47 research outputs found
Mechanism of action of fumonisin B1, beauvericin and ochratoxin A in kidney cells
Fumonizin B1 (FB1), bovericin (BEA) i okratoksin A (OTA) su svjetski raÅ”ireni mikotoksini koji izazivaju Äitav
niz patoloÅ”kih promjena in vivo i in vitro, a Äije je istodobno pojavljivanje u kukuruzu dokazano na podruÄju
Hrvatske. Stoga je u ovom radu ispitan mehanizam djelovanja FB1, BEA i OTA i njihove interakcije u PK 15
stanicama (epitel bubrega svinje), praÄenjem preživljavanja stanica, integriteta plazmatske i mitohondrijske
membrane, razine lipidne peroksidacije i antioksidacijskog kapaciteta stanice te stupnja apoptoze i
genotoksiÄnog uÄinka niskih koncentracija mikotoksina (0,05 g/mL, 0,5 g/mL i 5 g/mL), tijekom 24 i 48
sati. Fumonizin B1, BEA i OTA su citotoksiÄni za PK 15 stanice Å”to se oÄituje smanjenjem vijabilnosti i
poveÄanjem katalitiÄke aktivnosti laktat dehidrogenaze i glutamat dehidrogenaze ovisno o dozi mikotoksina. Sva
tri mikotoksina u najveÄoj koncentraciji znaÄajno stimuliraju lipidnu peroksidaciju, a veÄ pri nižim
koncentracijama smanjuju koncentraciju glutationa u PK 15 stanicama. Fumonizin B1, BEA i OTA uzrokuju
apoptozu PK 15 stanica pri Äemu se znaÄajnije morfoloÅ”ke apoptotiÄke promjene zamjeÄuju nakon 48-satnog
izlaganja. Okratoksin A ima jaÄe djelovanje na aktivnost kaspaze-3 veÄ nakon 24-satnog tretiranja u
koncentracijama od 0,5 i 5 g/mL, dok BEA i OTA uzrokuju znaÄajnije poveÄanje aktivnosti ovog enzima
nakon duljeg izlaganja. FB1 i BEA su genotoksiÄni za PK 15 stanice u koncentracijama od 0,5 i 5 g/mL, dok
OTA znaÄajno poveÄava broj mikronukleusa veÄ kod najmanje koncentracije. Sva tri mikotoksina imaju
klastogeni i aneugeni uÄinak pri Äemu je klastogeno djelovanje OTA i BEA viÅ”e izraženo. U koncentracijama
0,05 i 0,5 g/mL nakon 24- i 48-satnog izlaganja, kombinacije dva i sva tri mikotoksina mikotoksina pokazuju
uglavnom aditivane a manje sinergistiÄke interakcije Å”to se odražava na smanjenje vijabilnosti, naruÅ”avanje
integriteta staniÄne i mitohondrijske membrane, stimulaciju lipidne peroksidacije i smanjnje antioksidacijskog
kapaciteta te apoptozu i mutagenezu. Nakon 24-satnog izlaganja navedenim kombinacijama u koncentraciji 5
g/mL zapažen je aditivan, sinergistiÄki i antagonistiÄki uÄinak, a nakon 48-satnog tretmana u istoj koncentraciji
viŔe je izražen antagonizam. Istodobni unos niskih koncentracija ovih mikotoksina putem hrane negativno se
odražava na antioksidacijski sustav te može pridonijeti imunosupresiji, nefrotoksiÄnosti i karcinogenezi u ljudi i
životinja tijekom dulje izloženosti.Fumonisin B1 (FB1), beauvericin (BEA) and ochratoxin A are widespread mycotoxins, which cause a great
number of pathological changes in vivo and in vitro. Their co-occurrence was established in maize in Croatia.
