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Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.Okratoksin A(OTA) i citrinin (CTN) nefrotoksični su mikotoksini koji zajednički kontaminiraju žitarice. Cilj ovoga istraživanja bio je izmjeriti koncentraciju OTA-e i CTN-a u bubrezima i jetri štakora, tretiranih tim mikotoksinima, te provjeriti hoće li tretman resveratrolom (RSV) smanjiti koncentraciju mikotoksina u tkivima. Istraživanje je provedeno na mužjacima štakora soja Wistar, koji su 21 dan bili tretirani OTA-om (0,125 i 0,250 mg/kg t. m.), a dva dana CTN-om (20 mg/kg t. m.) ili kombinacijama tih mikotoksina. Dvije skupine štakora koje su tretirane mikotoksinima OTA+CTN dobivale su 21 dan RSV (20 mg/kg t. m.). Povećanje koncentracija OTA-e u bubrezima i jetri bio je u skladu s povećanjem doze. Tretman mikotoksinima OTA+CTN smanjio je nakupljanje OTA-e u bubrezima i jetri, a povećao je koncentraciju CTN-a. Tretman RSV-om povećao je koncentraciju OTA-e u bubrezima i jetri, ali je smanjio koncentraciju CTN-a u bubrezima tretiranih štakora. Koncentracija OTA-e značajno se smanjila u prisutnosti CTN-a, vjerojatno zbog kompeticije CTN-a i OTA-e za prijenosnike OAT1 i 3, koji služe za prijenos tih toksina kroz membrane u bubrezima.

Cardiopulmonary bypass (CPB) is an essential technique in cardiac surgery but is also associated with adverse effects, including the systemic inflammatory response syndrome that manifests itself as ischaemia-reperfusion injury and multi-organ dysfunction. The aim of this mini review is to take a look at the current knowledge of resveratrol, a stilbenoid and natural antioxidant believed to have many cardioprotective effects including vasodilation, lowering of blood pressure and reactive oxygen species levels, suppression of low-density lipoprotein peroxidation, and mitigation of ischaemia/-reperfusion injury. We mostly focus on its cardioprotective potential in patients undergoing cardiac surgery supported by CPB. Current findings, however, are still inconclusive and call for further research, including clinical trials, Kardijalna kirurgija je kirurška struka koja se vrlo brzo razvija, a u kojoj se pri radu koristi uređaj za izvantjelesni krvotok. Njegovo korištenje dovodi do sustavnog upalnog odgovora koji se prezentira kao ishemijsko-reperfuzijska ozljeda i multiorganska disfunkcija. Resveratrol, stilbenoid iz skupine fenola, prirodni je antioksidans koji se nalazi u grožđu, borovnicama, malinama, murvi i kikirikiju, a smatra se da ima neuroprotektivna, antidijabetička, antikarcinogena i kardioprotektivna svojstva. Prema dosadašnjim znanstvenim istraživanjima, resveratrol je moguće glavni čimbenik kardioprotektivnoga djelovanja vina, koje se očituje vazodilatacijom, smanjenjem količine reaktivnih radikala kisika i arterijskoga krvnog tlaka, zatim smanjenjem peroksidacije lipoproteina male gustoće i ublažavanjem ishemijsko-reperfuzijske ozljede. Cilj je ovoga pregleda sažeti trenutačno znanje o potencijalnim kardioprotektivnim svojstvima resveratrola u bolesnika koji se podvrgavaju kardiokirurškom zahvatu pri kojem se koristi uređaj za izvantjelesni krvotok. Resveratrol ima razne učinke na ljudsku kardiovaskularnu fiziologiju, od kojih mnogi još moraju biti istraženi.

Sterigmatocystin is a mycotoxin structurally similar to aflatoxin B1 and a precursor to its biosynthesis. It is synthesized by 55 different species of mould that belong to phylogenetically and ecologically diverse genera. Sterygmatocistin can be found in various foodstuffs, even after processing, as well as in indoor humid spaces and dust. The LD50 for oral exposure is 160 mg/kg b.w. The mechanism of action is associated with the formation of exo-epoxides and the formation of DNA adducts. Therefore, the assumed mechanism of action is related to the occurrence of oxidative stress, which can have a great impact on health. This study investigates the role of sterigmatocystin in the generation of oxidative stress in the kidneys of male Wistar rats. Rats were treated once with steigmatocystin at three different doses: 1/4, 1/8, and 1/16 LD50 (40, 20, and 10 mg/kg b.w.). Parameters of oxidative stress that have been measured are activities of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), the concentration of reduced glutathione (GSH) and malondialdehyde (MDA). Controls included solvent control corn oil and water. When compared to the controls, a statistically significant difference was observed in MDA concentration and SOD activity. The difference was observed for all three concentrations in MDA and 1/16 and 1/4 treatments in SOD. A significant difference in the activity of CAT and GPx and concentration of GSH was not noted in kidneys, although CAT showed an increasing and GPx decreasing trend. The results showed that there was a high occurrence of oxidative processes (SOD) and tissue damage (MDA) in the kidneys and that they were sensitive to the occurrence of oxidative stress caused by sterigmatocystin. However, further studies are needed to discover the mechanism of sterigmatocystin toxicity.

Mechanisms of aging, and cancer as one of main associated pathology are only on verge of understanding. Even in healthy aging gradually loss of function in excretion organs kidneys and liver appears. Previously, we did not find expected elevated tissue concentrations of reactive oxidative species (ROS), that are assumed to be present in old humans and experimental animals and that they can be reversed by antioxidants (AO) treatment. The new hypothesis was that the expression of ROS inducible, potent antioxidant and metal scavenging proteins, metallothioneins Mt1 and Mt2 (MTs), among others, increases in old age to maintain ROS levels and that they can be further modulated with AO. To that aim we studied male and female Wistar rats regularly aged together with these treated with melatonin and resveratrol. Starting from 3 months of age, for the next 9 or 21 months treated rats were drinking AO in water (~1 mg/kg b.w./day), whereas the control animals were drinking water or AO vehicle (0.01% ethanol). Trace elements (TE) were analysed by ICP-MS and the expression of MTs mRNA and proteins in kidneys and liver were determinate by end-point RT-PCR and immunochemical methods, respectively. AO did not change TE accumulation and expression of MTs in both sexes, but known iron accumulation in aged animals was followed by copper and toxic metal cadmium with known female domination. After 1 year, aging increased MTs expression in both sexes that did only slightly further increased after 2 years. We conclude that Fenton reactive metals accumulated in aged animals may influence MTs mRNA and protein expression to help retain physiological ROS level in old rats. (Croatian Science Foundation project IP-2013-11-1481).

Arctostaphylos uva-ursi je tradicionalna biljka koja se koristi u terapeutske svrhe za liječenje akutnih urinarnih infekcija, a njezina antiseptička svojstva mogu se pripisati hidrokinonu koji nastaje hidrolizom arbutina. Cilj ove studije bio je odrediti toksikološki profil hidrokinona na stanicama mokraćnog mjehura (T24). Kvantitativna i kvalitativna analiza ekstrakta i digestivna stabilnosti te bioraspoloživosti arbutina i hidrokinona provedena je HPLC testom i simuliranom in vitro probavom. Citotoksični učinak, indukcija reaktivnih kisikovih vrsta (ROS) i promjene u proteomu utvrđeni su Neutral red i DCFH-DA testom te spektrometrijom masa. Tijekom in vitro probave, arbutin je bio stabilan, a koncentracija hidrokinona se povećala nakon želučane probave pepsinom (109, 6%), a zatim se smanjila nakon faze tankog crijeva (65, 4%). Preporučene doze napitka imale su citotoksični učinak kada su svi konjugati hidrokinona bili prevedeni u slobodni hidrokinon (320 i 900 μg/mL), a toksični učinak je bio pojačan 24-satnim oporavkom. Jedna šalica terapijske doze imala je prooksidativni učinak nakon 4 h inkubacije, a kraće vrijeme tretmana (2 h) nije imalo utjecaja na indukciju ROS-ova. Proteomska analiza otkrila je 17 statistički znatno više eksprimiranih proteina u odnosu na kontrolu povezanih s odgovorom na oksidativni stres, signalizaciju prilagođenog odgovora na stres, „heat-shock“ odgovorom i inicijacijom translacije. Dobiveni rezultati su dokaz da biljni spojevi, iako iz prirodnog izvora, mogu pokazati negativna svojstva.

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3., Okratoksin A(OTA) i citrinin (CTN) nefrotoksični su mikotoksini koji zajednički kontaminiraju žitarice. Cilj ovoga istraživanja bio je izmjeriti koncentraciju OTA-e i CTN-a u bubrezima i jetri štakora, tretiranih tim mikotoksinima, te provjeriti hoće li tretman resveratrolom (RSV) smanjiti koncentraciju mikotoksina u tkivima. Istraživanje je provedeno na mužjacima štakora soja Wistar, koji su 21 dan bili tretirani OTA-om (0,125 i 0,250 mg/kg t. m.), a dva dana CTN-om (20 mg/kg t. m.) ili kombinacijama tih mikotoksina. Dvije skupine štakora koje su tretirane mikotoksinima OTA+CTN dobivale su 21 dan RSV (20 mg/kg t. m.). Povećanje koncentracija OTA-e u bubrezima i jetri bio je u skladu s povećanjem doze. Tretman mikotoksinima OTA+CTN smanjio je nakupljanje OTA-e u bubrezima i jetri, a povećao je koncentraciju CTN-a. Tretman RSV-om povećao je koncentraciju OTA-e u bubrezima i jetri, ali je smanjio koncentraciju CTN-a u bubrezima tretiranih štakora. Koncentracija OTA-e značajno se smanjila u prisutnosti CTN-a, vjerojatno zbog kompeticije CTN-a i OTA-e za prijenosnike OAT1 i 3, koji služe za prijenos tih toksina kroz membrane u bubrezima.

