Maja Šegvić Klarić

Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzyme-linked immunosorbent assay and thin-layer chromatography.

Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer Chromatography Aspergillus, Penicillium, and Fusarium species frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 μg kg-1), followed by FBs (27%, 3690 μg kg-1), AFs (24.3%, 4.6 μg kg-1), and OTA (16.2%, 9.8 μg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases. Određivanje aflatoksina, okratoksina A, fumonizina i zearalenona u žitaricama i krmivu primjenom kompetitivnoga direktnog imunoenzimatskog testa (CD-ELISA) i tankoslojne kromatografije (TLC) Vrste plijesni iz rodova Aspergillus, Penicillium i Fusarium česti su kontaminanti usjeva te na takvim supstratima tvore mikotoksine. Stoga su žitarice i krmiva često kontaminirana aflatoksinima (AFs), okratoksinom A (OTA), fumonizinima (FBs) i zearalenonom (ZEA) u različitim koncentracijama ovisno o mikroklimatskim uvjetima na polju i u skladištu. Konzumiranje hrane kontaminirane mikotoksinima često je povezano s akutnim ili kroničnim trovanjima, ali i s razvojem karcinoma. Cilj ovog rada bio je odrediti istodobnu pojavnost AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3) i ZEA u uzorcima žitarica i krme (N=37) koji su nasumično skupljeni u individualnim domaćinstvima na području endemske nefropatije (EN) u Hrvatskoj (2007). Za određivanje navedenih mikotoksina korišten je kompetitivni direktni ELISA-test (CD-ELISA) u kombinaciji s tankoslojnom kromatografijom (TLC). Najzastupljeniji mikotoksin bio je ZEA (92 %, srednja koncentracija 318.3 μg kg-1), nakon čega slijede FBs (27 %, 3690 μg kg-1), AFs (24.3 %, 4.6 μg kg-1) te OTA (16.2 %, 9.8 μg kg-1). Koncentracije AFs, FBs i ZEA određene CD-ELISA-testom statistički značajno koreliraju s rezultatima dobivenim s TLC. OTA je potvrđen metodom TLC samo u jednom uzorku zbog visokog limita detekcije. Dokazane koncentracije su ispod razina dopuštenih za krmiva, dok je 29 % uzoraka žitarica sadržavalo FBs, OTA ili ZEA u koncentracijama iznad dopuštenih u hrani za ljude. Kokontaminacija s dvama odnosno trima toksinima varirala je između 4.2 % i 54 % odnosno između 4.2 % i 7.6 %. Dugotrajni unos AFs, OTA, FBs i ZEA putem hrane može povećati rizik od razvoja različitih kroničnih bolesti zbog njihova mogućega sinergističkog djelovanja.

Deleterious Effects of Mycotoxin Combinations Involving Ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with carcinogenic properties. Its presence was detected in various foodstuffs all over the world but with significantly higher frequency and concentrations in areas with endemic nephropathy (EN). Even though food is often contaminated with more than one mycotoxin, earlier studies focused on the occurrence and toxicology of only OTA. Only a limited number of surveys showed that OTA co-occurs in food with mycotoxins (citrinin-CIT, penicilic acid, fumonisin B1-FB1, aflatoxins-AF) which exert nephrotoxic, carcinogenic or carcinogen-promoting activity. This review summarises the findings on OTA and its co-occurrence with the mentioned mycotoxins in food as well as experimental data on their combined toxicity. Most of the tested mycotoxin mixtures involving OTA produced additive or synergistic effects in experimental models suggesting that these combinations represent a significant health hazard. Special attention should be given to mixtures that include carcinogenic and cancer-promoting mycotoxins.