The mechanism of action of FB1, BEA and OTA, as well as their interactions, were studied in the PK 15 cells
(porcine kidney epithelial cells). We examined the effects of low concentrations (0.05, 0.5 and 5 g/mL) of
individual and combined mycotoxins on cell viability, mitochondrial and plasma membrane integrity, lipid
peroxidation, antioxidative status, and apoptotic and genotoxic events in PK 15 cells. Decrease of cell viability
and increase of catalytic activity of LDH and GLDH show that FB1, BEA and OTA are cytotoxic to PK 15 cell
in dose dependent manner. Individual treatment of cells with 5 g/mL of FB1, BEA and OTA significantly
stimulated lipid peroxidation, while lower concentrations significantly reduced levels of glutathione. Significant
morphological apoptotic changes were observed after 48 hours of exposure to individual mycotoxins. Ochratoxin
A activated caspase-3 already after 24 hours of treatment with 0.5 and 5 g/mL, while BEA and OTA
significantly affected this enzyme after prolonged exposure. Fumonisn B1 and BEA are genotoxic to PK 15 cells
in concentrations of 0.5 and 5 g/mL, while OTA significantly increased the number of micronuclei already after
treatment with lowest concentrations. All of three mycotoxins mainly act as clastogens. Combined treatment
with two or all of three mycotoxins in concentrations of 0.05 and 0.5 g/mL during 24 and 48 hours, showed
mainly additive and less synergistic interactions on cell viability, mitochondrial and plasma membrane integrity,
stimulation of lipid peroxidation and decrease of antioxidative capacity, apoptotic and genotoxic events in PK 15
cells. After 24 hours of exposure to combined mycotoxins in concentration of 5 g/mL, additive, synergistic and
antagonistic effects were observed, while after 48 hours of exposure to same concentration we observed
antagonism. Simultaneous prolonged exposure to mycotoxins in low dietary concentrations can negatively affect
antioxidant status contributing the immunosuppression, nephrotoxicity and carcinogenicity in animals and
humans
ToksiÄni uÄinci Ustilago maydis i fumonizina B1 u Å”takora
Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which was indicated by hyperactivity, brain hyperaemia and especially by decreased AChE activity. These pathological changes could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity. Pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group, indicating possible synergism. Increased activity of LDH in the serum was observed only in the US+FB1 group, which led to the conclusion that FB1 is responsible for cytotoxic effects. Such activity of FB1 could increase the toxicity of U. maydis and other co-contaminants of fungal origin that can be found in foods and feeds.ToksiÄno djelovanje kukuruzne snijeti Ustilago maydis i moguÄi sinergizam s fumonizinom B1 (FB1) ispitivani su na Å”takorima soja Fischer. ToksiÄni uÄinci su procijenjeni na temelju organoleptiÄkog pregleda organa žrtvovanih životinja i mjerenja biokemijskih parametara u serumu (LDH, ALT, GGT, ChE), te homogenatu tkiva jetre i mozga (AChE, ChE, GGT, ALP). U jednoj pokusnoj skupini (US) životinje su hranjene smjesom biomase i snijeti masenog udjela 70%, dok je druga pokusna skupina (US+FB1) primala smjesu biomase i snijeti masenog udjela 70%, te fumonizina B1 (1 mg po kilogramu hrane) tijekom 17 dana. Kontrolna skupina je hranjena standardnom laboratorijskom hranom. Tijekom pokusa životinje su bile ekscitirane. Masa pokusnih životinja bila je na kraju pokusa neÅ”to veÄa u odnosu na kontrolnu skupinu. UoÄene patoloÅ”ke promjene na jetri, pluÄima, uterusu i ovarijima bile su viÅ”e izražene u skupini US+FB1. SpecifiÄna katalitiÄka aktivnost AChE