Aspergilli section Nidulantes series Versicolores, producers of the mycotoxin sterigmatocystin (STC), dominated the fungi isolated from air and dust in flood-affected homes (Gunja) and control houses (Gornji Stupnik) in Croatia. The majority of isolates were able to produce STC and some isolates also produced its derivate 5- methoxysterigmatocystin (5-MET-STC). How can this affect human health? Here we present: a) Aspergilli series Versicolores species diversity in air and dust (calmodulin sequence-based methods) ; b) occurrence of STC and metabolites from the STC biosynthesis pathway in dust (LC- MS/MS) ; and c) immunomodulatory and genotoxic effects of STC and 5-MET-STC in lungs of male Wistar rats upon intratracheal instillation in concentrations found in dust. Most of the Versicolores isolates were from Gunja (91/125). Most isolates belonging to A. jensenii, A. creber, A. puulaauensis, A. tennesseensis, A. venenatus produced both STC and 5-MET-STC. A. amoenus, A. fructus, A. griseoaurantiacus, A. pepii and A. protuberus produced STC but not 5-MET-STC, while A. sydowii did not produce any of these two toxins. Among the 75 fungal compounds detected in dust, metabolites from the STC pathway including STC, 5-MET-STC, veriscolorin C, averufin and averantin were among the most abundant metabolites, which prevailed in repaired houses in Gunja. The highest concentrations of STC (0.59 µg/g) and 5-MET-STC (7.70 µg/g) in dust were detected in Gunja. In rat lungs, the immunomodulatory effects of STC (0.4 µg/mL) and 5- MET-STC (5 µg/mL) were insignificant. The levels of DNA damage were similar although STC was applied at a 10-fold lower dose. The results of combined treatment suggested possible antagonistic interactions. This work was fully supported by the Croatian Science Foundation under the project MycotoxA (IP-09-2014-5982).

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxic mycotoxins that frequently co-contaminate food and feed, thus representing a serious health threat to humans and animals. Studies have shown that these toxins deregulate an array of genes/proteins leading to their expressional changes. However, individual and combined effect of OTA and CIT on the expression of renal proteins facilitating the transport of biologically important molecules such as water and glucose, are poorly studied. In this study, we used immunochemical methods (immunofluorescence microscopy and Western blotting) to reveal the individual and combined effects of OTA (0.125 and 0.250 mg/kg b.w.) and CIT (20 mg/kg b.w.) on the localization/expression of renal water channels (rAqp1 & rAqp2) and sodium-glucose cotransporters (rSglt1 & rSglt2) after a 21-day subchronic oral exposure of Wistar male rats. Additionally, we investigated if antioxidant resveratrol (RSV ; 20 mg/kg b.w.) can counteract the OTA- and CIT-related nephrotoxic effects on the localization/expression of studied proteins. In the rat kidney, the higher individual doses of OTA significantly downregulated protein expression of rAqp1 glycosylated isoforms and upregulated protein expression of rSglt1, whereas combination of higher OTA dose and CIT downregulated expression of rAqp1 non-glycosylated isoforms. Interestingly, the protein expression of rAqp1 glycosylated isoforms and rSglt1 was significantly downregulated in the kidneys of rats exposed to the combination of both mycotoxins (OTA+CIT) and RSV. Thus, OTA and CIT seem to have a selective effect on protein expression of studied transporters in rat kidneys, whereas RSV does not counteract their nephrotoxic effects. (Croatian Science Foundation project IP-09-2014-5982).

Human studies on oxidative stress induced by general anaesthesia are rather scarce. It is important to study effects of anaesthetics on oxidative stress, particularly in surgical procedures with high ROS production, because their effect may be pivotal for the outcome of the surgery. In the presented study, patients with hydronephrosis underwent balanced anaesthesia or Total Intravenous Anaesthesia (TIVA) with target- controlled infusion (TCI) during surgery. Blood samples from patients (N=32) were taken before induction of anaesthesia, 30 minutes and 24 hours after beginning of the surgery. Measured parameters of oxidative stress were compared between two types of anaesthesia. Malondialdehyde (MDA) was measured using HPLC with fluorescence detector. Glutathione (GSH), reactive oxygen species (ROS), total antioxidant capacity (TOC) and protein carbonyl concentration were measured spectrophotometrically. Activity of superoxide dismutase (SOD) was measured spectrophotometrically using commercial kits. Observed data were analysed by mixed modelling with oxidative stress parameter as a dependent variable and sampling time nested within an anaesthetic group as fixed effects, and subject id as a random effect. Post-hoc were performed and the false discovery rate was controlled by the Benjamini-Hochberg method. SOD activity was significantly higher in patients that underwent balanced anaesthesia than anaesthesia with TIVA/TCI 30 minutes after beginning of the surgery. There was no statistically significant difference in other measured parameters between balanced and TIVA/TCI type of anaesthesia. Results of the majority of the measured parameters of oxidative stress were significantly different 30 minutes or 24 hours after the surgery from those measured before the anaesthesia (balanced and TIVA/ TCI). Results from this study contribute to general knowledge about oxidative stress caused by anaesthesia as an important factor in causing higher rates of postoperative complications and delayed recovery after surgery.

Previous studies have shown that transporters for both organic anions (Oat) and organic cations (Oct) provide a possible entrance pathway for mycotoxins including ochratoxin A (OTA) and citrinin (CIT) having the nephrotoxic effects in mammals. However, compared to Oats, individual and combined exposure effects of these mycotoxins on Oct expression in mammalian kidney is still unknown. Here, we used immunofluorescence microscopy and Western blot analysis to investigate individual and combined effects of OTA (0.125 and 0.250 mg/kg b.w.) and CIT (20 mg/kg b.w.) exposure on renal localization and protein expression of rat Oct (rOct1 and rOct2) following 21-day subchronic oral exposure of Wistar rats. In addition, we have studied the potential protective effects of resveratrol (RSV) (20 mg/kg b.w.) on renal localization and protein expression of studied rOcts. Results showed that protein expression of rOct1 (but not rOct2) was significantly downregulated by both individual OTA doses used in this study. Combination of both mycotoxins (OTA+CIT) had a downregulating effect exclusively on the rOct1 protein expression when the higher experimental OTA dose (0.250 mg/kg b.w.) was used. In individual manner, citrinin did not affect the expression of both rOct1 and rOct2 proteins. Interestingly, the combination of tested mycotoxins (OTA+CIT) and RSV caused a significant decrease of renal rOct1 and rOct2 protein expression. In conclusion, OTA has an Oct- and dose-dependent effect on the protein expression of studied rOcts whereas RSV does not protect, but potentially enhances the nephrotoxic effects of studied mycotoxins in rat kidneys. (Croatian Science Foundation project IP-09-2014-5982).

This study aimed to explore involvement of oxidative stress in sterigmatocystin (STC) toxicity in male Wistar rats. Animals were orally treated with a single STC dose (10, 20 and 40 mg/kg b.w.). Short-term treatment resulted in moderate oxidative stress determined by a significant increase of malondialdehyde (MDA ; all STC doses) and catalase (CAT ; 10 mg/kg b.w.) in plasma, a decrease of glutathione peroxidase (GPx ; 20 and 40 mg/kg b.w.) in the liver, and increase of MDA and superoxide dismutase (SOD) in kidneys (all STC doses). Heat shock protein (Hsp27 and Hsp70) expression was determined by Western blotting in rat liver and kidneys. Hsp27 expression was downregulated by STC, particularly in the liver (40 mg/kg b.w.). The lowest STC dose elevated the expression of Hsp70 in both liver and kidneys, while an increase in STC doses restored Hsp70 expression to control. Alterations in expressions of Hsp27 and Hsp70 could be only partially associated with oxidative stress. STC provoked a significant DNA damage in both liver and kidneys (alkaline comet assay), but the liver was more affected by a broader spectrum of DNA lesions. Oxidative DNA damage (hOGG1-modified comet assay) contribute to the overall mechanism of STC-induced DNA damage in both organs, but kidneys in general seem to be more susceptible to oxidative stress upon short-term exposure to sublethal doses of STC.

Introduction: Aging-related impaired body structure and functions are assumed to be, at least partially, caused by elevated oxidative stress (OS). A red wine polyphenol resveratrol may act as an antioxidant and anti-aging medium, but its actions in vivo are controversial (1). Here we report on sex- related effects of resveratrol treatment (RT) on blood/plasma parameters of DNA damage, oxidative status, and concentrations of sex hormones (SH) in a rat model of aging (2). Material & Methods: Starting from their age of 3-mo, for the next 9-mo or 21-mo male and female Wistar rats were drinking resveratrol in water (10 mg/L ; predicted intake: ~1 mg/kg b.m./day) or only water (controls). In their blood/plasma we performed the alkaline comet assay (3) and determined glutathione (4) and malondialdehyde (5) by the established methods. SH were determined by commercial kits. Results: Compared with controls, the 9-mo RT did not change any parameter in both sexes. After the 21-mo RT, the OS-related parameters remained unchanged, but testosterone levels in males were ⁓45% higher (P

Sterigmatocystin (STC), a precursor of aflatoxin B1, is mycotoxin which International Agency for Research on Cancer evaluated as Group 2B. STC was isolated in 1954 from Aspergillus versicolor and afterwards from other Aspergillus species producing it as aflatoxin B1 precursor or as final mycotoxin. Due to its structural similarity to carcinogen AFB1, STC carcinogenic potential was studied much more than its toxicity. The aim of this study was to evaluate the effect of STC on antioxidative enzymes and heat shock proteins (Hsp 70 and Hsp27) as parameters of oxidative stress. Male Wistar rats (N=5 per group) were treated with single oral STC doses of 1/16, 1/8, and 1/4 of LD50 (10, 20 and 40 mg kg-1 b.w.). Control group was treated with corn oil which was used as STC vehicle. Catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in plasma, kidneys and liver were measured on a plate reader using commercial kits. Expressions of heat shock proteins Hsp70 i Hsp27 were measured in kidneys and liver using Western blot methods. CAT activity did not change in organs of treated animals. GPx activity was significantly lower in liver of rats treated with 1/8 and 1/4 LD50. Activity of SOD was significantly increased in kidneys of rats given 1/16 and 1/4 LD50. Expression of Hsp70 was significantly increased in liver samples of rats treated with 1/16 LD50 ; in kidneys the same dose augmented expression of Hsp70 but without significant difference to control group. Highest STC dose returned Hsp70 expression to control values. Expression of Hsp27 in liver and kidneys was not significantly changed at any STC dose. Taken together our findings suggest that acute oral exposure to STC causes oxidative stress both in liver and kidneys.