Adverse Effects Of Combined Mycotoxins / Štetni Učinci Kombiniranih Mikotoksina

This article brings an overview of mycotoxin co-occurrence in foods in Croatia and neighbouring countries and experimental data from mycotoxin interaction studies involving Fusarium toxins, ochratoxin A (OTA), and aflatoxin B1 (AFB1). Only a few studies of combined mycotoxin toxicity have employed a mathematical/statistical design, while others have used common statistics in order to compare the effects of mycotoxin mixtures with effects of single toxins. So far, most studies have observed additive or synergistic effects, suggesting that these mixtures pose a signifi cant threat to human and animal health.Abstract This article brings an overview of mycotoxin co-occurrence in foods in Croatia and neighbouring countries and experimental data from mycotoxin interaction studies involving Fusarium toxins, ochratoxin A (OTA), and aflatoxin B1 (AFB1). Only a few studies of combined mycotoxin toxicity have employed a mathematical/ statistical design, while others have used common statistics in order to compare the effects of mycotoxin mixtures with effects of single toxins. So far, most studies have observed additive or synergistic effects, suggesting that these mixtures pose a significant threat to human and animal health U članku je prikazan pregled mikotoksinske kokontaminacije hrane u Hrvatskoj i susjednim zemljama te njihovih kombiniranih toksičnih učinaka na različitim eksperimentalnim modelima. Pritom su obuhvaćene studije interakcija koje uključuju fuzarijske mikotoksine, okratoksin A (OTA) i aflatoksin B1 (AFB1). Nekoliko je takvih istraživanja napravljeno na temelju matematičko-statističkog modela, dok je većina studija primijenila jednostavnu statističku analizu koja omogućava usporedbu učinaka kombiniranih mikotoksina u odnosu na učinke pojedinačnih. Općenito, većina dosadašnjih studija pokazuje da kombinacije mikotoksina u biološkom sustavu imaju sinergistički ili barem aditivni učinak, što znači da su velik rizik za zdravlje ljudi i životinja.

Adverse Effects of Combined Mycotoxins

This article brings an overview of mycotoxin co-occurrence in foods in Croatia and neighbouring countries and experimental data from mycotoxin interaction studies involving Fusarium toxins, ochratoxin A (OTA), and aflatoxin B1 (AFB1). Only a few studies of combined mycotoxin toxicity have employed a mathematical/statistical design, while others have used common statistics in order to compare the effects of mycotoxin mixtures with effects of single toxins. So far, most studies have observed additive or synergistic effects, suggesting that these mixtures pose a signifi cant threat to human and animal health.Abstract This article brings an overview of mycotoxin co-occurrence in foods in Croatia and neighbouring countries and experimental data from mycotoxin interaction studies involving Fusarium toxins, ochratoxin A (OTA), and aflatoxin B1 (AFB1). Only a few studies of combined mycotoxin toxicity have employed a mathematical/ statistical design, while others have used common statistics in order to compare the effects of mycotoxin mixtures with effects of single toxins. So far, most studies have observed additive or synergistic effects, suggesting that these mixtures pose a significant threat to human and animal health U članku je prikazan pregled mikotoksinske kokontaminacije hrane u Hrvatskoj i susjednim zemljama te njihovih kombiniranih toksičnih učinaka na različitim eksperimentalnim modelima. Pritom su obuhvaćene studije interakcija koje uključuju fuzarijske mikotoksine, okratoksin A (OTA) i aflatoksin B1 (AFB1). Nekoliko je takvih istraživanja napravljeno na temelju matematičko-statističkog modela, dok je većina studija primijenila jednostavnu statističku analizu koja omogućava usporedbu učinaka kombiniranih mikotoksina u odnosu na učinke pojedinačnih. Općenito, većina dosadašnjih studija pokazuje da kombinacije mikotoksina u biološkom sustavu imaju sinergistički ili barem aditivni učinak, što znači da su velik rizik za zdravlje ljudi i životinja.

Melatonin-loaded chitosan/Pluronic® F127 microspheres as in situ forming hydrogel: An innovative antimicrobial wound dressing.

