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The high production and consumption of pharmaceuticals nowadays have resulted in their constant release into the environment. Environmental problems associated with pharmaceuticals are mainly due to their longevity and critical biological activity. It is well known that conventional wastewater treatment processes are not capable of completely removing them from wastewater. Therefore, new and more efficient methods need to be developed. Several studies have shown that ionizing radiation is a successful and cost-effective method for the degradation of a variety of pharmaceuticals [1, 2]. In this study, we focused on the radiation-induced degradation of doxazosin (DOX) [3], a widely used antihypertensive drug found in raw water. The efficiency of degradation was studied under different absorbed doses, dose rates, pH values and in the presence of radical scavengers and inorganic ions. Removal of 10 mg L−1 DOX reached almost 100 % at irradiation doses of 200 Gy regardless of the dose rate used. The effect of saturated solutions with N2, N2O, air and the addition of radical scavengers such as 2-PrOH and thiourea on DOX degradation was investigated. The efficiency of degradation increased in the order: thiourea < 2-PrOH < N2 < air < N2O. DOX degradation was favorable in the neutral, slightly acidic range (pH = 6.5) and decreased under acidic or alkaline conditions. Despite the strong degradation of DOX up to 200 Gy, much higher doses are required for the overall mineralization process. Samples irradiated with 500 Gy resulted in higher toxicity testing on V. fischeri bacteria than samples irradiated with lower doses. High- resolution quadrupole time-of-flight (QTOF) mass spectrometry was used for detection and characterization of degradation products. The structures of degradation products were determined based on MS and MSMS spectra, and degradation pathways were proposed under different experimental conditions. Overall, irradiation technology could be a promising technique for the removal of micropollutants, such as pharmaceuticals in real water matrices.

Konvencionalne metode pročišćavanja otpadnih voda temelje se na kemijskim, fizikalnim ii biološkim procesima te često nisu dovoljno učinkovite u uklanjanju teško razgradivih zagađivala kao što su pesticidi, herbicidi i farmaceutici. Tretiranje vodenih otopina ionizirajućim zračenjem kao jednim od naprednih oksidacijskih procesa može biti efikasno u riješavanju problema otpadnih voda. Cilj ovog rada bio je istražiti utjecaj doze zračenja, brzine doze, anorganskih iona i pH na radiolitičku razgradnju često korištenog antihipertenziva, doksazosina. Utvrđeno je da su •OH glavne reaktivne vrste odgovorne za razgradnju doksazosina, a efikasnost razgradnje polaznog spoja raste s porastom doze zračenja. Prisutnost nitrata je imala značajniji utjecaj na razgradnju doksazosina u usporedbi s karbonatima i fosfatima. Optimalna pH vrijednost za razgradnju vodene otopine doksazosina je u neutralnom, blago kiselom području (pH = 6, 5), dok je pri izrazito kiselim i baznim uvjetima razgradnja bila slabija. Rezultati istraživanja ukazuju da je radijacijska tehnologija pogodna u procesu uklanjanja mikrozagađivala kao što su farmaceutici u realnim vodenim sustavima.

Pharmaceuticals are widely consumed in modern societies and after use active agents and their metabolites can enter the environment via different pathways [1]. Due to high stability of pharmaceuticals in water and incomplete removal during conventional wastewater treatment processes some pharmaceuticals end up in the environment mainly in surface waters, groundwater and also in drinking water. The presence of pharmaceuticals in the aquatic environment may have adverse effects on human health which include aquatic toxicity, development of pathogenic bacteria, genotoxicity and endocrine disrupter [1, 2]. Doxazosin (DOX), a selective alpha blocker, is widely used in medical therapy as an effective antihypertensive agent. The degradation scenario of DOX under TiO2 photocatalytic conditions, as one of the AOP processes, together with the degradation mechanism and toxicity of photoproducts have been studied recently [3]. In the present work we have focused on the degradation of doxazosin in aqueous solution by gamma irradiation. The efficiency of degradation was studied under different absorbed doses, dose rates, pH values and in the presence of radical scavengers and inorganic ions. The irradiated samples were analyzed by UV/VIS spectrophotometry and high performance liquid chromatography with diode array detector. The results indicate that •OH are the main reactive species responsible for the degradation of doxazosin, the efficiency of degradation being higher with increasing dose of gamma radiation (Figure 1.). The presence of nitrates had a stronger effect on doxazosin degradation compared to carbonates and phosphates. An optimal pH for doxazosin degradation was in the neutral, slightly acidic range (pH = 6.5), while degradation was slower under extremely acidic or alkaline conditions.

Over the last decade, pharmaceuticals have attracted increased attention as emerging contaminants due to their presence in natural waters and potential negative effect on the environment. It is known that conventional wastewater treatment processes are unable to completely remove them from wastewater, therefore there is a need for developing new and more effective processes, such as oxidation by generating highly reactive radicals by photochemical or ionizing radiation. Several studies indicated that gamma irradiation is a successful and cost-effective method of degradation for a wide variety of pharmaceuticals. Doxazosin (DOX) is a selective alpha blocker, used in the treatment of hypertension. Due to its widespread use, there is a possibility of its occurrence in aquatic environment. Therefore, development of suitable degradation method is of great importance. Photolytic and photocatalytic degradation od doxazosin was previously studied, and main degradation products were characterized. In this work, degradation of doxazosin by gamma irradiation in the presence of air and N2O was studied. High-resolution quadrupole time-of-flight (QTOF) mass spectrometry was used for detection and characterization of degradation products. Structures of degradation products were determined based on MS and MSMS spectra, and degradation pathways under different conditions were proposed.