Aspergilli section Versicolores, producers of mycotoxin sterigmatocystin (STC), dominated the fungi isolated from air and dust in flood-affected homes (Gunja) and control houses (Gornji Stupnik) in Croatia. As many as 70% of isolates were able to produce STC and some isolates also produced its derivate 5-methoxy-STC. How this could affect human health? Here we present: a) Aspergilli section Versicolores species diversity in air and dust (calmodulin sequence-based methods) ; b) occurrence of STC in contrast to other mycotoxins in dust (multitoxin HPLC/MS/MS) ; c) immunomodulatory effects (ELISA) and genotoxicity (alkaline comet assay) of STC vs STC-producing Aspergilli extracts in THP-1 cells macrophage-like cells and human lung A549 cells ; d) genotoxic effects of STC and 5-methoxy-STC in lungs of male Wistar rats upon intratracheal instillation in concentrations found in dust. Most of the Versicolores isolates were from Gunja (91/125). A. jensenii (31%) and A. creber (30%) were the most frequent, followed by A. puulaauensis (10 %), A. protuberus (9 %), A. tennesseensis (6 %), A. sydowii (5 %), A. amoenus (3 %), A. venenatus (2 %), A. fructus (1.6 %), A. griseoaurantiacus (1 %) and the recently described A. pepii (1 %). Among the 75 mycotoxins detected in dust, STC (70%) and 5-methoxy-STC (50%) were the most dominant. The highest concentrations of STC (0.59 mg/g) and 5-methoxy-STC (7.70 mg/g) in dust were detected in Gunja. Subcytotoxic concentrations of STC (0.032 and 0.32 µg/ml) induced a concentration-dependent increase of IL-1b, IL-6 and IL-8 and a decrease of TNF-α ; A. jensenii and A. creber extracts containing the same concentrations of STC did not significantly affect cytokine levels. STC evoked a concentration-dependent increase of DNA damage in A549 cells ; Aspergilli significantly increased DNA breaks, but a lower concentration provoked greater DNA damage. In rat lungs, levels of DNA damage were similar upon treatment with STC and 5-methoxy-STC, but STC was applied in a 10-fold lower dose. The results of combined treatment suggested possible antagonistic interactions. Taken together, differences in toxicity pattern of single STC and Aspergilli may be explained by the presence of 5-methoxy-STC and possibly other metabolites that antagonised STC toxicity.

Humans and animals are constantly exposed to mycotoxins mostly through the ingestion of contaminated food and feed, although other ways of exposure are also possible. Food is usually contaminated with several mycotoxins due to co- contamination by various molds but also because some mold strains may produce various mycotoxins under modified conditions of growth. There are about 400 known mycotoxins that, using very sensitive methods, can be found in food and feed, but only some have been found to have a toxic effect in mammals. Unfortunately, even fewer have known toxicokinetics and mechanisms of toxicity. The mechanisms of toxicity of mycotoxins are rarely studied on experimental animals, while the results of in vitro studies on cell cultures are often contradictory. In the available literature on the effect of co-exposure to mycotoxins either in experimental animals or in in vitro studies, many different end-points were measured which creates difficulties in the interpretation of the results. In animals these were changes in body weight, parameters of kidney, liver and immune system lesions, diuresis, activity of plasma, organs and urine enzymes, teratogenicity and mortality rate. In various cell cultures, cell viability, reactive oxygen species production, apoptosis and parameters of oxidative stress were measured. It was found that the same combinations of mycotoxins may have an antagonistic, additive or synergistic effect, depending on the dose of mycotoxins. Although some mycotoxins in combination with others have always had a clear antagonistic effect at any tested dose, the best protection against them is the prevention of their overall appearance.

Mycotoxins are secondary metabolites produced by many mould species. More than 300 mycotoxins are known and most of their toxicity mechanisms are unknown. Humans can be exposed to mycotoxins found in food, through skin or inhalation. Sterigmatocystin (STC) is a mycotoxin produced by many Aspergillus mould species which can contaminate wet buildings. STC is a precursor and has similar structure as a carcinogenic mycotoxin aflatoxin B1. The International Agency for Research on Cancer classified OTA as a Group 2B (possible human carcinogen) carcinogen. So far, STC exposure was connected to liver and kidneys damage and DNA damage of certain cell lines. The aim of the study was to determine the role of oxidative stress in mechanism of STC toxicity. Experiments were performed on male adult Wistar rats because of their sensitivity to STC exposure. Animals were treated with single oral STC doses of 1/4, 1/8, and 1/16 of LD50. After 24 hours rats were sacrificed and blood, liver, kidney, brain and lung samples were collected. The experiment was approved by the Ethic Committee of the Institute for Medical Research and Occupational Health and the Ministry of Agriculture of Republic of Croatia in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU). Parameters of oxidative damage of lipids, proteins and DNA were measured in different tissues. Levels of different oxidative stress parameters were not dose dependent and the kidney were more affected with STC treatment than other organs. This work has been fully supported by the Croatian Science Foundation under the project MycotoxA (HRZZ-IP-09-2014-5982).

Water is the most abundant molecule in living cells. Aquaporin protein channels (AQPs) facilitate the passive movement of water across cell membranes. In mammalian kidneys, AQP1 and AQP2 facilitate water reabsorption from primary urine across the apical membrane of proximal and distal nephron tubules. Studies indicate that renal functional/structural changes in humans and experimental animals develop with age. Aging promotes fluid loss in tissues which was suggested to reflect changes in the expression/activity of AQPs. Here we investigated the potential age-ameliorating effects of two potential geroprotector substances, melatonin (endogenous hormone) and resveratrol (plant flavonoid), on AQP1 and AQP2 expressions in the rat model of experimental aging. During 21 months, rats of both sexes were given melatonin or resveratrol via drinking water (~1 mg/kg b.w./day), whereas control animals drank vehicle (0.01% ethanol in drinking water). The expression of both AQPs in the kidneys of control and treated animals was determined using immunofluorescence cytochemistry in tissue cryosections and Western blot analysis in isolated cell membranes. Results showed that melatonin and resveratrol treatment had no effect on renal AQP1 expression in both sexes. AQP2 expression in male and female kidneys of resveratrol-treated rats also did not change compared to controls. However, only in female rats, AQP2 expression was upregulated by melatonin in the inner stripe and papilla kidney regions. Therefore, in 24-month old rat kidneys, chronic oral intake of melatonin and resveratrol had no effect on AQP1, whereas melatonin exerted sex-dependent upregulation of AQP2. (Supported by Croatian Science Foundation project IP-2013-11-1481).

Mycotoxins are products of moulds that contaminate all kinds of food and feed. Humans are continuously exposed to mycotoxins mostly by ingestion, but other ways of exposure (such as respiratory and dermal) are also possible. Foods of plant origin, such as grains and bread, coffee, grapes and vine, beer and spices are contaminated following infection with mycotoxin-producing moulds, while food of animal origin contains mycotoxins if animals are fed with contaminated feed. Mycotoxins may cause intoxications (either acute or chronic) called mycotoxicoses with high variability of target organs. These may be hepatotoxic, nephrotoxic, immunotoxic, neurotoxic, cardiotoxic, haematotoxic and also have carcinogen and teratogen properties. The severity of mycotoxicoses depends on the toxicity of the mycotoxin, extent of exposure and nutritional status of the individual and possible synergistic effect of other chemicals to which an individual is exposed. The toxic effects of mycotoxins are mostly known from veterinary practice. Mycotoxicoses, which can occur both in industrialized and developing countries, appear when environmental, social and economic conditions are combined with meteorological conditions (humidity and temperature) that favour the growth of moulds. Mycotoxins have highly variable chemical structures, but being organic compounds of low molecular mass, they do not cause allergies. Out of about 400 mycotoxins, only a few have known toxic effects in experimental animals, and even fewer possess effects known to be toxic in humans. Most mycotoxicoses were first recognized in domestic animals and it is difficult to diagnose them in humans. In humans, mycotoxicosis should be considered in case when a disease appears in several persons, with no obvious connection to a known etiological agent, such as microorganisms. Unfortunately, medical doctors are not trained well to diagnose them, and mycotoxicoses very often remain unrecognized. There are several international organizations and agencies (such as World Health Organization, International Agency for Research on Cancer and European Food Safety Agency) that deliver scientific opinions on various chemicals in food. The scientific opinion is based on results published in scientific literature on the effects of a toxin in laboratory animals. Researchers have realised that, in the field of mycotoxin toxicology, a particular problem is co- contamination of food with several mycotoxins with antagonistic, additive and synergistic toxic effects. Unfortunately, studies on the combined exposure to two or more mycotoxins are rare and inconclusive.

Telomeres are the main guardians of genome stability. During DNA replication, progressive shortening of telomeres takes place with each cell division. Critically short telomeres induce cell cycle arrest or apoptosis. Oxidative stress also plays an important role in telomere dynamics. Recent evidence highlights direct molecular connection between telomere attrition and mitochondrial dysfunction. Although reactive oxygen species (ROS) are normal products in intermediary metabolism, and are crucial for different intracellular signalling pathways, a disbalance resulting in ROS excess induces oxidative stress and accelerates telomere shortening. It is assumed that with ageing, production of ROS may be increased causing faster telomere shortening, and that both may be ameliorated by antioxidants. To test this hypothesis, we treated male and female Wistar rats for 9 or 21 months with melatonin and resveratrol and investigated their effect on telomere attrition rate in liver and kidney (various zones) tissues. Telomere length was assessed by Southern blotting of genomic DNA. The results in 1- and 2- year-old male and female rats showed an absence of significant differences in telomere length in the tissues from both organs after treatment with antioxidants compared to control (vehicle-treated) animals (N=4 per group). Based on obtained results, we did not observe beneficial properties of tested antioxidants towards age-dependent telomere shortening.

Ochratoxin A (OTA) is a nephrotoxic and carcinogen mycotoxin that contaminates different food commodities. Its toxicokinetics and accumulation in plasma, kidneys and liver are species-specific. The European brown bear (Ursus arctos L.) is the largest terrestrial mammal in Croatia and most its diet consists of plant food. Maize from supplemental feeding sites makes up an important part of the bears’ diet, especially in periods and years of low natural food resources. This supplemental maize is often of poor quality to begin with and is further adulterated by weather conditions, which enhance the production of OTA. We hypothesized that the OTA level in kidneys would reflect the intake of OTA-contaminated maize. A total of 56 kidney samples were collected (19 samples in 2013 and 37 samples in 2015) from bears hunted according to the Brown Bear Management Plan in Croatia. After liquid- liquid extraction, OTA concentration was measured using HPLC with a florescent detector. Regression analysis revealed that OTA concentration was significantly lower (13.82 ± 12.55 ng g-1 tissue ; median 12.25) in samples collected during 2013 than in samples collected during 2015 (142.30 ± 237.53 ng g-1 tissue, median 53.31), while controlling for age, sex and season of collection (b=0.60, p

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic and hepatotoxic mycotoxins commonly found together in grain. There is the EU legal obligation to monitor OTA in various commodities but there is no such an obligation for CTN. Recent research on the presence of CTN concentration in grains showed high contamination (50-75 % samples) with a considerably large concentration range (0 – 400 µg kg-1). Target organs of OTA and CTN toxicity are kidney and liver. The aim of this work was to see the fate of OTA and CTN in kidney and liver of rats co-treated with both mycotoxins. Adult male Wistar rats were treated with OTA (0.125 and 0.250 mg kg-1 b.m.) daily for three weeks and with CTN (20 mg kg-1 b.m.) for two last days of experiment. In addition to the control group, we also had a group receiving OTA alone and CTN alone (in the same doses as above). OTA showed a dose-dependent increase in both organs compared to control. In animals receiving combined treatment, liver and kidney OTA was significantly lower than in animals receiving OTA alone. In contrast, CTN liver and kidney concentrations in the OTA + CTN-treated animals were three to six times higher than in animals given CTN alone. These findings could be explained by higher affinity of the organic anion transporter 1 (OAT1) and OAT3 for CTN than for OTA, which led to lower OTA accumulation in the kidney. This is the first animal study of combined exposure to OTA and CTN to confirm earlier in vitro findings that CTN decreases OTA accumulation. It also suggests that mycotoxins when combined may compete for the same binding site and change each other's accumulation in target organs for better or for worse.