u homogenatu jetre i mozga bila je znaÄajno smanjena u US skupini (61, odnosno 63%) u odnosu na kontrolu (p < 0,05), Å”to ukazuje na neurotoksiÄno djelovanje snijeti. Osim toga, zabilježena je znaÄajno smanjena aktivnost ovog enzima i ALP u homogenatu jetre US+FB1 skupine (p < 0,05). SpecifiÄna katalitiÄka aktivnost LDH u serumu bila je znaÄajno poveÄana u US+FB1 skupini (165%, odnosno 166%) u odnosu na US i kontrolnu skupinu, Å”to ukazuje da su oÅ”teÄenja staniÄne membrane rezultat djelovanja FB1. NeurotoksiÄni uÄinci u Å”takora soja Fischer vjerojatno su posljedica djelovanja alkaloida U. maydis i poremeÄaja metabolizma sfingolipida uslijed djelovanja FB1
Beauvericin: Chemical and Biological Aspects and Occurence
Bovericin (BEA) cikliÄki je heksadepsipeptid koji sintetiziraju Beauveria bassiana, Paecilomyces fumosoroseus, Paecilomyces tenuipes i Polyporus sulphureus te velik broj vrsta Fusarium. Ovaj mikotoksin ima antimikrobno, insekticidno, citotoksiÄno i apoptotsko djelovanje, a specifiÄni je inhibitor kolesterolaciltransferaze. BioloÅ”ko djelovanje BEA povezano je s njegovom moguÄnoÅ”Äu tvorbe kompleksa s esencijalnim kationima (Ca2+, Na+, K+) Äime poveÄava propusnost membrane za te ione i naruÅ”ava ionsku homeostazu. Bovericin je naÄen u uzorcima kukuruza iz razliÄitih zemalja, ukljuÄujuÄi i Hrvatsku te je utvrÄena ko-kontaminacija s drugim fuzariotoksinima (fumonizini, moniliformin) i okratoksinom A. BioloÅ”ka aktivnost BEA mogla bi poveÄati toksiÄnost drugih mikotoksina koji se istodobno pojavljuju u hrani. Uloga BEA u razvoju mikotoksikoza u ljudi i životinja joÅ” je uvijek nepoznata.Beauvericin (BEA) is a cyclic hexadepsipeptide produced by Beauveria bassiana, Paecilomyces fumosoroseus, Paecilomyces tenuipes, Polyporus sulphurous, and a variety of Fusarium species. This mycotoxin shows antimicrobial, insecticidal, cytotoxic, and apoptotic activity. It is the most potent specific inhibitor of cholesterol acyltransferase and possesses ionophoric properties. BEA increases ion permeability in biological membranes by forming a complex with essential cations (Ca2+, Na+, K+), which may affect the ionic homeostasis. BEA has been frequently found in maize samples in Europe, USA and Africa and co-contamination with other Fusarium toxins such as fumonisins, and moniliformin was also found. There is only one report of BEA occurrence and co-occurrence with fumonisin B1, fumonisin B2 and ochratoxin A in Croatia. Biological activity of BEA may increase the toxicity of other mycotoxins that co-occur with BEA in food. The role of BEA in the development of human and animal mycotoxicosis is still unknown
NeurotoksiÄnost fumonizina B1 u Å”aranske mlaÄi (Cyprinus carpio L.)
For years scientists have suspected that the environment plays a role in neurodegenerative diseases such as Alzheimerās disease, Parkinsonās 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-bas d 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 fi sh brain. After staining with hemalaun-eosin, histology of the fi sh brain revealed vacuolated, degenerate, or necrotic neural cells, scattered around damaged blood capillaries and in the periventricular area. These fi ndings 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.Odavno je poznato da okoliŔ ima važnu ulogu u razvoju neurodegenerativnih bolesti kao Ŕto su Alzheimerova
i Parkinsonova bolest te multipla skleroza. Mikotoksin fumonizin B1 (FB1) tvori nekoliko vrsta Fusariuma, najÄeÅ”Äe F. verticillioides, koja je najuÄestaliji 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 kroniÄne eksperimentalne toksikoze mlaÄi Å”arana (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 znaÄajne histopatoloÅ”ke promjene na mozgu životinja ukljuÄujuÄi vakuolizaciju, degeneraciju