Rosuvastatin je lijek skupine statina koji se koristi za regulaciju visoke razine kolesterola u ljudskom tijelu. Isto tako, rosuvastatin i ostali statini pokazuju zaštitnu ulogu protiv oksidativnog stresa uzrokovanog slobodnim radikalima. Cilj ovog istraživanja bio je identificirati krajnje produkte koji su nastali radikalnom razgradnjom rosuvastatina. Kako bi se potaknula radikalna razgradnja, vodena otopina rosuvastatina ozračena je različitim dozama gama zračenja (50-1000 Gy) pri oksidativnim uvjetima. Rosuvastatin i srodni degradacijski produkti odvojeni su nanoC18 kolonom gradijentnom elucijom, a identifikacija je provedena pomoću vezanog sustava nanoUPLC i nanoESI-QTOF. Pomoću točno mjerenih masa, zajedno s algoritmom usporedbe izotopnih raspodjela, provedena je elementna analiza kojom je identificirano devet degradacijskih produkata. U ovom je istraživanju po prvi puta provedena gama-inducirana razgradnja rosuvastatina te detaljno opisana kemijska struktura, MS/MS fragmentacija te mehanizam nastanka pojedinog degradacijskog produkta. Priloženi rezultati doprinose razumijevanju razgradnog puta rosuvastatina i ostalih statina pri gama zračenju.

The main objective of this work was to identify the most common bacterial strains in the Nin peloid mud, their biochemical profile, and their fatty acid composition.

Sterilizacija je postupak u kojem se uništavaju svi mikroorganizmi pa i oni najrezistentniji poput bakterijskih spora. Kontrola sterilizacije provodi se fizikalnim, kemijskim i biološkim postupcima. Međutim, kemijski i fizikalni parametri sterilizacije ne pokazuju uspješnost procesa sterilizacije već potvrđuju da je materijal bio podvrgnut sterilizaciji odabranim postupkom. Jedna od najpouzdanijih, najprikladnijih i najjednostavnijih načina kojim se dokazuje učinkovitost sterilizacije je biološka kontrola. Jedna od često korištenih metoda za sterilizaciju različitih materijala i proizvoda poput medicinskog pribora za jednokratnu upotrebu, farmaceutskih sirovina, gotovih proizvoda i ambalaže je gama zračenje. To je efikasna, brza i sigurna metoda za inaktivaciju i eliminaciju svih bioloških kontaminanata u svim životnim ciklusima. U suradnji Nastavnog zavoda za javno zdravstvo Andrija Štampar i Instituta Ruđer Bošković provedeno je istraživanje djelotvornosti ionizirajućeg zračenja u postupku sterilizacije TSA i Sabouraud Dextrose agar Petrijevih zdjelica uz praćenje utjecaja gama zračenja na njihove fizikalno-kemijske parametre i mikrobiološke karakteristike u prisustvu bioloških indikatora koji sadrže spore Bacillus pumilus. Rezultati provedenih ispitivanja su pokazali da je primjena gama zračenja za postizanje sterilnih Petrijevih zdjelica kao gotovog proizvoda učinkovita budući su zahtjevi za sterilnost sukladno smjernicama EU i odgovarajućim europskim normama, u potpunosti zadovoljeni. Biološki indikatori koji su pritom bili upotrijebljeni dokazuju da su vegetativni oblici spora uništeni u procesu sterilizacije.

The group of new pollutants, so called emerging pollutants, includes diverse groups of pharmaceuticals and pharmaceutical active substances used in human and veterinary medicine (such as painkillers, antibiotics, hormones, anti-inflammatory drugs, lipid regulators, beta bolckers, anti-cancer drugs) and personal care products. Continuous release of pharmaceuticals to the environment as complex mixtures can result in higher concentrations, long-term and negative effects on aquatic and terrestrial organisms. For these reasons biological, physical and chemical methods have been developed for removal of pharmaceuticals from aqueous solution. Doxazosin, a selective alpha blocker, is widely used in medical therapy as an effective antihypertensive agent. It is very frequently prescribed drug and for this reason, environmental and ecotoxicological research is of great importance in terms of exposure and risk for both aquatic species and humans. In this study we focused on photolytic and TiO2 photocatalytic degradation processes of doxazosin under different simulated conditions, with the emphasis on identification of degradation products. TiO2 photocatalytic and photolytic (without TiO2) experiments were performed in photoreactor equipped with polychromatic low-pressure mercury lamps (1.6 mW/cm2 of UVA (300 - 400 nm)) as well as with UVC gemicidal lamps (254 nm). In the case of photolytic experiments, doxazosin aqueous solution was irradiated for 120 min in the presence and absense of oxygen, while photocatalytic degradation of doxazosin aqueous solution have been carried out under constant oxygen flow. Selected samples have been further concentrated for LC-MS/MS analysis, which proved the formation of several degradation products, depending on reaction conditions (presence, absence of oxygen, photocatalysis). The detailed degradation pathway has been proposed and will be presented.

One of the largest health issue in modern society is increased frequency of hard tissues chronic diseases. Often the only treatment of such diseases is implementation with the aim to regenerate damaged or diseased tissue. Considering that the main inorganic component of hard tissue is biological apatite, form of calcium phosphate (CaP), composite materials based on calcium phosphate and different inorganic nanomaterials attracts attentions as possible new and innovative implant materials. Among different nanomaterials, TiO2 nanomaterials stand out because they can improve mechanical properties of CaP. Albumine is one of three soluble proteins which adsorbs on the surface of implant materialsright after its implantation and affect their behavior in vivo. But, its role in formation of calcium phosphates on titanium implants is still not clarified. Therefore, the aim of this study is to investigate the adsorption of bovine serum albumin (BSA) on nanocomposites of calcium phosphate (CaP) and TiO2 nanoparticles (CaP/TiNP) or nanotubes (CaP/TiNT). In order to do that, the kinetics of BSA adsorption kinetics of BSA adsorption on TiO2 nanoparticles (TiNP), titanate nanotubes (TiNT), calcium deficient hydoxiapatite (CaDHA) obtained in control system, CaP/TiNP and CaP/TiNT was measured in situ on UV/VIS scanning spectrophotomer. Samples obtained after adsorption were filtered and given for further analysis which included Fourier- transform infrared apectroscopy (FTIR), powder x-ray diffraction (XRD) and scanning electron microscopy (SEM). The results of kinetics measurements were modeled using kinetics models of pseudo-first, pseudo-second rate and interparticle diffusion model. From the results of various kinetic models, the adsorption kinetics were found to follow pseudo-second- order rate kinetic model for CaP/TiNT nanocomposites, respectively pseudo-second- order rate and interparticle diffusion model for CaP/TiNP nanocomposites. Results obtained from FTIR, XRD and SEM analysis showed that albumin did not influence the composition and solid phase morphology