Many industrialized countries are facing demographic changes that manifest themselves in population aging. It is estimated that roughly one third of Europe’s population is 50+ with this share likely to increase. Therefore, the investigation of aging molecular mechanisms is a priority to facilitate healthy aging and improve the productivity/longevity of the aged population in both men and women. Within the frame of project Aging-related Expression of Membrane Transporters in Rats (AGEMETAR), aging is investigated in the Wistar rat model over 24 months, which corresponds to the elder age of humans. The aim of the current study was to investigate baseline DNA damage using the comet assay in kidneys, liver and blood mononuclear cells (BMCs) of adult 3-month-old rats of both sexes. In order to establish the influence of sex-hormones on baseline DNA damage, gonadectomized rats of both sexes were compared to sex-matched sham-operated animals (N=10/group). The sex-related differences of baseline DNA damage were not observed for any tissue studied in 3-month-old rats ; the percentage of comet DNA was in: BMCs 0.62±0.13% (male) compared to 0.63±0.20% (female) ; kidneys 1.00±0.24% (male) compared to 1.28±0.49% (female) ; and liver 0.86±0.21% (male) compared to 0.84±0.26% (female). Similarly, castration in males or ovariectomy in females had no effect on baseline DNA damage in: BMCs 0.55±0.13% (castration) and 0.53±0.14% (ovariectomy) ; kidneys 0.92±0.25% (castration) and 1.00±0.39% (ovariectomy) ; and liver 0.83±0.20% (castration) and 0.93±0.15% (ovariectomy) when compared to sex-matched sham-operated animals. Altogether, these results indicate that sex hormones have no effect on baseline DNA damage in the BMCs, kidneys and liver of 3-month-old rats. Furthermore, they provide useful information for further investigation of sex- and age-related differences of baseline DNA damage in the Wistar rat animal model of aging. (Supported by the Croatian Science Foundation Grant No. IP-2013-11-1481 ; AGEMETAR).

Animals and humans are continuously exposed to a variety of mycotoxins produced by food- contaminating molds. Despite the efforts of scientists to understand the mechanisms of mycotoxin toxicity, an insight has been gained for only 10 of the 400 known mycotoxins. Only a few human and animal diseases have been associated with mycotoxin exposure for certain. Research started with aflatoxins and continued with ochratoxins, trichotecenes, and fumonisins, but all these compounds were analyzed separately. Recently, some already known mycotoxins that had not been considered important, such as citrinin and sterigmatocystin, have returned in the focus of mycotoxin research because they seem to increase the toxic effect if combined with other mycotoxins. With highly sophisticated technology that can measure many mycotoxins in food at the same time, we have become aware of combined exposure but still do not understand the toxicological significance of extremely low concentrations of dozens of mycotoxins in food. So many mycotoxins in one place are expected to have additive effects in mammals, but there have been reports on antagonistic interaction between mycotoxins, which probably explains while we are still alive. Unfortunately, the requirement of the EU to switch research from in vivo to in vitro studies may result with unrealistic ideas about combined toxicity because cell cultures have different metabolism from mammals. Other difficulties involve a very high cost of mycotoxins and equipment, disagreement between scientists about the research end-points, and high variation in experimental designs. The last two issues stymie the comparison of the toxic effects of mycotoxin co-exposure.

Levels of elements in brown bear (Ursus arctos) blood are unknown. Previously, we discovered that brown bears in Croatia have the highest tissue levels of toxic metals among hunted terrestrial wild mammals. However, associations with other related biomarkers of exposure and/or effect have not been studied. Considering that oxidative stress has been suggested as one of the pathways of toxic damage caused by metals, its biomarkers could be useful indicators of metal-related health effects. Our aim was to study biomarkers of oxidative stress (lipid peroxidation, antioxidative enzyme SOD activity) and metal exposure, (metallothionein, MT), in association with toxic metal levels in the blood of free- living brown bears from Croatia (N=8) and Poland (N=11). Metal content was measured using ICP- MS, products of lipid peroxidation (malondialdehyde, MDA) using HPLC, activity of antioxidative enzyme using spectrophotometry, and MT using differential pulse voltammetry. Median (range) mass fractions of cadmium (Cd) and mercury (Hg) in brown bear blood were 0.356 (0.110-1.34) and 7.23 (1.09-13.5) µg/kg, and showed no differences between the two populations, as well as MDA and MT. On the contrary, lead (Pb) was higher (p=0.041) in the Polish (median 145 ; range 104-192 µg/kg) compared to the Croatian (101 ; 58.9-184 µg/kg) bears, while SOD activity was lower (p=0.007) in the Polish (738 ; 21.6-2328 U/L) than in the Croatian (1428 ; 1008-2326 U/L) bears. Age, gender and season of collection had no significant influence on the measured parameters. Results present valuable contribution to toxicological studies of European brown bear and the first data describing metals and biomarkers in the blood of this species.

Mycotoxin fumonisin B1 (FB1) is produced by moulds from Fusarium genera and is found as a contaminant on cereals all around the world. FB1 is toxic to domestic and experimental animals and development of some human diseases is associated with exposure to this mycotoxin. International Agency for Research on Cancer classified FB1 as a group 2B carcinogen. Although FB1 is found in environment, its impact on plants is not studied thoroughly. The aim of this study was to test toxicity and oxidative stress as mechanism of toxicity of mycotoxin FB1 on duckweed (Lemna minor L.). Duckweed was treated with FB1 (0.5 and 1 mg L-1) for 3 days. Toxicity was assessed in terms of growth rate (based on either frond number or fresh weight) and content of photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids). Malondialdehyde (MDA), non-protein thiols, antioxidant enzymes activities, level of glutathione and reactive oxygen species in situ were evaluated as parameters of oxidative stress. FB1 reduced grow rate for 16% and 21%, at 0.5 and 1 mg l-1 treatment, respectively (p

Statins are drugs widely used to treat hypercholesterolemia and reduce the risk of cardiovascular morbidity and mortality in patients with coronary heart disease. The mechanism of their action may be the inhibition of oxidant formation and blocking the effects of reactive oxygen species (ROS). As reported in recent studies, rosuvastatin has a beneficial effect on the plasma lipid profile and can reduce serum cholesterol and ROS formation in diabetic patients. The aim of this work was to determine the influence of rosuvastatin on the prooxidant- antioxidant balance in rat kidneys. This study was performed on male normolipidemic Wistar rats (N=36) divided into two control (treated with saline 5 mg kg-1 per day) and two experimental rosuvastatin-treated (5 and 10 mg kg-1 per day) groups. Animals were treated orally for 21 days. Malondialdehyde (MDA) – parameter of lipid peroxidation was measured in the kidney tissue homogenate of control and treated animals using the method by Drury et al. (1997). Data were analysed using t-test. Results are expressed as Mean±STD. MDA concentration was significantly lower in both experimental groups compared to controls: 0.41±0.05 vs. 0.62±0.08 µmol g-1 tissue (5 mg kg-1 per day vs. control ; p

Aging is a physiological process characterized by a reduced ability to respond to environmental stressors. Despite many aging theories, this process is still poorly understood. As evidenced in recent studies, aging may be associated with increased oxidative stress (OS), which may affect an organ’s functional and morphological characteristics. Reactive oxygen species can change the structure and function of membrane lipids, inhibit enzyme activities and oxidative phosphorylation, and oxidize nucleic acids and proteins. This study used gonadectomised rats (N=5) as an experimental model of aging and sham-operated rats as the controls to investigate if sex hormones affect OS parameters in the major internal organs. Experiments were performed on three-month old Wistar rats of both sexes six weeks after the surgical procedures. Glutathione (GSH) was measured in the kidneys, liver, brain and plasma. Significant difference in GSH concentration was observed only in plasma of sham-operated females (3.62±0.34 nmol g-1 tissue) compared to sham-operated males (4.93±0.68 nmol g-1 tissue, mean±SD ; P

Mycotoxins ochratoxin A (OTA) and citrinin (CTN) are fungal secondary metabolites often present together in food and feed. OTA is widely found mycotoxin and since its discovery it has been intensively studied. A number of OTA toxic effects are well known and target organs of its toxicity are kidney and liver. It is known that OTA induce oxidative damage in experimental animals. Although, OTA and CTN can co- occure in food and feed, data on CTN toxicity are rather scarce and the experiments are performed mostly on cell cultures. The purpose of our study was to check whether CTN treatment and combined treatment with CTN and OTA induce oxidative stress in kidney and liver of experimental animals. Adult male Wistar rats (N=6) were treated either with OTA (0.125 mg kg- 1 b.w., p.o., 21 d) or CTN (20 mg kg-1 b.w., p.o. 2 d) given separately or administered together. Spectrophotometer was used to measure glutathione (GSH) concentration while malondialdehyde (MDA) concentration was measured using HPLC with UV detector. Treatment with OTA alone significantly increased MDA concentration in kidney (145% compared with 100% of controls) which is in accordance with fact that kidney is primarily target of OTA toxicity. CTN is less toxic and treatment with CTN alone did not have significant effect on kidney and liver tissue. When animals were treated with both mycotoxins at the same time, MDA concentration in kidney and liver were significantly increased (194%, 160%, respectively) compared to controls. Treatment with either OTA or CTN did not significantly affect GSH concentration. When animals were treated with OTA and CTN at the same time, GSH was significantly increased in kidney and decreased in liver (70%) compared to controls (100%) which is in accordance with the fact that GSH is mostly synthetized in liver and then transported to other organs.