i nekrozu neurona, posebice u blizini oÅ”teÄenih krvnih kapilara i u periventrikularnoj regiji. Ova saznanja pokazuju da FB1, kao hidrofi lna molekula, prolazi kroz krvno-moždanu barijeru mladih Å”arana uzrokujuÄi oÅ”teÄenje neurona
JednogodiŔnje istraživanje razine plijesni u zraku unutarnjih prostora
This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air during one year in Zagreb, Croatia. The incidence of Aspergilli from sections Flavi, Nigri, and Versicolores was also assessed. Airborne fungi were collected using an air-sampler and DG-18 agar plates. The average concentrations of airborne fungi in the grain mill ranged from 14,310 to 40,000 cfu m-3, which was above the hazardous level (104 cfu m-3), whereas the values statistically estimated using Fellerās correction were up to six times higher. Concentrations in the apartment (163-1244 cfu m-3) were lower than in outdoor air (286-2090 cfu m-3) and lower than in the basement (697-1203 cfu m-3), except in the warmer period of the year when they were similar. The most abundant species throughout the year were Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), and Alternaria spp. (10-100 %), which are common for temperate climates. Aspergilli from the Flavi (50-100 %) and Nigri (15-40 %) sections as well as A. ochraceus (15-60 %) and Eurotium spp. (85-100 %) were the most abundant at the grain mill and were rarely found in outdoor air. In the basement, Aspergilli (Versicolores) were more abundant than in the apartment. The excess of aeromycoparticles in the grain mill throughout the year may have represented a serious health risk to mill workers. This is the first Croatian one-year study of indoor airborne fungi in a grain mill and dwellings; however monitoring should continue over a longer period.Cilj rada bio je ispitati varijacije uÄestalosti (%) i koncentracija (cfu m-3) plijesni u zraku mlina žitarica i Äetiriju stambenih prostora (dva stana i dva podruma) u odnosu na vanjski zrak tijekom jedne godine u Zagrebu (Hrvatska). UÄestalost Aspergillus vrsta iz sekcija Flavi, Nigri i Versicolores takoÄer je ispitana. Plijesni iz zraka su uzorkovane pomoÄu ureÄaja Air-sampler MAS 100 Eco u kojem su postavljene DG-18-agarske ploÄe. ProsjeÄna koncentracija aerogenih plijesni u mlinu bila je izmeÄu 14.310 i 40.000 cfu m-3, Å”to je veÄe od koncentracije (104 cfu m-3) koja se smatra opasnom za zdravlje. Procijenjene vrijednosti koncentracija plijesni u zraku mlina, dobivene Felerovom korekcijom, Äak su Å”est puta veÄe od izmjerenih koncentracija. U stanovima (163-1244 cfu m-3) koncentracije aerogenih plijesni bile su manje nego u vanjskom zraku (286-2090 cfu m-3) i podrumima (697-1203 cfu m-3), izuzev u toplijim mjesecima kada su izmjerene vrijednosti bile sliÄne. Tijekom godine dominirale su vrste iz rodova Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), i Alternaria spp. (10-100 %), koje su uobiÄajene u podruÄjima s umjerenom klimom. Aspergile iz sekcija Flavi (50-100 %) i Nigri (15-40 %), A. ochraceus (15-60 %) i Eurotium spp. (85-100 %) dominirali su u zraku mlina, a u uzorcima vanjskog zraka rijetko su detektirane. Vrste Aspergillus iz sekcije Versicolores s veÄom uÄestaloÅ”Äu naÄene u podrumima nego u stanovima. Veliko optereÄenje mlina aerogenim plijesnima tijekom cijele godine može biti opasno za zdravlje radnika. Ujedno, ovo je prvo jednogodiÅ”nje ispitivanje sezonske varijabilnosti u sastavu plijesni u zraku mlina žitarica i stambenih prostora u Hrvatskoj. MeÄutim, monitoring treba nastaviti tijekom dužeg razdoblja
Tvorba verukulogena u kliniÄkih i iz zraka izoliranih sojeva vrste Aspergillus fumigatus Fres.