Gamma irradiation method is efficient, safe and fast method for inactivation of all biological contaminants at all stages of their life cycle. This method is based on the ability of high-energy photons generated by radioactive 60Co to induce chemical DNA damage in biological contaminants such as insects, moulds, yeasts and bacteria. Therefore, γ-irradiation finds its use in sterilization of different materials such as medical supplies and pharmaceuticals and in food quality preservation. The aim of this research work was to investigate the physico-chemical parametars and microbiological characteristics of Tryptone soya and Sabouraud Dextrose agar media on Petri dishes after irradiation in dependence on the irradiation dose. Microbiological media (20 Petri dishes) packed in thermalshrinking foil were put in cardbox in the presence of biological indicators (spores of Bacillus pumilus). The three irradiation cycles were performed on the panoramic 60Co source at the Ruđer Bošković Institute. Dosimetry were established with the ethanol- chlorobenzene dosimetry system (ISO/ASTM 51538: 2017). Quality control of microbiological media was perfomed before and after irradiation according HRN EN ISO 11133. Physico-chemical parameters (appearance, color, homogeneity, agar consistency) and microbiological characteristics of culture media (sterility and productivity) were tested. Since the results of performed tests meet the requirements in accordance of guidelines of EU and corresponding standards we recommend these plates to be used for microbiological methods, validation of aseptic procedures control of safe operation under clean conditions. This research is result of collaboration of the Radiation Chemistry and Dosimetry Laboratory and the Department of Clinical Microbiology, Andrija Štampar Teaching Institute of Public Health in Zagreb

The goal of this study was to examine the effect of 1-week of high salt (HS) intake and the role of oxidative stress in changing the mechanisms of flow- induced dilation (FID) in isolated pressurized middle cerebral arteries (MCA) of male Sprague-Dawley rats (N=15-16/per group). Reduced FID in the HS group was restored by intake of the superoxide scavenger TEMPOL (HS+TEMPOL in vivo group). Nitric oxide synthases (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME), COX inhibitor indomethacin (INDO) and selective inhibitor of microsomal CYP450 epoxidase activity N- (methylsulfonyl)-2-(2-propynyloxy)- benzenehexanamide (MS-PPOH) significantly reduced FID in the LS group, while FID in the HS group was mediated by NO only. COX-2 mRNA (but not protein) expression was decreased in the HS and HS+TEMPOL in vivo groups. HIF-1α and VEGF protein levels were increased in the HS group but decreased in the HS+TEMPOL in vivo group. Assessment by direct fluorescence of MCA under flow revealed significantly reduced vascular NO levels and increased superoxide/reactive oxygen species levels in the HS group. These results suggest that HS intake impairs FID and changes FID mechanisms to entirely NO- dependent, in contrast to the LS group where FID is NO, prostanoid and epoxyeicosatrienoic acids (EET's) dependent. Those changes were accompanied by increased lipid peroxidation products in the plasma of HS-fed rats, increased vascular superoxide/reactive oxygen species levels and decreased NO levels ; together with increased expression of HIF-1α and VEGF.

Background of the study Various metals and alloys are commonly used in medicine. As such they are often exposed to aggressive environments and thus subjected to corrosion, i.e. dissolution. Thus, modification of the metal surface is interesting since this way the resistance to corrosion can be increased and the durability of the object can be prolonged. Medical equipment, in most cases, must be sterilized and a common method is using ionizing radiation. While ionizing radiation does not markedly influence the properties of the metal it can influence the protective layer that is formed on its surface. Fatty acids are non-toxic compounds which have an affinity for self-assembling on metals and therefore lately they have been investigated as possible protective layers on metals. The influence of ionizing radiation on the formation of such a layer is needed to determine weather it changes the properties of the protective layer or maybe even enhances them. Methodology Procedures for adsorption of self-assembling layers of fatty acids on metals exist in literature. This work studies influence of ionizing gamma-radiation on the process of assembling such a layer and the efficiency of the outcoming layers in corrosion protection. Various variables are studied. The most important variables are the applied dose of gamma irradiation, as well as the dose rate at which it is applied. The quality and efficiency of the obtained layers in corrosion protection are studied by electrochemical methods. Results The results have shown that the layer of fatty acid that forms on the surface of the metal using ionizing radiation offers additional protection to the bare metal making it more resistive to corrosion. Conclusion For successful formation of a protective layer on the metal it is essential to determine the proper molecule which has good adhesive properties and to irradiate with an appropriate dose, at an optimal dose rate.

Reactive oxygen species, especially hydrogen peroxide (H2O2), contribute to functional molecular impairment and cellular damage, but also are necessary in normal cellular metabolism, and in low doses play stimulatory role in cell proliferation and stress resistance. In parallel, reactive aldehydes such as 4-hydroxynonenal (HNE), are lipid peroxidation breakdown products which also contribute to regulation of numerous cellular processes. Recently, channeling of H2 O2 by some mammalian aquaporin isoforms has been reported and suggested to contribute to aquaporin involvement in cancer malignancies, although the mechanism by which these membrane water channels are implicated in oxidative stress is not clear. In this study, two yeast models with increased levels of membrane polyunsaturated fatty acids (PUFAs) and aquaporin AQY1 overexpression, respectively, were used to evaluate their interplay in cell's oxidative status. In particular, the aim of the study was to investigate if HNE accumulation could affect aquaporin function with an outcome in oxidative stress response. The data showed that induction of aquaporin expression by PUFAs results in increased water permeability in yeast membranes and that AQY1 activity is impaired by HNE. Moreover, AQY1 expression increases cellular sensitivity to oxidative stress by facilitating H2 O2 influx. On the other hand, AQY1 expression has no influence on the cellular antioxidant GSH levels and catalase activity. These results strongly suggest that aquaporins are important players in oxidative stress response and could contribute to regulation of cellular processes by regulation of H2O2 influx.