Ochratoxin A (OTA) is nephrotoxic, hepatotoxic and carcinogen mycotoxin with relatively low acute toxicity. In food it appears in all climatic zones very often together with other mycotoxins such as fumonisin B1 (FB1), zearalenone, aflatoxins (AFs) and citrinin (CTN). Because of very long half-life OTA in organism of mammals the other mycotoxins even with much shorter halflife may affect its toxicity. It is hard to predict the toxic interactions of mycotoxins based on the toxic effect of a single mycotoxin. The exposure of kidney cells to OTA and primarily hepatotoxic AFs causes decreased cell viability, increased DNA fragmentation, p53 activation and decreased expression of antiapoptotic factor bcl-2. These two mycotoxins caused increased toxicity in chicks. The weight loss, increased mortality, decrease in egg production, abnormalities in chicken embryos, atrophy of lymphoid organs was in contrast to biochemical parameters (cholesterol, albumin, total proteins, creatinine, uric acid and blood urea nitrogen) which were less affected than in animals treated with AFs alone. Synergism in the increase of mortality was noticed in chicken and mice treated with OTA and patulin (PA). Experimental nephropathy in chicken and pigs very similar to spontaneous cases of nephropathy in Bulgaria was induced by low doses of these mycotoxins. In mononuclear cells of human peripheral blood no synergistic effect was noticed. The combined effects of OTA and FB1 caused the additive or synergistic toxic interactions in vitro and in vivo. Although it was found on rats that these two mycotoxins have additive effects on oxidative stress, in another studies on turkey poults, rabbits and rats the type of interaction depended significantly on the applied dose. Simultaneous exposure of cells and treatment with OTA and CTN resulted in antagonistic, additive and synergistic effects. In PK-15 cells synergism in apoptotic effect was dose- and timedependent, increase of nuclear buds was additive while increase of the micronucleus rate was antagonistic. These mycotoxins in rat kidney and liver cause significant increase of malondialdehyde and glutathione concentrations, as well as oxidative DNA damage. Most studies on OTA combined with other mycotoxins were performed on cell cultures aimed to resolve the problem of the mechanism of combined toxicity. Studies of metabolism and toxicokinetics of single mycotoxin (when combined with another) are lacking.

Thyroid cancer is one of the fastest growing types of cancer in the world with annual percent change between 0.8 and 24.2. Presented results are part of three-phase-study that aims to improve cancer prevention. In the phase one of the study, the genome integrity is biomonitored using battery of tests in patients with thyroid diseases. Phase two was done in order to determine impact of oxidative stress on the levels of DNA damage. Finally, in the phase three, evaluation of expression of variant B-Raf and Ret proteins was done. The study population consisted of 83 volunteer patients (70 female:13 male, average age 52.00±12.63 years, 22 smokers) diagnosed with follicular adenoma (33), papillary cancer (26), and other thyroid diseases (goitre and thyroiditis, 24). Compared to healthy population consisted of 83 volunteers (matched for gender, age, and smoking status) the population with thyroid diseases showed 1.51- fold higher number of chromosome aberrations, 1.78-fold higher percentage of DNA in comet’s tails, and 2.35-fold higher number of micronuclei. Furthermore, malondialdehyde as the parameter of lipid peroxidation was assessed and 1.65-fold higher blood concentration in the patients’ group was detected. On the contrary the levels of glutathione were 1.49-fold higher in the control group. Finally, 82.50% of thyroid tissues from diseased volunteers showed positive expression of B-Raf, while only 21.25% of thyroid tissues was positive for Ret protein. The results of this study suggest that patients with thyroid diseases have more DNA damage and worse oxidative status. Overall results will provide great basis for detection of biomarkers that could be used for better risk assessment of cancer diseases and therefore for improvement of cancer prevention.

Mycotoxicoses are acute and chronic diseases caused by exposure to mould toxins that contaminate food and feed across the world. In Croatia and other temperate zone countries, the most frequently found mycotoxins of toxicological concern are aflatoxins B1 and B2 (AFB1, AFB2), ochratoxin A (OTA), fumonisin B1 (FB1), and zearalenone (ZEA). Symptoms of a single mycotoxicosis depend on the intrinsic toxicological features of mycotoxins, such as toxicokinetics and target organs of toxicity that show high variability. For instance, the target organ of AFB1 and AFB2 is the liver and aflatoxins cause acute or chronic liver injury ; OTA is nephrotoxic and a possible causative agent of Balkan Endemic Nephropathy (BEN) ; FB1 impedes the closure of neural tubes during embryonic development causing brain and spinal cord malformations ; whereas ZEA interacts with estrogen receptors disturbing the reproductive system. The severity of symptoms depends on the dose and length of exposure. Acute mycotoxicoses, which result from exposure to high doses of mycotoxins, are yet to be recorded in Croatia, while chronic mycotoxicoses have been suspected of occurring. Among mycotoxins, only aflatoxins have been proven to be carcinogenic (cause primary liver carcinoma), while OTA and FB1 are carcinogen in experimental animals but have not been shown to present a threat to humans. However, OTA is suspected to be the causative agent of very frequent urinary tract tumors in the BEN-region, and FB1 to be the causative agent of esophageal tumors in some regions of Africa and Italy as well as the primary liver carcinoma in China.

Od svih toksina plijesni (mikotoksina) najtoksičniji i jedini sigurni karcinogeni su aflatoksini koje je Međunarodna agencija za istraživanje karcinoma godine 2002. svrstala u I. skupinu karcinogena (što znači da postoji dovoljno dokaza da su karcinogeni za ljude). U skupini od dvadesetak aflatoksina najvažniji su aflatoksin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) i M1 (AFM1). Ljudi su izloženi ovim mikotoksinima putem hrane koja, posebno u tropskim krajevima, može sadržavati visoke koncentracije tih spojeva. Bolesti koje uzrokuju mikotoksini nazivaju se mikotoksikozama koje mogu biti akutne ili kronične. Mikotoksikoze uzrokovane aflatoksinima nazivaju se aflatoksikoze. Njihovo je glavno obilježje oštećenje jetre iako aflatoksini imaju i druga neželjena svojstva (imunosupresivnost, mutagenost, teratogenost i karcinogenost). Čak i u XXI. stoljeću akutne se aflatoksikoze pojavljuju kao epidemije u tropskim krajevima, a njihov je uzrok izloženost visokim koncentracijama aflatoksina u hrani. Obično su prvi simptomi aflatoksikoze povišena temperatura, a za nekoliko se dana pojavljuje žutica koja brzo napreduje. U trbušnoj se šupljini nalazi tekućina (ascites) a jetra i slezena su znatno povećane (hepatosplenomegalija). Ako su bolesnici i nakon pojave ovih simptoma i dalje izloženi aflatoksinima, pojavljuju se masovna krvarenja koja su uzrok velike smrtnosti od aflatoksikoza (10-60 % u različitim epidemijama). Kod kroničnih je aflatoksikoza ciljni organ također jetra, no simptomi nisu tako brzo vidljivi. Dugotrajna izloženost niskim koncentracijama aflatoksina može uzrokovati kronične bolesti i maligne tumore jetre. Učestalost primarnih karcinoma jetre posebno je velika u osoba koje su izložene aflatoksinima, a preboljele su virusni hepatitis. Posebni je problem izloženost djece prije rođenja (putem majčine krvi) i nakon rođenja (tijekom dojenja majčinim mlijekom) metabolitu aflatoksina M1 koji nastaje iz AFB1. Ta djeca slabije napreduju od ostale djece, sklona su upalama zbog smanjenog imuniteta i njihova je smrtnost u dječjoj dobi veća. Zbog vrlo ozbiljnih oštećenja zdravlja ljudi i životinja europski su zakonodavci postavili vrlo nisku dozvoljenu granicu aflatoksina u hrani, a naročito u mlijeku jer je mlijeko osnovna namirnica u dječjoj dobi.

Airborne fungi have been incriminated to cause several respiratory disorders based on different immunological and toxic pathways including hypersensitivity pneumonitis, asthma, rhinitis, chronic bronchitis and organic dust toxic syndrome. Mycotoxins in fungal spores have been proposed to cause adverse health effects particularly after chronic exposure to high levels of airborne fungi in indoor environments. Recent study (January 2012-November 2012) taken in grain mill (GM), apartments, and basements (Zagreb, Croatia) showed that average concentration of airborne fungi in GM was about 30 times higher (up to 40000 cfu/m3) than in other locations (up to 1244 cfu/m3). At GM species of Aspergillus, Penicillium and Eurotium dominated over other detected airborne fungi. Among Aspergilli and Eurotia, A. ochraceus and E. herbariorum were constantly present in the samples taken at GM. Aspergillus ochraceus (AO) is well-known producer of ochratoxin A (OTA) and Eurotium herbariorum (EH) produces a wide range of metabolites with poorly investigated toxicity. In the current investigation OTA was detected (HPLC, immunoaffinity columns) in spore extracts (0.3-28 µg/mL) of 5/6 AO strains isolated in November 2012, while none of tested EH strains were able to produce OTA. Taking into account maximum detected concentration of OTA in the spores, daily intakes of OTA by inhalation were calculated to be 0.08 and 3 ng/kg b.w., which is below the tolerable daily intake of OTA (17 ng/kg b.w.). Cytotoxicity of pure OTA, OTA-positive (AO-OTA+) and OTA- negative (AO-OTA-) spore extracts, as well as spore extract of EH, was tested on human lung adenocarcinoma cells A549, individually and in combination, using colorimetric MTT test (570 nm). Concentrations that decreased the cell viability by 50% (IC50) were as it follows: pure OTA (53 μg/mL) ; AO-OTA+ (mass concentration 934 μg/mL corresponds to 10.5 μg/mL of OTA in spore extract) ; and 2126 μg/mL for EH. Highest applied concentration of AO-OTA- spore extract (4940 g/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Combinations EH + AO-OTA+ and EH + AO-OTA- applied in subtoxic concentrations showed dominant additive interactions. Also, synergistic effect was obtained for one combination of EH and AO- OTA+. Despite the low calculated daily intake of OTA by inhalation, chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose significant threat to human health due to their possible additive and/or synergistic interactions.