Among airborne aspergilli sampled in outdoor air of the Zagreb area (2002/2003), Aspergillus niger (v. Teigh.) and A. fumigatus (Fres.) were the most abundant species (20-30%), with low mean annual concentrations (0.21-1.04 CFU m-3). Higher concentrations of A. fumigatus were observed in autumn and winter (0.5-1.05 CFU m-3) than in spring and summer (0-0.4 CFU m-3). On the other hand, A. fumigatus was found to be the most frequent isolate from upper and/or lower respiratory tracts of imunocompromised patients in many studies. This species produces several mycotoxins including the tremorgenic mycotoxin verruculogen that can be found in spores and during myceliar growth. Verruculogen production ability was tested by biosynthesis on 30 airborne and 33 clinical isolates of A. fumigatus. In both groups, high percentage of verruculogen-producing strains was noticed (84% of airborne and 91% of clinical isolates). Verruculogen production was not significantly different in the groups of airborne isolates (0.34 Ā± 0.16 mg mL-1) and clinical isolates (0.26 Ā± 0.19 mg mL-1, p > 0.05).Uzorkovanje spora plijesni na podruÄju grada Zagreba obavljeno je tijekom godine 2002./2003. Pri tome su Aspergillus niger (v. Teigh.) i A. fumigatus (Fres.) bile najuÄestalije (20ā30%) meÄu aerogenim Aspergillus vrstama. Srednje godiÅ”nje koncentracije njihovih spora bile su izrazito niske (0,21ā1,04 CFU mā3). VeÄe koncentracije spora A. fumigatus zabilježene su tijekom jeseni i zime (0,50ā1,05 CFU mā3) u odnosu na koncentracije izmjerene tijekom proljeÄa i ljeta (0ā0,40 CFU mā3). S druge strane, mnogobrojne studije ukazuju na visoku uÄestalost A. fumigatus izolata iz gornjih i donjih diÅ”nih puteva imunokompromitiranih bolesnika. Ova plijesan tvori nekoliko razliÄitih mikotoksina, ukljuÄujuÄi i tremorgeni mikotoksin verukulogen koji se može naÄi u sporama i miceliju vrste A. fumigatus. Tvorba verukulogena ispitana je u 30 aerogenih i 33 kliniÄka izolata A. fumigatus vrste. U obje skupine dokazana je visoka uÄestalost verukulogen-pozitivnih sojeva (84% aerogenih i 91% kliniÄkih izolata). Potencijal tvorbe verukulogena u aerogenih (0.34 Ā± 0.16 mg mLā1) i kliniÄkih sojeva (0.26 Ā± 0.19 mg mLā1) nije se znaÄajnije razlikovao
Genotoxicity of Fumonisin B1, Beauvericin and Ochratoxin A in Porcine Kidney PK15 Cells: Effects of Individual and Combined Treatment
After 24- and 48-hour treatment with 0.05, 0.5 and 5 mg/mL of fumonisin (FB1), beauvericin (BEA) and ochratoxin A (OTA) alone or combinations of two or all three mycotoxins in equal concentrations, Giemsa-stained binucleated PK15 cells were scored for the presence of micronuclei (MN), nuclear buds (NBs) and nucleoplasmic bridges (NPBs). Single mycotoxins induced MN and NPBs in a dose-dependent manner, showing predominantly clastogenic effect. OTA showed stronger genotoxic potential than FB1 and BEA, since 0.05 and 0.5 mg/mL OTA increased the frequency of MN and NPBs, respectively. Mycotoxin combinations increased the frequency of MN and NPBs, mostly in additive manner. A dose-dependent increase in NBs was observed after 24-hour exposure to single mycotoxins. Prolonged treatment with 5 mg/mL of each toxin alone as well as combined exposure to all mycotoxin concentrations resulted in a decrease in NBs, which could be related to the extrusion of micronuclei and/or saturation of the genotoxic effect. This is the first report on genotoxicity of BEA
OCHRATOXIN A AND ZEARALENON: CEREALS AND FEED CONTAMINATION IN CROATIA (1977-2007) AND INFLUENCE ON ANIMAL AND HUMAN HEALTH
