidence interval (95% CI) = 0.04-0.29). A CD4+ cell count of < 100 x 10(6)/L was an independent risk factor for patients surviving up to 2 years (adjusted HR = 1.96, 95% CI = 1.1-3.43, p = 0.02). Patients with tuberculosis or fungal infections had a longer survival when compared to other diagnosis (adjusted HR = 0.53, 95% CI = 0.32-0.90, p = 0.01). However, despite dramatic survival benefit of combination antiretroviral therapy, mortality at six months following the first AIDS event was similar in the two study periods and the one-year probability of death was still substantial (27.2%) in the period 1997-2000

Calorimetric Study of Ligand Binding to Fumarase

A microcalorimetric method has been used to study the binding of inhibitors, succinate and citrate, to pig heart fumarase at single conditions, i.e. 37 °c, pH 7.4 and I\u27/2 0.03, but by using three buffer systems with different heats of ionization. Binding processes with succinate and citrate were endothermic and the same general pattern was obtained for the inhibitors used (~G0 - 37 or 38 kJ. mol-1). It has been shown that 0.1 or 0.2 moles of H+ per mol of enzyme was absorbed when the enzyme was saturated with succinate or citrate, respectively. Binding processes in buffer systems with different heats of ionization made possible the calculations of enthalpies of binding (~H0 \u27 - 16.4 or 16.8 kJ moi-1) in a hypothetical buffer system with zero. heat of ionization