The aim of this study was to develop melatonin-loaded chitosan based microspheres as dry powder formulation suitable for wound dressing, rapidly forming hydrogel in contact with wound exudate. Microspheres were produced by spray-drying method. Fractional factorial design was employed to elucidate the effect of formulation and process parameters (feed flow rate, inlet air temperature, chitosan concentration, chitosan/melatonin ratio and chitosan/Pluronic® F127 ratio) on the product characteristics related to process applicability (production yield, entrapment efficiency and product moisture content) and microsphere performance in biological environment (microsphere mean diameter and surface charge). Appropriate formulation and process parameters for the establishment of efficient drying process resulting in fine-tuned chitosan and chitosan/Pluronic® F127 microspheres (efficient melatonin encapsulation, small diameter positive surface charge and low moisture content) were identified. Microspheres were characterized by appropriate flowability and high rate and extent of fluid uptake. Incorporation of Pluronic® F127 in microsphere matrix resulted in high melatonin amorphization and consequent higher melatonin release rate. Entrapment of melatonin in chitosan/Pluronic® F127 microspheres has potentiated chitosan antimicrobial activity against Staphylococcus aureus and five clinical isolates S. aureus MRSA strains. Microspheres were shown to be biocompatible with skin keratinocytes and fibroblasts at concentrations relevant for antimicrobial activity against planktonic bacteria.

Spray dried microparticles for controlled delivery of mupirocin calcium: Process–tailored modulation of drug release

Spray dried microparticles containing mupirocin calcium were designed as acrylic matrix carriers with modulated drug release for efficient local drug delivery at minimum daily dose. Particle generation in spray drying and its effect on release performance were assessed by varying drug : polymer ratios with consequently altered initial saturations. Narrow-sized microparticles with mean diameters of 1.7–2.5 µm were obtained. Properties of the generated solid dispersions were examined by X-ray, thermal (thermogravimetric analysis, modulated differential scanning calorimetry) and spectroscopic (Fourier transformed infrared, Fourier transformed Raman) methods and correlated with drug loading and in vitro release. The best control over mupirocin release was achieved for 2 : 1 (w/w) drug : polymer ratio and found to be strongly process-dependent. For a particular ratio, increased feed concentration (>4%) boosted while increased inlet temperature (≥100°C) reduced drug release. Antimicrobial activity testing confirmed that encapsulated drug preserved its antibacterial effectiveness. Conclusively, spray drying was proven as a suitable method for preparing structured microparticles which can control drug release even at exceptionally high drug loadings.

«Suspects» in Etiology of Endemic Nephropathy: Aristolochic Acid versus Mycotoxins

Despite many hypotheses that have been challenged, the etiology of endemic nephropathy (EN) is still unknown. At present, the implications of aristolochic acid (AA) and mycotoxins (ochratoxin A—OTA and citrinin—CIT) are under debate. AA-theory is based on renal pathohistological similarities between Chinese herbs nephropathy (CHN) and EN, findings of AA-DNA adducts in EN and in patients with urinary tract tumors (UTT), as well as the domination of A:T®T:A transversions in the p53 mutational spectrum of UTT patients, which corresponds with findings of such mutations in AA-treated rats. However, exposure pathways of EN residents to AA are unclear. Experimental studies attempting to deduce whether nephrotoxins OTA and CIT appear at higher frequencies or levels (or both) in the food and blood or urine of EN residents support the mycotoxin theory. Also, some molecular studies revealed the presence of OTA-DNA adducts in the renal tissue of EN and UTT patients. In this review, data supporting or arguing against AA and mycotoxin theory are presented and discussed.

Fumonisin B1 Neurotoxicity in Young Carp (Cyprinus carpio L.)