Hydrogen sulfide is endogenous gasotransmitter that is known to exert effects in cardiovascular and central nervous systems. Different H2S-derived radicals originating either naturally within the body or coming from the environment can be involved in inflammation during radical stress and in cell signaling. Generally, the double-sward character of thiol species towards biomolecules has been increasingly addressed in the last decade. There are two important characteristics of thiyl radicals: their ability to abstract hydrogen atoms from the molecules such as lipids, initiating lipid damage due to lipid peroxidation and to catalyse the double bond isomerization in polyunsaturated fatty acid. The reactions of thiyl radicals as isomerizing agents are not well known especially with the interplay of lipid peroxidation process. The aim of this study is to investigate the role of H2S as a precursor of diffusible simple S-centered radicals on lipid peroxidation and geometrical isomerization processes in biomimetic lipid model system. In this study lipid peroxidation via the formation of lipid hydroperoxides, and geometrical trans-isomerization were determined under biomimetic conditions. Thiyl radicals were generated by γ-radiation of simple model micellar system containing linoleic acid and H2S. Model lipid systems were irradiated up to the dose of 400 Gy, in equilibrium with air or after saturation with N2O at room temperature using panoramic 60Co source at the Ruđer Bošković Institute. Accurate dose rates in the irradiating positions were established with the ethanol-chlorobenzene dosimetry system (ISO/ASTM 51538:2009). The concentrations of hydroperoxides of linoleic acid were determined by the spectrophotometric ferric thiocyanate method and the geometrical isomerization of LH methyl ester was studied by capillary gas chromatography. In addition, we investigated the concentration effect of thiols on the outcome of the two lipid modification processes. We believe that this study could contribute to the understanding of H2S reactions which take place in biomembranes that could be essential to rationalize the biological properties of this newly recognized signaling molecule in biological systems.

Background of the study. Polyunsaturated fatty acids (PUFA), major constituents of biological membranes are sensitive to a free radical attack. The reactions of PUFA with free radicals are known to occur via two main processes: (i) lipid peroxidation and (ii) cis– trans isomerization. Lipid peroxidation can be inhibited by thiols, due to their H-donation ability. On the other hand, thiyl radicals are known to catalyse the double bond isomerization in PUFA. Both processes have damaging potential that must be carefully considered for its consequences in a biological systems. Therefore, the protection against lipid degradation under oxidative and free radical conditions is of special interest. The aim of this study is to elucidate the influence of different naturally occurring antioxidants on lipid peroxidation and cis–trans isomerization processes in biomimetic model system under different conditions. Methodology. Model system containing mixed surfactant micelles and buffer was prepared by slow solubilization of linoleic acid (LH) in non-ionic surfactant micelles previously formed by mixing Tween®-20 and PB, pH 6.5. The composition of the investigated systems was typically 0.5 mM LH, 0.28 mM Tween®-20 and 5 mM PB (pH ~ 5). Ascorbic acid (AscH), -tocopherol (α-TOH) and resveratrol (ResOH)) of defined concentrations were added during preparation of model systems. The addition of the amphiphilic 2.8 mM 2- mercaptoethanol was added to previously prepared micelles just before irradiation. Model lipid systems were irradiated up to the dose of 400 Gy, in equilibrium with air or after saturation with N2O at room temperature using panoramic 60Co source at the Ruđer Bošković Institute. Accurate dose rates in the irradiating positions were established with the ethanol-chlorobenzene dosimetry system (ISO/ASTM 51538:2009). The concentrations of hydroperoxides of linoleic acid were determined by the spectrophotometric ferric thiocyanate method, and the geometrical isomerization of LH methyl ester was studied by capillary gas chromatography. Results. Under air-equlibrated conditions, the addition of different natural occuring antioxidants retarded the process of lipid peroxidation among which ResOH showed the best antioxidative property. In model systems where process of cis–trans isomerization prevailed, the presence of antioxidants influenced on a decrease of the trans isomerization level ; AscH was the the most effective inhibitor of radiation-induced trans- isomer formation. Among the natural compounds analyzed in this work an amphiphilic ResOH has proven to be the most effective antioxidant, also significant inhibitor for cis–trans isomerization process in conditions of low irradiation dose rates with formation of low initial radical species concentration. Conclusion. Results indicate that the cis–trans isomerization and lipid peroxidation processes level could be dependent on the hydrophilic/lipophilic properties of particular antioxidant and its localization in model lipid system. This study contributes to the understanding of the role of antioxidants in radical processes which are not only able to prevent peroxidation but can also behave as anti-isomerizing compounds.

Trefoil factor family proteins (Tff1, Tff2, Tff3) are small and compact peptides containing one or two so called trefoil domains. TFFs are predominanatly expressed in gastrointestinal tract where they have important role in maintenance of epithelial barrier integrity during the process of restitution upon mucosal injury. Moreover their role in other systems started to emerge. Tff3 gene was identified as one of the genes involved in liver steatosis. Hepatic Tff3 expression levels are decreased in obese and high-fat diet induce mice compared to control mice. The aim of this study was to determine hepatic neutral lipid accumulation, fatty acid profile in the liver and serum of Tff3 knock-out mice as well as the serum lipid content. Mice were anesthetized at the age of 12 weeks and blood was collected by cardiac puncture. Following mice were sacrificed by cervical dislocation and liver tissues were collected for histological analysis (HE, Oil red O). Total lipids for fatty acid profiling were extracted from tissue homogenates and serum which were analyzed by gas chromatography (Varian 450-GC). Serum lipids were analysed in routine laboratory using Architect c8000 (Abbott Diagnostic). Microscopic analysis of liver sections revealed normal tissue architecture (HE). Oil red O staining of liver sections from Tff3 deficient mice showed accumulated neutral lipids. Tff3 knock-out mice exhibited statistically changed level of numerous fatty acids in the liver and serum compared to WT. The level of saturated and polyunsaturated fatty acids were elevated and the level of monounsaturated fatty acids were decreased in liver of Tff3 deficient mice. In addition, the n-3/n-6 ratio was elevated in liver and decreased in serum of Tff3 deficient mice compared with wild type strain. However, total serum levels of the cholesterol, triglycerides, HDL or LDL cholesterol did not differ among the groups. Our results revealed that Tff3 is involved in fatty acid metabolism and as such it could be a potential target to develop therapeutic strategies against metabolic disorders.

