Search

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.

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).