Mikotoksini kao sekundrani metaboliti plijesni usko su povezani s distribucijom plijesni, njihovim vrstama i toksinogenim sojevima, mikroklimom, naÄinom sabiranja i skladiÅ”tenja usijeva. NaÅ”a istraživanja u proteklih 30 godina pokazuju da Fusarium i Penicillium vrste podjednako (40-60%) dominiraju u usjevima zbog Äega su žitarice i krmiva Äesto kontaminirani okratoksinom A (OTA) i zearalenonom (ZEA). OTA je nefrotoksin s potencijalnim karcinogenim djelovanjem u ljudi, koji se dovodi u vezu s razvojem endemske nefropatije (EN). ZEA je mikotoksin s uterotropnim, estrogenim i anaboliÄkim djelovanjem u domaÄih životinja s potencijalnim negativnim uÄinkom na ravnotežu spolnih hormona u ljudi. Tijekom 30-godiÅ”njih istraživanja OTA i ZEA su u prosjeku dokazani u 20%, odnosno u 30% uzoraka. Znatno veÄe koncentracije ovih mikotoksina naÄene su tijekom izuzetno vlažne 1978. (68900 Ī¼g OTA/kg i 275800 Ī¼g ZEA/kg) i 1980. (4700 Ī¼g OTA/kg), za vrijeme domovinskog rata (3200 Ī¼g OTA/kg i 19900 Ī¼g ZEA/kg) te u izrazito hladnoj (temperaturni stres) i vlažnoj 2004. (29430 Ī¼g ZEA/kg). Tijekom ostalih godina koncentracije ovih mikotoksina kreÄu se od 0,26 do 220 Ī¼g/kg (OTA) te od 0,39 do 3000 Ī¼g/kg (ZEA). U posljednje vrijeme sve se viÅ”e važnosti pridaje ko-kontaminciji usjeva mikotoksinima zbog njihovog moguÄeg sinergistiÄkog uÄinka. Tako je posljednjih godina dokazana ko-kontaminacija kukuruza i drugih žitarica sa OTA i ZEA u 37% odnosno 13,5% uzoraka. MeÄutim, zadnjih godina utvrÄene koncentracije su niže u odnosu na prethodna razdoblja Å”to se može pripisati relativno suÅ”nom razdoblju. Iako je zbog toga smanjen rizik trovanja to ne iskljuÄuje sustavni unos subtoksiÄnih koncentracija mikotoksina u organizam te njihov moguÄi toksinopatogeni sinergizam koji se negativno odražava na enzimatske i imunoloÅ”ke funkcije u ljudi i životinja.Mycotoxins are secondary metabolites produced by various mould species. Contamination of cereals and feeds with mycotoxins depends on distribution of toxigenic mould strains, micro-climate, harvesting techniques and storage conditions. Our research in the past 35 years shows that Fusarium sp. and Penicillium sp. dominated in the crops (40-60%). Consequently, ochratoxin A (OTA) and zearalenone (ZEA) were frequently found in cereals and feed. OTA is a nephrotoxin with a potential carcinogenic activity in humans and is connected with development of endemic nephropathy (EN). ZEA shows uterotropic, estrogenic and anabolic activity in domestic animals and it has been implicated in hormonal interruption in humans. In the past three decades OTA and ZEA were detected in 20% and 30% of samples. High concentrations were found in extremely humid and/or cold years; in 1978 (68900 Ī¼g OTA/kg and 2758000 Ī¼g ZEA/kg), in 1980 (4700 Ī¼g OTA/kg), and in 2004 (29430 Ī¼g ZEA/kg) as well as during wartime in Croatia (3200 Ī¼g OTA/kg and 19900 Ī¼g ZEA/kg). In other years of sampling concentrations were between 0.26 and 220 Ī¼g/kg (OTA) and between 0.39 and 3000 Ī¼g/kg (ZEA). Recently, many reports were focused on mycotoxin co-contamination of crops, since they could produce synergistic toxic effects in animals and humans. In the past few years simultaneous occurrence of OTA and ZEA were detected in 37% and 13.5% of samples, respectively. However, levels of these toxins are significantly lower compared to previous sampling which is probably a consequence of relatively dry microclimatic conditions. Even low
concentrations of mycotoxins reduce the risk of acute poisoning, chronic intake of subtoxic concentrations and their possible synergistic interactions could have an impact on biochemical and immunological functions in animals and humans