Gamma irradiation is a physical method for materials processing that involves their exposure to high energetic gamma photons emitted by 60Co. Food irradiation up to dose of 10 kGy (and above in some cases) has been considered as an effective technology for microbial decontamination in comparison with traditional pasteurisation methods. Recently, the industrial hemp Cannabis sativa L. subsp. sativa has been grown in Croatia by several individual farmers. Thus, mycobiota of C. sativa was poorly investigated so far in this part of Europe. The aim of this pilot study is to check total mould count and mycobiota composition of two samples of C. sativa prior and after gamma irradiation at doses 1-10 kGy. The irradiation was performed at panoramic source of 60Co in RCDL of RBI. Samples were serially diluted in peptone water and plated in duplicate onto Dichloran Glycerol (DG18) agar and Dichloran Rose Bengal Chloramphenicol (DRBC) agar plates. The number of mould colony-forming unit (CFU) was determined upon incubation 5-7 days at 25°C, and the data were expressed as the number of CFU/g. Upon irradiation at 1, 3, 5, 7 and 10 kGy at dose rate 120 Gy/min and ambient conditions, samples were plated onto DG18 and DRBC and CFU/g was checked on 7th, 14th and 28th day. Total mould count was 7.8×105 CFU/g (DRBC) and 2.5×105 CFU/g (DG18). Fusarium spp. and Cladosporium spp. were dominant natural contaminants which comprised 60% and 20% of hemp mycobiota. All applied irradiation doses suppressed more than 90% of initial mould contamination. Mould count that survived irradiation was between 2000 and 4000 CFU/g which is approximately 0.25 and 0.5 % of initial count. Moulds that survived g-irradiation were Fusarium spp. Cladosporium spp., Aspergillus fumigatus and Phoma spp. In the next experiment hemp samples were sterilized at 25 kGy and inoculated with moulds (2 ml, 102 CFU/ml of water) that survived previous irradiation. Samples were inoculated with single moulds as well as with mixture of all four species, while control was inoculated with 2 ml of water. All samples were incubated 7 days at 25°C and than irradiated with doses of 10, 12 and 15 kGy. After 10 days samples were analysed on DRBC as described previously. All plates remained sterile during 28 days period. In conclusion, gamma irradiation up to 10 kGy can be applied for significant reduction of mould contamination but higher doses are required for complete elimination of particular mould species.

Uzročno-posljedične veze između slobodnih radikala u biološkim sustavima i različitih patoloških stanja omogućavaju da se radijacijsko-kemijske metode koriste za pripravu i praćenje njihovih reakcija u kontroliranim uvjetima. U ovom su radu istraživani radikali višenezasićene masne kiseline, prekursori za dvije vrste procesa, peroksidaciju i, posebno u prisustvu biološki važnih tiola, cis, trans-izomerizaciju na dvostrukoj vezi linolne kiseline, LiH. Pritom je razvijen modelni sustav micela LiH u kojemu se radikalski procesi mogu selektivno pratiti u uvjetima koji se, za razliku od fizioloških uvjeta, mogu prilagođavati potrebama eksperimentiranja. Prvi put se istražila povezanost tih dvaju radikalima induciranih procesa u biomimetičkim uvjetima te je pokazano da hidroperoksidi (LiOOH) i transkonfiguracija LiH nastaju i u oksidacijskim uvjetima. Biomimetički model priređen je s dodatkom 2-merkaptoetanola kao amfifilnog tiola, a inicijacija radikalskih procesa provedena gama zračenjem s dozama zračenja do 400 Gy u različitim uvjetima. U uvjetima ravnoteže sa zrakom uz značajnu koncentraciju LiOOH (~105 mol dm3) odvijao se i proces cis, transizomerizacije s katalitičkim ciklusom 370. Kako bi se predvidio utjecaj samoorganiziranih supramolekulskih struktura LiH na ispitivane procese istražen je i utjecaj veličine micela te prisutnost prirodnih antioksidansa. The cause-effect relationship between free radicals in biological systems and the various pathological conditions enable us to use the radiation-chemical methods for preparation and determination these reactions under controlled conditions. In this paper we investigated polyunsaturated fatty acid radicals which are precursors for the two types of processes, peroxidation and, especially in the presence of biologically relevant thiol, cis, transisomerization of linoleic acid (LiH). A model micellar system was developed in which radical processes of LiH were selectively determined under conditions that, in contrast to the physiological, could be adapted to the experiments requirements. These two radical-induced processes were compared for the first time under biomimetic conditions demonstrating that hydroperoxides (LiOOH) and trans-lipids can be formed under oxidizing conditions. Biomimetic model was prepared with the addition of 2-mercaptoethanol as amphiphilic thiol, and radical processes initiation was performed by -radiation up to 400 Gy under different conditions. In air-equilibrated solutions, together with substantial amount of LiOOH (~10-5 mol dm3), the cis, trans-isomerization process was also observed with a catalytic cycle of 370. In order to envisage the impact of self-organized supramolecular organization of LiH on the outcome of lipid peroxidation and cis, trans-isomerization processes, the influence of the micelle size and the presence of natural antioxidants were also studied.