Fumonisin B1 Neurotoxicity in Young Carp (Cyprinus Carpio L.) For years scientists have suspected that the environment plays a role in neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, and multiple sclerosis. Mycotoxin fumonisin B1 (FB1) is produced by several Fusarium species, mainly by Fusarium verticilioides, which is one of the most common fungi associated with corn worldwide. Fumonisins are known to cause equine leukoencephalomalacia, a disease associated with the consumption of corn-based feeds contaminated with FB1. Here we have reported chronic experimental toxicosis in one-year-old carp (Cyprinus carpio L.) receiving feed containing 100 mg kg-1 or 10 mg kg-1 of added FB1 for 42 days. We focused on fumonisin toxicity in the fish brain. After staining with hemalaun-eosin, histology of the fish brain revealed vacuolated, degenerate, or necrotic neural cells, scattered around damaged blood capillaries and in the periventricular area. These findings suggest that fumonisin, although it is a hydrophilic molecule, permeated the blood-brain barrier of young carp and had a toxic effect on neuronal cells. Neurotoksičnost fumonizina B1 u šaranske mLađi (Cyprinus carpio L.) Odavno je poznato da okoliš ima važnu ulogu u razvoju neurodegenerativnih bolesti kao što su Alzheimerova i Parkinsonova bolest te multipla skleroza. Mikotoksin fumonizin B1 (FB1) tvori nekoliko vrsta Fusariuma, najčešće F. verticillioides, koja je najučestaliji kontaminant kukuruza. Ovaj mikotoksin odgovoran je za leukoencefalomalaciju konja, mula i magaradi povezanu s konzumacijom kukuruza kontaminiranog s FB1. U ovom su radu prikazani rezultati kronične eksperimentalne toksikoze mlađi šarana (Cyprinus carpio L.) koji su u hrani primali 100 mg kg-1 i 10 mg kg-1 FB1 tijekom 42 dana. Nakon bojenja hemalaun-eozinom zabilježene su značajne histopatološke promjene na mozgu životinja uključujući vakuolizaciju, degeneraciju i nekrozu neurona, posebice u blizini oštećenih krvnih kapilara i u periventrikularnoj regiji. Ova saznanja pokazuju da FB1, kao hidrofilna molekula, prolazi kroz krvno-moždanu barijeru mladih šarana uzrokujući oštećenje neurona.

Differential activation of MAPKs by individual and combined ochratoxin A and citrinin treatments in porcine kidney PK15 cells

The aim of this study was to investigate the underlying mechanisms of OTA and CTN individual and combined toxicity in porcine kidney PK15 cells of proximal tubule origin. Activation and expression of mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 were determined by Western blot analysis. MAPKs were differentially activated by single or dual OTA and CTN treatments. Single OTA and CTN stimulated transient ERK and prolonged JNK activation, while phospho-p38 signal was more persistent after OTA treatment. Mycotoxin mixture provoked significant down-regulation of ERK activation, more prolonged phospho-p38 signal, and two-stage JNK phosphorylation pattern. In order to define the role of particular MAPKs in mycotoxin(s) cytotoxicity, we performed MTT assay with specific MAPKs inhibitors. In both individual and combined treatments JNK and p38 inhibition significantly induced cell survival. When cells were exposed to toxin mixture, inhibition of ERK also promoted cell survival, although to a lesser extent that JNK and p38 inhibition. Next we investigated the association between calcium (Ca(2+)) and MAPKs after OTA and/or CTN treatments, and we employed Ca(2+) chelator BAPTA-AM. We demonstrated that p38 activation was significantly down-regulated in cells treated with CTN alone or OTA + CTN suggesting the role of Ca(2+) in mycotoxin-induced cell death.

Disturbed Hsp70 and Hsp27 expression and thiol redox status in porcine kidney PK15 cells provoked by individual and combined ochratoxin A and citrinin treatments

The aim of this study was to explore the oxidative properties of ochratoxin A (OTA) and citrinin (CTN) as a possible underlying mechanism of their individual and/or combined cytotoxicity. Metabolic activity of PK15 porcine kidney cells was significantly reduced with OTA and CTN co-exposures, with synergistic cytotoxic interactions. Single CTN increased both reduced (GSH) and oxidized (GSSG) glutathione after 24 h. However, GSH was significantly lowered with all OTA and CTN combined applications in synergistic manner after 12 and 24 h. GSH/GSSG ratio was reduced in most single and dual treatments, which suggested the presence of oxidative stress. In addition, OTA and CTN exposures significantly decreased concentrations of total thiols, with mycotoxins interactions being synergistic or antagonistic. The expression levels of Hsps were differentially affected by single and dual mycotoxin(s) applications. Single OTA provoked significant down-regulation of Hsp70 and Hsp27 expressions, while CTN stimulated Hsps expressions. Hsps were also up-regulated by dual treatments, and this induction was much stronger then with single CTN. In conclusion, significant alterations in cellular redox status (glutathione, thiols) and protective mechanisms (Hsps) suggest that those disturbances might be involved in OTA and CTN individual and combined mechanisms of cytotoxicity.