Mikotoksini su toksični proizvodi plijesni koji mogu biti karcinogeni, genotoksični, mutageni i teratogeni. Pri umjerenoj klimi najčešće se u hrani za životinje nalaze aflatoksini, trihoteceni, fumonizini, zearalenon i okratoksin A, a sintetiziraju ih najčešće plijesni rodova Aspergillus, Penicillium i Fusarium. Pogodovni čimbenici za nastanak mikotoksina na žitaricama su zaraza toksinogenim plijesnima, ugljikohidratna podloga i učestale vegetacijske godine s izraženijim promjenama temperature i količine oborina. Nastanku mikotoksina na žitaricama u polju pogoduje korištenje hibrida koji daju visoke prinose uz smanjenu otpornost na stres, reducirana osnovna obrada, povećana gustoća sklopa, uzgoj u monokulturi i povećana zakorovljenost. Kontaminiranosti pogoduje mehanizirana žetva s povećanim lomom zrna, vlažnost zrna, naseljavanje štetnika tijekom skladištenja i skladištenje usjeva u velikim skladišnim prostorima što neminovno dovodi do miješanja zdravog i zaraženog usjeva. Hrvatska agencija za hranu provela je opsežno istraživanje s ciljem prikazivanja stanja zaraženosti dominantnih krmiva (sačma soje, kukuruz) koja količinski čine najveći udio u svim krmnim smjesama kako bi se procijenio rizik za životinje, a posljedično i za ljude. Uzorci sačme soje i kukuruza sakupljeni su kod 20 najvećih proizvođača hrane za životinje u Republici Hrvatskoj. Obim i intenzitet zaraženosti aflatoksinom, zearalenonom, deoksinivalenolom i fumonizinom B1 analiziran je na 300 uzoraka (181 kukuruza, 119 sačma soje) iz vegetacijske godine 2011. metodom ELISA (Enzyme-Linked Immunosorbent Assay). U kukuruzu je koncentracija aflatoksina iznad granice detekcije nađena u 23 % uzoraka (raspon 1, 0-10, 0 μg/kg), deoksinivalenola u 25 % (55- 500 μg/kg), fumonizina u 55 % (220-2000 μg/kg), a zearalenon je nađen samo u jednom uzorku. U sačmi soje aflatoksini su nađeni u 16 % uzoraka (raspon 1, 0-2, 0 μg/kg), deoksinivalenol u 30 % (55-200 μg/kg), zearalenon u 29 % (16-100 μg/kg) dok niti jedan uzorak nije bio kontaminiran fumonizinima iznad granice detekcije. Dobiveni rezultati ukazuju na učestalu kontaminaciju mikotoksinima. Koncentracije aflatoksina B1 znatno su niže od maksimalno dozvoljenih koncentracija propisanih Pravilnikom o nepoželjnim tvarima u hrani za životinje (NN 80/2010). I ostali analizirani mikotoksini nađeni su u koncentracijama koje su niže od onih koje je preporučila Europska komisija.

Ochratoxin A (OTA) and citrinin (CTN) are secondary metabolites of moulds that can be found in cereals and other commodities all over the world. The mechanism of OTA and CTN toxicity is not fully understood, although the involvement of oxidative stress is suspected. Resveratrol is antioxidative compound naturally found in red wine. In our study the adult male Wistar rats were treated with OTA (0, 125, 250 ug/kg, daily for 21 days) with or without CTN (20 mg/kg for two days two last days of OTA treatment). Groups of animals treated with OTA with or without CTN were also treated with resveratrol (20 mg/kg for 21 days). 24 hours after the last treatment, samples of blood were collected and catalytic activities of antioxidant enzymes were measured spectrophotometrically. Superoxide dismutase (SOD) and catalase (Cat) were measured in plasma and erythrocytes of treated animals and compared with control animals, while activity of glutathione peroxidise (GPx) was measured in whole blood. Activity of SOD was decreased in plasma of animals treated with higher dose of OTA (250 ug/kg), lower dose of OTA (125 ug/kg)+CTN, as well as with OTA (250 ug/kg)+CTN+RSV (p

In order to protect European consumers from the exposure of all kinds of possible lesions and diseases of food origin the European Commission (EC) founded the independent scientific advisory board called European Food Safety Authority (EFSA). In 2003, EFSA organized Panel on Contaminants in the Food Chain (CONTAM Panel) with the mandate to deliver scientific opinions on “contaminants in food and feed, associated areas and undesirable substances such as natural toxicants, mycotoxins and residues of non-authorized substances not covered by another Panel”. CONTAM Panel receives the requests from the EC (95 %), Member States (1 %) and the European Parliament (1 %). Since 2003 CONTAM Panel produced 107 opinions (55 on contaminants in food, 43 in feed and 9 combined assessments in food and feed), 15 out of them on mycotoxins. Evaluation of risk assessment of mycotoxins is specific because of their natural origin. Mycotoxins are products of moulds that contaminate food and feed all over the world. Low concentrations of mycotoxins are unavoidable in commodities of plant and animal origin. There are about 400 known mycotoxins, but only some of them are known to be toxic, mutagen or carcinogen in animals and humans. Mycotoxins have rather different chemical structure and their metabolism, which determines their untoward effects in organisms in most cases are not elucidated. They are stable compounds, resistant to heating and food production does not destroy them although in some cases it decreases their concentration. Risk assessment is the procedure of evaluation whether or not a hazardous chemical contaminant or undesirable substance in food is likely to be associated with adverse health effects in the population. Risk assessment is based on two components: the potential of substance to cause adverse effects (hazard) and knowledge about the human exposure to the substance. In the procedure of hazard identification in case of mycotoxins all available data regarding the evaluated compound in the scientific literature are taken into account. These data include reports on the human and animal intoxication, studies on experimental animals as well as on the cell cultures. For the evaluation of human exposure CONTAM Panel relies on data on occurrence of contaminants submitted by Member States upon the calls for data. Risk assessment for animals is based on less reliable feed consumption data and therefore contains more uncertainties.

Ageing is characterized by an impaired functional and structural integrity of mammalian kidneys. A diminished handling of organic compounds, drugs, and water, as observed in the kidneys of older humans and experimental animals, indicates that ageing may affect the expression and/or activity of the renal transporters that mediate excretion and/or reabsorption of organic anions and cations, glucose, and water. We compared the expression of relevant transporters in the kidneys of 3-month- old and 24-month-old male Wistar rats, including Na/K-ATPase, organic anion transporters Oat1 (Slc22a6), Oat2 (Slc22a7), Oat3 (Slc22a8), and Oat5 (Slc22a19), organic cation transporters Oct1 (Slc22a1) and Oct2 (Slc22a2), glucose transporters Sglt1 (Slc5a1) and Sglt2 (Slc5a2), and water channels AQP1 and AQP2. These transporters/channels reside in specific cell membrane domains along the nephron. Protein expression was studied by immunofluorescence cytochemistry (IC) in tissue cryosections and Western blotting (WB) in isolated membranes, whereas expression of mRNA in the tissue was tested by RT-PCR. The protein and mRNA expression of Oat1, Oat3, Oct1, Oct2, and AQP1 was significantly weaker in senescent animals. However, the expression of Na/K-ATPase, Oat2, Oat5, Sglt1, Sglt2, and AQP2 at both protein and/or mRNA levels was not affected by age. Our findings imply that the cellular uptake and excretion of endogenous and/or exogenous compounds in the kidneys can be compromised due to the ageing-related downregulation of certain renal transporters. This can lead to an impaired ability to maintain the homeostasis of specific compounds and an increased susceptibility to drug interaction and toxicity in kidneys and other organs.

Mikotoksini su toksični proizvodi plijesni koji mogu biti karcinogeni, genotoksični, mutageni i teratogeni. Pri umjerenoj klimi najčešće se u hrani za životinje nalaze aflatoksini, trihoteceni, fumonizini, zearalenon i okratoksin A, a sintetiziraju ih najčešće plijesni rodova Aspergillus, Penicillium i Fusarium. Pogodovni čimbenici za nastanak mikotoksina na žitaricama su zaraza toksinogenim plijesnima, ugljikohidratna podloga i učestale vegetacijske godine s izraženijim promjenama temperature i količine oborina. Nastanku mikotoksina na žitaricama u polju pogoduje korištenje hibrida koji daju visoke prinose uz smanjenu otpornost na stres, reducirana osnovna obrada, povećana gustoća sklopa, uzgoj u monokulturi i povećana zakorovljenost. Kontaminiranosti pogoduje mehanizirana žetva s povećanim lomom zrna, vlažnost zrna, naseljavanje štetnika tijekom skladištenja i skladištenje usjeva u velikim skladišnim prostorima što neminovno dovodi do miješanja zdravog i zaraženog usjeva. Hrvatska agencija za hranu provela je opsežno istraživanje s ciljem prikazivanja stanja zaraženosti dominantnih krmiva (sačma soje, kukuruz) koja količinski čine najveći udio u svim krmnim smjesama kako bi se procijenio rizik za životinje, a posljedično i za ljude. Uzorci sačme soje i kukuruza sakupljeni su kod 20 najvećih proizvođača hrane za životinje u Republici Hrvatskoj. Obim i intenzitet zaraženosti aflatoksinom, zearalenonom, deoksinivalenolom i fumonizinom B1 analiziran je na 300 uzoraka (181 kukuruza, 119 sačma soje) iz vegetacijske godine 2011. metodom ELISA (Enzyme-Linked Immunosorbent Assay). U kukuruzu je koncentracija aflatoksina iznad granice detekcije nađena u 23 % uzoraka (raspon 1, 0-10, 0 μg/kg), deoksinivalenola u 25 % (55- 500 μg/kg), fumonizina u 55 % (220-2000 μg/kg), a zearalenon je nađen samo u jednom uzorku. U sačmi soje aflatoksini su nađeni u 16 % uzoraka (raspon 1, 0-2, 0 μg/kg), deoksinivalenol u 30 % (55-200 μg/kg), zearalenon u 29 % (16-100 μg/kg) dok niti jedan uzorak nije bio kontaminiran fumonizinima iznad granice detekcije. Dobiveni rezultati ukazuju na učestalu kontaminaciju mikotoksinima. Koncentracije aflatoksina B1 znatno su niže od maksimalno dozvoljenih koncentracija propisanih Pravilnikom o nepoželjnim tvarima u hrani za životinje (NN 80/2010). I ostali analizirani mikotoksini nađeni su u koncentracijama koje su niže od onih koje je preporučila Europska komisija.

The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on sterigmatocystin (STC) in food and feed. STC is a polyketide mycotoxin that shares its biosynthetic pathway with aflatoxins. Following an EFSA call for data, analytical results from 247 food and 334 feed samples were submitted. In food, analytical results on STC were reported to be all below the limit of detection or limit of quantification. In feed, only four quantified results were reported. Therefore, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) concluded that the available occurrence data are too limited to carry out a reliable human and animal dietary exposure assessment. Acute oral toxicity of STC is relatively low, and liver and kidneys are the target organs. STC is mutagenic in both bacterial and mammalian cells after metabolic activation and forms DNA adducts. Tumourigenicity has been observed after oral, intraperitoneal, subcutaneous and dermal administration resulting in hepatocellular carcinomas, haemangiosarcomas in the liver, angiosarcomas in brown fat and lung adenomas. Since no exposure data were available, the margin of exposure approach for substances that are genotoxic and carcinogenic could not be applied for STC, and thus the CONTAM Panel could not characterise the risk for human health. Regarding animals, the Panel noted that STC is hepatotoxic in poultry and pigs, and nephrotoxic in poultry and toxic in several fish species. However, in the absence of exposure data for livestock, fish and companion animals, and given the limited knowledge on the adverse effects of STC, the CONTAM Panel could not characterise the risk for animal health. More occurrence data on STC in food and feed need to be collected to allow dietary exposure assessment. For food, methods with a limit of quantification of less than 1.5 μg/kg should be applied.