For a long time lipid peroxidation has been a process of considerable interest because of its significant negative effects on biological systems resulted during oxidative stress. A much recent research deals with geometrical isomerization of polyunsaturated fatty acids which can occur due to radical stress. Free radical reactivity of thiol compounds is the common link between two processes, since lipid peroxidation is inhibited by thiols whereas geometrical isomerization is catalysed by S-centered radical (RS•). We have established the simplest biomimetic model of cell membrane, the micelles of linoleic acid in which we followed two processes in parallel.1, 2 Furthermore, we investigated the influence of several natural occurring antioxidants on radical induced lipid reactions. The systems were irradiated by gamma radiation up to 400 Gy under a variety of conditions where RS• are the main reactive species. The results have shown that lipid peroxidation was retarded by ascorbic acid and alpha-tocopherol, whereas this process was strongly inhibited by resveratrol. At the same time, the process of trans-isomerization was found to increase under anaerobic conditions along the series: resveratrol  -tocopherol  ascorbic acid. This study contributes to the understanding of the role of antioxidants in radical processes which are not only able to prevent peroxidation but can also behave as anti-isomerizing compounds. References: [1] B. Mihaljević, I. Tartaro, N. Filipović Vinceković, Eur. J. Lipid Sci. Technol. 114 (2012) 1292–1303. [2] B. Mihaljević, I. Tartaro, C. Ferreri, C. Chatgilialoglu, Org. Biomol. Chem. 9 (2011) 3541–3548.

Lipidna peroksidacija i geometrijska izomerizacija na dvostrukoj vezi višenezasićenih masnih kiselina su dva važna procesa potaknuta radikalima lokaliziranim na S-atomu koji se normalno generiraju u živim organizmima. Navedeni procesi uzrokuju trajnu promjenu strukture lipida kao gradivnih komponenata staničnih membrana i kao posljedicu brojne poremećaje njenih normalnih funkcija. Ova dva konkurentna procesa proučavana su u jednostavnom modelnom lipidnom sustavu pripremljenim dodatkom linolne kiseline (LiH), neionskog surfaktanta (Tween-20) i 2-merkaptoetanola .Istražen je utjecaj dodatka askorbinske kiseline, α-tokoferola odnosno resveratrola na procese lipidne peroksidacije, s posebnom pažnjom usmjerenom i na pojavu procesa geometrijske izomerizacije koja do sada nije bila praćena paralelno s peroksidacijom lipida. Navedeni procesi inducirani su γ-zračenjem modelnih lipidnih sustava u kontroliranim uvjetima u kojima su tiilni radikali bile primarne inicijalne čestice.

Recent investigation has shown that TFF2 deficient mice are protected from high fat diet-induced obesity, accumulating less weight and fat depots than WT animals, while keeping a normal lean mass. In addition to, TFF3 gene was identified as one of the genes involved in liver steatosis. The aim of this study was to determine the rate of fatty acids in the liver of Tff2 and Tff3 knock-out mice, hepatic proteomic profile as well as the serum glucose and lipid content. TFF2 knock-out, TFF3 knock-out and wild type mice were used. Mice were sacrificed at the age of 12 weeks and the liver tissues are collected for regular histological analysis (HE, PAS). Total lipids were extracted from tissue homogenates and analyzed by gas chromatography. Serum lipids and glucose were analyzed in routine laboratory using Architect c8000. Proteome profiles of different groups were analyzed by 2D-PAGE and differentially displayed proteins were identified by MALDI TOF/TOF MS. TFF2 and TFF3 deficient mice compared to WT mice have significantly higher amount of PUFAs. Total serum levels of the cholesterol, triglycerides, HDL or LDL cholesterol did not differ among the groups. Microscopic analysis of the liver sections revealed normal tissue architecture (HE) and glycogen reserves (PAS). Proteomic analysis revealed several proteins involved in lipid, carbohydrate, citric acid cycle and protein metabolism. TFF2 and TFF3 deficient mice have altered caloric metabolism. Our results show that TFF2&TFF3 are involved in energy metabolism and as such they could be a potential target to develop therapeutic strategies against metabolic disorders.

Lipid peroxidation and geometrical isomerization are two main classes of reactions that are known in the context of reactivity of lipids toward free radicals. Both processes peroxidation and isomerization were detected in biomimetic model of linoleic acid in the presence of oxygen and thiols.1 Thiols are known repairing agents in case of lipid peroxidation, due to the H-donation towards peroxyl radicals. In doing so, thiols are precursors of thiyl radicals that can act as isomerizing agents for the fatty acid double bonds. The double-sward character of thiols towards biomolecules has been increasingly addressed in the last decade. Whether these processes can occur together or influence each other in the presence of some common antioxidant, such as aromatic amines and phenols, is presently unknown. We were interested in extending the investigation of the reactivity of thiols involving diphenylamine in aerobic conditions. We investigated the interaction of diphenylamine and 2-mercaptoethanol in the radical stress induced to linoleic acid under biomimetic conditions. As a measure of lipid peroxidation in model systems of linoleic acid we determined hydroperoxides of linoleic acid initiated by thiyl radicals which were generated by gamma radiolysis.2 The role of diphenylamine in protecting from linoleic acid geometrical isomerization was determined simultaneously by gas chromatography.3 The mechanistic scenario was defined contributing to a better understanding of complex interactions related to free radical stress and antioxidant activity, as well as of the mechanisms of aromatic amines reactivity. References: [1] B. Mihaljević, I. Tartaro, C. Ferreri, C. Chatgilialoglu, Org. Biomol. Chem. 9 (2011) 3541–3548. [2] B. Mihaljević, B. Katušin-Ražem, D. Ražem, Free Radical Biol. Med. 21 (1996) 53–63. [3] C. Ferreri, S. Kratzsch, O. Brede, B. Marciniak, C. Chatgilialoglu, Free Radical Biol. Med. 38 (2005) 1180–1187.

Blood-brain barrier (BBB) represents the main obstacle to the effective treatment of central nervous system (CNS) diseases. Even though lipid peroxidation end products are recognized as important factors in the development and onset of numerous neurodegenerative diseases, their role still needs to be clarified. In the field of brain tumors, 4-hydroxynonenal (HNE) is the most intensively studied aldehyde. It was proven as a valuable prognostic factor in astrocytomas. Besides, co-expression of HNE and CD133 in brain tumor cells, mesenchymal stroma and blood vessels emphasizes the role of HNE in tumor propagation into higher grade. Accelerated CNS tumor progression can be a consequence of increased BBB integrity, thus being aggravating factor in disease treatment. Equally, controlled increase of BBB permeability would result in easier drug flow to the brain. Hence, HNE and micelles of linoleic acid (a major source of essential arachidonic acid in the brain) were used in order to modulate permeability and integrity of BBB in vitro model. The model comprised rat brain endothelial cells (RbE4 cell line) and astrocytes grown on opposite sides of cell culture insert. Changes in permeability and integrity of BBB model were observed through TEER (trans endothelial electrical resistance) measurement and sodium fluorescein passage. Antioxidative status and lipid peroxidation were also assessed. Lipid profiles of the endothelial cells exposed to different treatments were determined. Results revealed that BBB integrity and permeability can be effectively modulated by HNE and linoleic acid micelles. These treatments caused changes in amounts of HNE-modified proteins present in cells constituting the BBB. Antioxidative status of these cells was also affected due to treatments.