COMPARISON BETWEEN ELISA AND TLC/HPLC METHODS FOR DETERMINATION OF ZEARALENON AND OCHRATOXIN A IN FOOD AND FEED
Fusarium i Penicillium vrste plijesni javljaju se kao najuÄestaliji kontaminanti žitarica u podruÄju umjerene klime. Kao posljedica se mikotoksini zearalenon (ZEA) i okratoksin A (OTA) Äesto nalaze u takvim supstratima u veÄim ili manjim koncentracijama ovisno o naÄinu pospremanja usjeva, skladiÅ”tenju i mikroklimatskim uvjetima. Cilj rada je bio odrediti pojavu ZEA i OTA tijekom 2007. godine u nasumiÄno uzimanim uzorcima žitarica i krmiva (N=37) u individualnim domaÄinstvima na podruÄju endemske nefropatije (EN) u Hrvatskoj. Za odreÄivanje ZEA i OTA koriÅ”ten je kompetativni direktni ELISA test (Veratox_) prema preporukama proizvoÄaÄa Neogen Europe Ltd. te modificirana metoda visokouÄinkovite tekuÄinske kromatografije (HPLC) prema Frisvad i Thrane (1987) i tankoslojna kromatografija (TLC). Usporedbom metoda odreÄene su granice detekcije i kvantifikacije te korelacije sadržaja ispitivanih mikotoksina. ZEA je dokazan u 91,9% uzoraka u rasponu od 12,5 Āµg/kg do 1182 Āµg/kg (srednja koncentracija 318,3 Āµg/kg), dok je OTA naÄen u 16,2% uzoraka s rasponom od 2,5 Āµg/kg do 31,7 Āµg/kg (srednja koncentracija 9,8 Āµg/kg). Kokontaminacija je dokazana u 13,5 % uzoraka. UtvrÄene vrijednosti ZEA i OTA u hrani i krmivu su ispod najveÄih dopuÅ”tenih koliÄina prema Pravilniku (Narodne novine br. 118. iz 2007.) te odgovaraju i preporukama Europske komisije (EC/576/2006) i EC/1881/2006). Usporedbom metoda, utvrÄeno je da najnižu granicu detekcije ima ELISA, a slijedi ju HPLC. Izmjerene koncentracije mikotoksina statistiÄki znaÄajno koreliraju izmeÄu ELISA i HPLC metode. Prednost ELISA metode je u jednostavnosti izvedbe (pogotovo za veliki broj uzoraka), osjetljivosti, ekonomskoj isplativosti te ekoloÅ”koj prihvatljivosti. Prednost HPLC metode je u analizi viÅ”e mikotoksina u jednom uzorku.Fusarium and Penicillium are the most common mould species that contaminate cereals in the mild climatic zone. Therefore, zearalenone (ZEA) and ochratoxin A (OTA) are frequently detected in these substrates in different concentrations depending on storage and microclimatic conditions. The purpose of this study was to determine the frequency and concentrations of ZEA and OTA in cereal and feed samples randomly collected in individual households of endemic nephropathy region (EN) in Croatia. Direct competitive ELISA test kit was used for ZEA and OTA determination according to manufacturer instructions. Results of ELISA method as well as limits of detection (LOD) and quantification were compared with high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) methods. Correlation between results was also analysed. ZEA was detected in 91.9% of samples ranging between 12.5 and 1168Āµg/kg (mean 318.3 Āµg/kg), while OTA was recovered from 16,2% of samples in concentrations ranging from 2.5 Āµg/kg to 31.7 Āµg/kg
(mean 9.8 Āµg/kg). ZEA and OTA simultaneously occurred in 13.5 % of samples. Detected values of mycotoxins were under the permissible levels according to Croatian law (Narodne novine No. 118/2007 and European Commission (EC/576/2006 and EC/1881/2006). The lowest LOD was obtained by ELISA followed by HPLC. Mycotoxin concentrations detected by ELISA significantly correlate with results obtained by HPLC. ELISA provide an attractive and promising method for OTA and ZEA detection in food due to high specificity, sensitivity, simplicity (analysis of high number of samples) and it is ecologically more acceptable than chromatographic methods. Advantage of HPLC method is multiple mycotoxin detection and quantification in one sample