Aging is an inevitable physiological process characterized by a gradual decline in the functional capabilities regarding personal and environmental needs. In the absence of underlying diseases that accelerate aging (diabetes, cancer, etc…), kidneys are crucial organs for „healthy aging“. Aging kidneys exhibit functional, structural, and genomic losses. The impaired secretory and reabsorptive functions in handling water, organic compounds, drugs, toxins, and the increased tendency to drug-drug interactions, drug-induced adverse reactions, and drug (nephro)toxicity indicate that aging affects various renal transporters, but relevant studies on their activity and/or expression are missing. To initiate such studies, we compared various biochemical, immunochemical, and mRNA parameters in young (Y ; 3 months old) and old (O ; 2 years old) male rats. In comparison with Y, the O rats had higher body mass, lower blood testosterone, lower urine flow and excretion of inorganic ions, and unchanged excretion of glucose. The O rats exhibited lower expression of organic anion (Oat1, Oat3) and cation (Oct1, Oct2) transporters, Na+-phosphate cotransporter NaPi2, and water channel AQP1. The expression of Na/K-ATPase, Oat2, Oat5, Na+-glucose cotransporters (Sglt1, Sglt2), and AQP2 was similar in Y and O rats. We concluded that aging in rats selectively downregulates the renal transporters that are androgen-dependent.

Mycotoxicoses are acute or chronic diseases of humans and animals caused by mycotoxins, toxic compounds produced by moulds. Of about 400 known mycotoxins only a small number are known to cause mycotoxicoses in humans. Organs that are most targeted are those in which mycotoxins are metabolised, that is, the liver and kidneys, but the lesions may affect the neurological, respiratory, digestive, haematological, endocrine, and immune systems as well. The epidemics of mycotoxicoses are often connected with times of famine, when population consumes food that would not be consumed in normal circumstances. Mycotoxicoses have infl uenced human history, causing demographic changes, migrations, or even infl uencing the outcomes of wars. Fortunately, epidemics affecting so many persons and with so many fatalities belong to the past. Today they only appear in small communities such as schools and factory canteens. This paper presents epidemics and pandemics of mycotoxicoses that infl uenced human history.

Individual differences in DNA damage response and lipid peroxidation level after exposure to ionising radiation (IR) are also due to single nucleotide polymorphism (SNP) in genes involved in DNA repair pathways. In order to estimate the influence of SNP on malondyaldehyde (MDA), as a product of lipid peroxidation, 58 individuals professionally exposed to low doses of IR were genotyped for hOGG1 (8-oxoguanined DNA glycosylase, Ser 326Cys) and XRCC1 (x-ray repair cross-complementing protein-group 1, Arg194Trp). Blood samples were irradiated with 2 Gy to 4 Gy (CO60). One or two polymorphic alleles were combined in one group (POL). T-test showed no significance between groups before and after radiation in MDA (Mean ± S.E.): (7.30±0.39), (7.36±0.46), 7.02±0.38) μmol L-1. After comparison before, and after 2 Gy and 4 Gy, results were XRCC1 POL: 7.02±0.47), (7.20±0.52), (7.04±0.47) μmol L-1 ; XRCC1 HO: (7.74±0.70), (7.61±0.87), (6.99±0.65) μmol L-1) ; hOGG1 POL: (7.80±0.53), (7.1±0.70), (6.30±0.56) μmol L-1 ; hOGG1 HO: 6, 90±0.57), (7.58±0.63), (7.61±0.50) μmol L-1. We found significant correlation among XRCC1 POL before and after both doses, while for XRCC1 HO there was correlation between the results before and after 2 Gy, and between 2 Gy and 4 Gy. HOGG1 POL correlated before and after 4 Gy ; and between the results before and after 2 Gy, and between 2 in all three groups. HOGG1 POL correlated before and after 4 Gy ; and between 2 Gy and 4 Gy radiation. hOGG1 HO correlated in all three groups. After t-test and Mann Whitney U-test comparison, there was a difference between the groups but it was not statistically significant. ANOVA showed an almost significant difference for hOGG1 groups (p=0.069), but not for XRCC1. Our conclusion is that the group was simply too small to estimate the real risk of exposure and conclude whether MDA is or is not a good biomarker for estimating occupational exposure to IR.

Toksini plijesni, zvani mikotoksini, često se nalaze u hrani i krmivu u svim krajevima svijeta. Mikotoksini mogu na različite načine štetiti ljudskom zdravlju jer osim bolesti uzrokovanih njihovom toksičnošću koje se u ljudi i životinja nazivaju mikotoksikozama, mogu biti karcinogeni, genotoksični i mutageni. Akutne mikotoksikoze, koje nastaju pri izloženosti ljudi i životinja velikoj koncentraciji mikotoksina, česte su u zemljama s tropskom klimom gdje su povoljniji uvjeti za rast plijesni i sintezu mikotoksina. U zemljama s umjerenom klimom akutne su mikotoksikoze velikih razmjera danas rijetke, iako su u povijesti nekoliko puta poharale stanovništvo Europe. Kronične su mikotoksikoze posljedica dugotrajne izloženosti nekom mikotoksinu i također su češće u tropskim zemljama. Povezanost nastanka bolesti s izloženošću nekom mikotoksinu vrlo je teško dokazati jer su ljudi i životinje izloženi istovremeno različitim mikotoksinima. Ljudi su u umjerenim klimatskim zonama, pa i u Hrvatskoj, često izloženi mikotoksinima fumonizinu B1 (FB1), okratoksinu A (OTA) i zearalenonu (ZEA), dok je izloženost aflatoksinu B1 (AFB1) rjeđa. Na pokusnim je životinjama dokazano da je ciljni organ toksičnog djelovanja za AFB1 jetra, za OTA bubreg, a za ZEA reproduktivni sustav. Vrlo je vjerojatno da su to ciljni organi njihovog djelovanja i kod ljudi. FB1 pokazuje znatnu razliku ciljnog organa kod različitih životinjskih vrsta, pa je teško pretpostaviti koji je njegov ciljni organ u ljudi. Dokazano je da izloženost mikotoksinu AFB1 uzrokuje maligne tumore u ljudi, a prema istraživanjima na pokusnim životinjama pretpostavlja se da bi FB1 i OTA mogli biti karcinogeni i za ljude. U radu su opisani akutni i kronični učinci mikotoksina AFB1, FB1, OTA i ZEA jer se ti mikotoksini mogu naći u hrani i krmivu u Hrvatskoj pa su ljudi trajno izloženi tim mikotoksinima.

Humans and animals are continuously exposed to mycotoxins ochratoxin A (OTA) and citrinin (CTN) which are often found together because they are produced by the same strains of molds . One of the possible mechanisms of their toxicity is the increase of reactive oxygen species and induction of oxidative stress. It is already known that one of the best known antioxidants is resveratrol (RSV) – a polyphenol found in grapes and red wine. The aim of our study was to check whether RSV treatment could decrease the oxidative stress in OTA and CTN treated animals. In this study adult male Wistar rats were given orally daily for 21 days with RSV and combination of RSV, OTA and CTN. The results were compared with those obtained in animals given the combination of OTA and CTN. . All animals were sacrificed 24 hours after the last treatment and liver tissue was collected and frozen until the analysis of malondialdehyde (MDA). The concentration of MDa in liver of rats treated with RSV was lower than in controls given 51mM NaHCO3 (72.40% of MDA concentration of controls). MDA concentration in liver of animals treated with mycotoxins and RSV was higher than in rats treated only with RSV (111.60%, compared to RSV treated), but lower than in control rats treated with 51mM NaHCO3 (80.80%, compared to controls). These results indicate that RSV may significantly reduce the oxidative lipid damage caused by OTA and CTN.

Najvažnijim mikotoksinima hrane za životinje smatraju se aflatoksini, trihoteceni, fumonizini, zearalenon i okratoksin A, a sintetiziraju ih najčešće plijesni rodova Aspergillus , Penicillium i Fusarium. Razvoj i intenzitet pojavnosti mikotoksina uvjetovana je mikroklimatskim čimbenicima u različitim dijelovima svijeta, te uz pogodovne ekološke prilike trajno ugrožavaju proizvodnju i skladištenje hrane za životinje. Pogodovni čimbenici pojačanog razvoja mikotoksina su toksinogene plijesni na odgovarajućoj podlozi (žitarice, ugljikohidratna podloga) i učestale vegetacijske godine s izraženijim promjenama temperature i količinom oborina. Nadalje, današnji hibridi selekcionirani su na visoke prinose, a smanjene biološke otpornosti na stres, i zbog toga lakše prijemčivi na razvoj mikotoksina. Agrotehnika koja podrazumijeva reduciranu osnovnu obradu, povećanu gustoću sklopa, uzgoj u monokulturi te povećanu zakorovljenost pogoduju razvoju mikotoksina još na polju. Mehanizirana žetva pri povećanoj vlazi i povećanom lomu zrna mogu biti tzv. „ulazna vrata“ izvoru zaraze. Skladištenje usjeva u velikim skladišnim prostorima neminovno dovodi do miješanja zdravog i zaraženog usjeva, a nije rijetko korištenje improviziranih skladišta s povećanim naseljavanjem štetnika. Osim toga, geografska rasprostranjenost mikotoksina vezana je uz klimatske uvjete rasta pojedinih vrsta plijesni. Mikotoksini mogu izazivati niz štetnih učinaka na ljude i životinje (karcinogenost, imunotoksičnost, probavne smetnje, neurotoksičnost, hepatotoksičnost, nefrotoksičnost, reproduktivna i razvojna toksičnost, i dr.), pri čemu često mogu istovremeno djelovati na različite načine i na više ciljnih mjesta u organizmu, što ovisi o samoj tvari, dozi i vremenu izloženosti. Višegodišnja istraživanja u Hrvatskoj pokazuju da mikotoksini učestalo kontaminiraju žitarice, te je sustavna kontrola mikotoksina u hrani i hrani za životinje neophodna kako bi se izbjegli negativni učinci na zdravlje kao i ekonomski gubici u poljoprivredi. U Hrvatskoj se na temelju godišnjeg plana uzorkovanja provodi sustavna kontrola namirnica biljnog i životinjskog podrijetla na mikotoksine u ovlaštenim laboratorijima za kontrolu zdravstvene ispravnosti hrane. Kako bi procijenila rizik za životinje, a posljedično i za ljude, Hrvatska agencija za hranu je provela istraživanje pod nazivom „Studija pojavnosti mikotoksina u krmivima i krmnim smjesama u RH“ koje je, uz ostale stručne i znanstvene reference, služilo kao temelje za izradu ovog znanstvenog mišljenja. Dobiveni rezultati studije ukazuju na učestalu kontaminaciju pretraživanim mikotoksinima, ali u koncentracijama znatno nižim od maksimalno dozvoljenih koncentracija aflatoksina B1 propisanih Pravilnikom o nepoželjnim tvarima u hrani za životinje (NN 80/2010). Također, ostali analizirani mikotoksini (zearalenon, fumonizini, deoksinivalenol) utvrđeni su u koncentracijama koje su ispod preporuka Europske komisije. Rizik za ciljne životinje konzumacijom krmiva pripravljenih od analiziranih sirovina – minimalan je. Rizik za potrošače proizvoda animalnog podrijetla koji potječu od životinja hranjenih ovim krmivima zanemariv je zbog niske razine kontaminacije hrane za životinje i općenito niskog prelaska ispitivanih mikotoksina u jestiva tkiva. S obzirom na donesene zaključke dane su i preporuke za kontinuirano praćenje i analizu ključnih sirovina i gotovih krmih smjesa u okviru specifičnih projekata kao i u okviru Državnog programa monitoringa rezidua i Državnog plana službenih kontrola i monitoringa hrane za životinje te uključivanje okratoksina A, T-2 i HT- 2 toksina u buduća istraživanja pri čemu je potrebno analizirati veći broj različitih sirovina i krmnih smjesa. Analiza specifičnih krmnih smjesa za pojedine životinje treba biti usredotočena na mikotoksine na koje su te životinje posebno osjetljive.