The aim of this study was to determine the rate of monounsaturated fatty acids (MUFAs) in the liver of Tff3 knock-out mice, as well as the serum glucose and lipid content. We found that: Tff3 knock-out mice compared to WT mice have significantly lower amount of MUFAs in the liver Total serum levels of the cholesterol, triglycerides, HDL or LDL cholesterol did not differ between WT and TFF3 defcient mice. Postprandial glucose levels in Tff3 knock-out mice were higher than in controls but did not reach statistical significance. Microscopic analysis of the liver sections revealed normal tissue architecture (HE) and glycogen reserves (PAS staining).

Physical and emotional stress, metabolic alterations, infections, carcinogenesis or inflammation are conditions that can trigger overproduction of reactive oxygen species. Increase of reactive oxygen species, free radicals and/or decreased antioxidant defense result in the misbalance in the cell redox reactions i.e. in the state termed the oxidative stress. The reactive oxygen species are continuously formed in small quantities during the normal metabolism of cell, however their overproduction is cytotoxic and damages macromolecules (DNA, proteins, sugars and lipids). Polyunsaturated fatty acids that are the main constituents of cell membranes are subject to free radicals induced-lipid peroxidation resulting in the destruction of biomembranes. Oxidative stress triggers a cellular stress response thereby activating a number of the redox-sensitive signaling cascades. A low levels oxidative stress induces protective effects but at high levels it may lead to more damaging effects. Quantification of lipid peroxidation or determination of the concentrations of lipid oxidation products is essential to assess the role of oxidative injury in pathophysiological disorders. One of initial products are lipid hydroperoxides (LOOH) which are well known to initiate further autoxidations during reactions of their production and decomposition. One of the pathways for excessive production of free radicals is decomposition of LOOH catalyzed by iron. The small pool of non-bound ferrous iron in cells is known to provide one of the active species involved in probably the most important route for free radical formation leading to increased levels of lipid peroxidation. Although both reactions of LOOH decomposition in the presence of iron realize in biological systems, reduction of LOOH with Fe2+ is more feasible than oxidation of LOOH with Fe3+. Spectrophotometric quantitative determination of LOOH is used to follow accurately the initial or early stages of the lipid peroxidation process. The spectrophotometric method of analysis of LOOH is based on the oxidation of ferrous to ferric ion and subsequent complexation of ferric ion by thiocyanate.1 The aim of this work was a reestimation of the oxidation of ferrous ion by hydroperoxide of oleic and linoleic acid to optimize the assay conditions with special attention given to the rate of oxidation of ferrous ion with alkyl hydroperoxide affected by acid type and its concentration, thus improving acceleration of the oxidation and the sensitivity of the method. Special attention was given to the measurements of very low LOOH concentrations from biological materials.2 The lowest detectable limit was estimated to be about 170 pmol LOOH/ml of analyzed solution, which corresponded to about 50 pmol LOOH/mg lipid in complex natural mixtures.

The interest in the study of lipid peroxidation processes in model systems has been motivated by aspects of the adverse consequences associated with peroxidation of unsaturated lipids in different natural and biological systems. In an attempt to elucidate the complex process responsible for lipid peroxidation in these systems, various simple model systems including those with surfactants were used. Surfactant supramolecules (micelles, vesicles, lyotropic mesophases) generally serve as models mimicking complex biological systems.1 An additional advantage of the surfactant supramolecules used as a model system is the possibility to investigate lipid peroxidation under acidic conditions. Low fatty acid solubility under acidic conditions can be bypassed by lipid solubilization into surfactant supramolecules using them as nanoreactors. We have chosen a nonionic surfactant, TWEEN®-20, whose stability and relative non-toxicity allows for its use as a suitable model system for biological media.2 This model system allows lipid reactivity to be studied in the context of the organization of the lipid molecules within the system, as well as their possible interactions with other types of molecules in their immediate vicinity, which can influence the lipid processes.3 A simple system with mixed nonionic surfactants TWEEN®-20/LA micelles as the model for the PUFA oxidation susceptibility assay, also as a very useful tool for studying thiyl radical-catalyzed cis-trans isomerization of unsaturated lipids will be described.

Biomimetic models provide basic molecular mechanisms studies that are closer to the biological environment such as living organism. There are two main classes of reactions that are known in the context of reactivity of polyunsaturated fatty acids toward free radicals. One of the well known process is lipid peroxidation and the second one is the process of geometrical isomerization of unsaturated lipids which can be catalysed by thiyl radicals (RS•). It has been shown that one process can be parallel with the occurence of the other and that lipid hydroperoxides and trans lipids can be the resulting effect of oxidative free radical conditions.1 Both processes have damaging effects on cell membranes of living organisms. In this study, the biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol (RSH) were irradiated by gamma radiation under a variety of conditions where RS• are the main reactive species. The study of these processes has been extended in the presence of several natural occuring antioxidants which have shown their protective properties against lipid peroxidation and trans isomerization. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol and the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol. Under anaerobic conditions, where lipid peroxidation is suppresed, the presence of antioxidants have influenced on a decrease of the trans isomerization level along the series resveratrol  -tocopherol  ascorbic acid. All results indicate that trans isomerization depends on the localization of the particular antioxidant in model system. In order to protect lipid structures from the thiyl radical damage a new role of the essential antioxidants can be assessed.

Utvrđeno je da se u anaerobnim uvjetima u micelarnoj otopini LH u prisustvu tiola odvijala samo cis-trans izomerizacija, mehanizmom adicije/eliminacije tiilnog radikala na dvostrukoj vezi LH, te je katalitički ciklus iznosio 420. U otopinama LH u ravnoteži sa zrakom utvrđeno je da se cis-trans izomerizacija još uvijek odvija s katalitičkim ciklusom 370, uz značajnu koncentraciju proizvedenih hidroperoksida LH. U ovom radu prikazat ćemo i utjecaj veličine micela LH na pojavu istraživanih procesa. Rezultati su pokazali da postoji razlika u raspodjeli nastalih mono trans izomera čime je potvrđeno postojanje supramolekulske organizacije u ispitivanom modelnom micelarnom sustavu.

The modeling of free radical reactions under naturally occurring conditions has become a basic step in the research of fundamental mechanisms of biological processes. The far most known process regarding polyunsaturated fatty acids (PUFA) is the peroxidation process which is generally considered to be the major mechanism of cell injury in organisms subject to oxidative stress. A much recent research deals with the geometrical isomerization of PUFA, evidencing that the cis double bond configuration is a inherent characteristic of membrane lipids which can be altered by radical stress. In view of the fact that these processes in lipids are the most relevant chemical processes occurring to PUFA, it is very useful to perform model studies where both processes occur. We have established the simplest biomimetic model of cell membrane, the micelles of linoleic acid (LH), prepared by addition of a non-ionic surfactant (TWEEN®-20) and the resulting solutions were irradiated by ionizing radiation up to 400 Gy under a variety of conditions where thiyl radicals are the main reactive species. The irradiation-induced peroxidation and trans isomerization in our model systems under controlled oxidation conditions have been studied. The concentrations of hydroperoxide of linoleic acid (LOOH) were determined using the spectrophotometric ferric thiocyanate method, while geometrical isomers were analyzed by GC using known conditions for the separation of cis and trans isomers. Data on relative importance of these processes in this competitive environment have been considered. This lecture will demonstrate that hydroperoxy- and trans-PUFA can be the resulting effect of oxidative free radical conditions. While under anaerobic conditions only the cis-trans isomerization was observed, in air-equilibrated solutions a substantial amount of LOOH was produced and the cis-trans isomerization process was still observed, e.g., irradiation of 500 M of LH at 400 Gy and dose rate of 4.6 Gy/s gave 20 M of LOOH and 10% conversion of LH into mono-trans isomers. The effect of micelle size will be discussed in order to gather data on the effect of supramolecular organization for the outcome of the two processes, and in particular, to envisage any positional preference of the two double bond.

The modeling of free radical reactions under naturally occurring conditions has become a basic step in the research of fundamental mechanisms of biological processes. Our current research is focused on the relationships between the structures and functions of lipids as affected by free radicals processes. The far most known process regarding polyunsaturated fatty acids (PUFA) is lipid peroxidation which is generally considered to be the major mechanism of cell injury in organisms subject to oxidative stress. A much recent research deals with the geometrical isomerization of PUFA. The double bond structures of natural unsaturated lipid molecules have almost invariably the cis geometry, which determines their properties and various biological activities. The conversion of lipids from cis to trans form occurs by a free radical process, which makes the examination of the underlying mechanism a challenging task accessible to radiation-chemical approach. In this context, in view of the fact that these processes in lipids are the most relevant chemical processes occurring to PUFA, studies of the lipid modification induced by irradiation can be carried out with biomimetic models that allow molecular processes of free radical reactions to be studied in a less complex environment, but respectful of the biological characteristics. We have established the simplest biomimetic model of cell membrane, the micelles of linoleic acid, prepared by addition of a non-ionic surfactant (TWEEN®-20) and the resulting solutions were irradiated by ionizing radiation up to 500 Gy under a variety of conditions where thiyl radicals are the main reactive species. The irradiation-induced peroxidation and trans isomerization in our model systems under controlled oxidation conditions represent a topic of our investigations. The concentrations of hydroperoxide of linoleic acid are determined using the spectrophotometric ferric thiocyanate method, while geometrical isomers are analyzed by GC (Rtx-2330 capillary column, FID) using known conditions for the separation of cis and trans isomers. Relative importance of these processes in this competitive environment will be considered. The effect of the natural antioxidants on the micelles supramolecular organization for the positional preference of the double bond isomerization will be also studied.

The far most known process regarding polyunsaturated fatty acids (PUFA) is the peroxidation process which is generally considered to be the major mechanism of cell injury in organisms subject to oxidative stress. A much recent research deals with the geometrical isomerization of PUFA, evidencing that the cis double bond configuration is a inherent characteristic of membrane lipids which can be altered by radical stress. In view of the fact that these processes in lipids are the most relevant chemical processes occurring to PUFAs, it is very useful to perform model studies where both processes occur. Lipid reactivity can be addressed by biomimetic chemistry. We have established the simplest biomimetic model of cell membrane, the mixed nonionic/anionic surfactant micelles of linoleic acid. The irradiation-induced peroxidation and trans isomerization in our model systems under controlled oxidation conditions have been studied. Data on relative importance of these processes in this competitive environment have been considered. This lecture will demonstrate that hydroperoxy- and trans-PUFA can be the resulting effect of oxidative free radical conditions.

Thiols are well known for their role in cellular redox homeostasis, while aromatic amines and phenols are the best known classes of chain-breaking antioxidants. On the other hand, thiyl radicals are known to catalyse the double bond isomerization in PUFA. We investigated the role and interplay of 2-mercaptoethanol and diphenylamine in the parallel processes of peroxidation and cis-trans isomerization of linoleic acid (LA) during gamma radiolysis, both in solution and micelles. Both compounds, used alone were able to protect LA from oxidation; however pro-oxidant activity and enhanced isomerization was observed when they were used together, depending on the experimental settings. Instead, alpha-tocopherol protected LA from both oxidation and isomerization in the presence of thiols under any tested settings. The mechanistic scenario is discussed highlighting the role of diphenylaminyl radicals in promoting thiyl-radical-induced cis-trans isomerization in the presence of oxygen. (C) 2015 Elsevier Ltd. All rights reserved.