Citrinin is a mycotoxin produced by several species of the genera Aspergillus, Penicillium and Monascus and occurs mainly in stored grains. The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on the health risks from citrinin in food and feed. For the exposure assessment, occurrence data submitted to EFSA in response to a call for data and from the literature were examined. Followin an EFSA call for data, analytical results from only 30 samples were submitted by one Member State. The reported citrinin concentrations varied widely in grains both when intended for food (up to 420 µg/kg) and for feed (up to 998 µg/kg), but in general, a high proportion (up to > 90 %) of left censored data were observed. The available occurrence data either submitted to EFSA in response to the call for data or from the literature were not adequate to carry out human and animal dietary exposure assessments. Hazard characterization revealed that citrinin is nephrotoxic and a no-observed-adverse-effect level (NOAEL) of 20 µg/kg body weight (bw) per day was identified from a 90-day study in rats. Due to the limitations and uncertainties in the database, the derivation of a health-based guidance value was not considered appropriate but a level of no concern for nephrotoxicity of 0.2 µg/kg bw per day could be determined. However, based on the available data a concern for genotoxicity and carcinogenicity could not be excluded at the level of no concern for nephrotoxicity. In the absence of adequate exposure data, characterisation of the risk of citrinin as a food contaminant was based on the estimate of the citrinin concentrations in grains and grain-based products that would result in an exposure equal to the level of no concern for nephrotoxicity. For high consuming toddlers, other children and adults this citrinin concentration is between 9 and 53 µg citrinin/kg and between 19 and 100 µg citrinin/kg for average consumers, respectively. For animals, risk characterisation was based on the estimate of the citrinin concentration in grains that would result in exceedance of the NOAEL of 20 µg/kg b.w. per day for pigs, which ranged between 640 and 1 173 µg/kg. The CONTAM Panel concluded that the impact of uncertainties on the risk assessment is large, and more data regarding the toxicity and the occurrence of citrinin in food and feed in Europe are needed to enable refinement of the risk assessment of citrinin.

Although the most threatening features of mycotoxins are their carcinogenicity and mutagenicity, they may also cause diseases, called mycotoxicoses. Mycotoxicoses, that may have even fatal outcome, occur mostly as the consequence of the ingestion of contaminated food, but the other ways of exposure, such as dermal and respiratory, are also possible. Mycotoxicoses are more frequent in tropical zones where climatic conditions favor the moulds growth and mycotoxins production but due to the international trade of cereals, the possibility of human exposure to these toxins increases also in mild climatic zones. Human mycotoxicoses are rarely diagnosed if a single person or a small group of persons is affected because unfortunately medical doctors are not educated to recognize them. Out of about 400 known mycotoxins only a small number of them are known to cause mycotoxicoses in humans. The target organs of mycotoxins are most frequently liver and kidney where they are detoxified, but the lesions may be seen in all other organs. The history of mankind was sometimes severely affected by the epidemics of mycotoxicoses because several times they spread over the whole regions or several countries (such as ergotism in the Middle Ages, trichothecene mycotoxicoses that caused the depopulation of the whole regions of Russia and the several fatal mycotoxicoses that appeared after the separation of Pakistan from India). Such epidemics with very high percentage of fatalities are nowadays rare, but particularly in tropical zones they appear in small communities such as schools and factory canteens. Although it seemed that with the better control of food production epidemics of mycotoxicoses should disappear, recent severe epidemics in 21st century indicate that they will appear as long as the wars, famine and other catastrophes will be present.

Ochratoxin A (OTA) is a mycotoxin produced by Penicillium and Aspergillus moulds. It contaminates cereals, cereal products, coffee, nuts, meat, milk and other commodities like wine, beer and cheese. Studies on laboratory and domestic animals have shown that OTA is nephrotoxic, carcinogenic, genotoxic, and immunotoxic. One of the possible mechanisms of OTA toxicity is the production of free radicals and consequent oxidative stress. In this study oxidative stress in plasma and different tissues of rats treated with OTA was investigated by measuring the levels of glutathione (GSH) with spectrophotometric method and malondialdehyde (MDA) with HPLC method. Adult male Wistar rats in groups by six were treated orally with NaHCO3 (control group), 250 g OTA/kg b.w. and 500 g OTA/kg b.w. every day for eight days. Animals were sacrificed 24 hours after the last treatment and plasma, liver, kidney and brain tissues were collected and frozen until analyzed. Concentrations of GSH measured in kidney of controls (196.2 nmol/g) and treated animals (190.5 and 201.9 nmol/g in animals treated with 250 g OTA/kg b.w. and 500 g OTA/kg b.w, respectively) was not statistically significant. Although kidney is the target organ of OTA toxicity, the lack of the effect of OTA treatment on kidney GSH concentration may be explained with the relatively short time of exposure. In liver of animals given 0, 250, 500 g OTA/kg b.w, GSH concentrations were 89.2, 71.5 and 62.4 nmol/g tissue, respectively, and in plasma 11.0, 7.3 and 8.5 µmol/L, respectively. The effect of OTA on plasma GSH concentration is probably the consequence of effect of OTA on liver, the most important detoxifying organ. Contrary to expectations, OTA treatment caused slight increase of GSH in brain tissue of rats. The concentration of MDA, which is the final product of lipid peroxidation, in brain and plasma was significantly higher in OTA- treated animals than in controls, confirming that OTA increases lipid peroxidation.

The aim of this study was to determine the cytotoxic and genotoxic combined effects of three mycotoxins : citrinin CIT, ochratoxin A OTA and fumonisin FB. The studies were conducted (i) in cells culture and (ii) in vivo on rat or pig fed OTA and/or CIT ; and/or FB. The decrease of viability (measured by MTT test) of human kidney cells (HK2) induced by OTA is considerably enhanced by the simultaneous presence of CIT & FB. Expression of biotransformation enzymes (CYP, COX, LIPOX) in HK2 is differently modulated depending of the combination. Induction of COX2 and LIPOX by CIT increased genotoxicity of OTA measured by DNA adducts formation detected by P32 post-labeling. DNA adducts patterns of rat kidney after a 3-weeks feeding by either CIT alone or OTA alone or both together, are similar to those obtained on cell cultures. The main OTA DNA-adduct, found in human tumours, identified as C8 dG-OTA is increased by simultaneous presence of CIT and OTA. In the same way, in in vivo studies on rat and pig fed simultaneously by OT and FB in feed we observed increased OTA specific DNA adducts including C-C8dG OTA adduct and the both OTHQ related adduct. These specific adducts are found in human urothelial tumours, notably in Balkan regions. This data is particularly interesting as we previously shown that families suffering BEN eat food more frequently contaminated by OTA, CIT and FB. In blood cell and kidney of human and rat we isolated OTA and several OTA derivatives identified by HPLC ms/ms, including quinone OTA (OTHQ), GSH-OTA, GSH-OTHQ, DC-OTHQ. The data indicate clearly that exposure to low concentration of mycotoxin which is considered as safe when they are present together can lead to dramatic effect. Until now, regulation does not take into account co-contamination.

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins both produced by several fungal strains belonging to the genera Penicillium and Aspergilus. Both mycotoxins contaminate cereals and are usually found together. The mechanism of OTA toxicity is much studied in contrast to the mechanism of CTN toxicity. Although they are frequently found together, studies of their common toxicity are rare and the results controversial. In this study adult male Wistar rats in groups by six were treated orally as follows: 1. OTA (125 µg/kg b.w. daily for 21 days), 2. CTN (20 mg/kg b.w. for two days), 3. OTA (125 µg/kg b.w. daily for 21 days) + CTN (20 mg/kg b.w. for the last two days of OTA treatment), 4. controls (treated with vehicle - 51mM NaHCO3). Animals were sacrificed 24 hours after the last treatment ; brain tissue was collected and frozen until parameters of oxidative stress were analyzed. The concentration of glutathione (GSH) and malondialdehyde (MDA) was measured using spectrophotometric and HPLC methods, respectively. OTA and OTA+CTN treatment caused insignificant decrease in concentration of GSH as compared to controls (0.46±0.04 and 0.40±0.04 µmol/g tissue ; 0.53±0.07 and 0.47±0.05 µmol/g tissue) while CTN treatment did not change GSH concentration. The concentration of MDA was higher in OTA- (5.27±0.68 and 5.42±1.01 nmol/g tissue) and CTN-treated animals (6.62±0.22 and 8.25±0.93 nmol/g tissue) than in controls. When animals were treated with both mycotoxins, the MDA concentration increased significantly (6.64±0.56 and 8.46±0.87 nmol/g tissue ; P