Ana-Marija Domijan

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia.

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB1(100%), followed by ZEA (84%) and OTA (39%), while FB2 was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB1, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg−1, respectively, and the concentrations of FB2 in three positive samples were 68.4, 109.2 and 3084.0 µg kg−1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.

The occurrence of ochratoxin A in blood in general population of Croatia

The exposure of general population in Croatia to mycotoxin ochratoxin A (OTA) was investigated in five cities: Split, Rijeka, Varazdin, Osijek, and Zagreb. In June 1997, blood donors from each of these cities gave 50 samples of 3 ml plasma each. The mean concentration of OTA, determined using high-pressure liquid chromatography (HPLC), was 0.39 ng/ml of plasma. The highest frequency of OTA-positive samples (>0.2 ng/ml plasma), and the highest number of samples with the concentration exceeding 1.0 ng/ml, were found in Osijek. This difference is probably due to the higher consumption of fresh and dried pork by population of Osijek. The calculated daily intake of OTA, estimated from the mean OTA concentration of all samples in each town (in the range from 0.24 to 0.91 ng/kg b.w. found in Rijeka and Osijek, respectively) is lower than the tolerable daily intake proposed by Joint FAO/WHO Expert Committee on Food Additives (1995) of 16.0 ng OTA/kg b.w.

Prolonged seizure activity impairs mitochondrial bioenergetics and induces cell death.

The mechanisms underlying neuronal death following excessive activity such as occurs during prolonged seizures are unclear, but mitochondrial dysfunction has been hypothesised to play a role. Here, we tested this with fluorescence imaging techniques in rat glio-neuronal neocortical co-cultures using low Mg2+ levels to induce seizure-like activity. Glutamate activation of NMDA receptors resulted in Ca2+ oscillations in neurons and a sustained depolarisation of the mitochondrial membrane potential, which was cyclosporine A sensitive, indicating mitochondrial permeability and transition pore opening. It was also dependent on glutamate release and NMDA receptor activation, because depolarisation was not observed after depleting vesicular glutamate with vacuolar-type H+-ATPase concanamycin A or blocking NMDA receptors with APV. Neuronal ATP levels in soma and dendrites decreased significantly during prolonged seizures and correlated with the frequency of the oscillatory Ca2+ signal, indicative of activity-dependent ATP consumption. Blocking mitochondrial complex I, complex V or uncoupling mitochondrial oxidative phosphorylation under low-Mg2+ conditions accelerated activity-dependent neuronal ATP consumption. Neuronal death increased after two and 24 hours of low Mg2+ levels compared with control treatment, and was reduced by supplementation with the mitochondrial complex I substrate pyruvate. These findings demonstrate a crucial role for mitochondrial dysfunction in seizure-activity-induced neuronal death, and that strategies aimed at redressing this are neuroprotective.

EXPERIMENTAL MYCOTOXICOSIS IN CHICKENS INDUCED BY OCHRATOXIN A AND PENICILLIC ACID AND INTERVENTION WITH NATURAL PLANT EXTRACTS

The combined toxic effect of ochratoxin A (OTA) and penicillic acid (PA) on the body mass, the weight and pathomorphology of some internal organs was studied in 85 broiler chickens fed a mouldy diet containing 130, 300 or 800 ppb OTA and 1000–2000 ppb PA. The main pathomorphological changes were cloudy swelling and granular degeneration in the epithelium and mononuclear cell proliferation and activation of capillary endothelium in the kidney and liver; degenerative changes and depletion of lymphoid cells in lymphoid organs (bursa of Fabricius, thymus and spleen) were also seen. Protective effects of 5% total water extract of artichoke and a new natural phytosubstance Rosallsat against these pathomorphological changes were observed. A significant decrease in body mass and relative weight of lymphoid organs was found after 6 weeks of exposure and a greater decrease after 10 weeks of exposure to OTA and PA, and a protective effect of artichoke extract and a slight effect of Rosallsat against that decrease was observed. A significant increase in relative weight of liver and kidneys was also observed as well as a protective effect of artichoke extract against that increase. The quantity of OTA and the percentage of positive samples were significantly lower in tissues of chickens treated with artichoke extract or Rosallsat in addition to OTA than in those treated with only OTA.

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na+] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets.

Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex.

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50-250 microg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 microg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

Assessment of cytogenetic damage and oxidative stress in personnel occupationally exposed to the pulsed microwave radiation of marine radar equipment

Due to increased usage of microwave radiation, there are concerns of its adverse effect in todays society. Keeping this in view, study was aimed at workers occupationally exposed to pulsed microwave radiation, originating from marine radars. Electromagnetic field strength was measured at assigned marine radar frequencies (3 GHz, 5.5 GHz and 9.4 GHz) and corresponding specific absorption rate values were determined. Parameters of the comet assay and micronucleus test were studied both in the exposed workers and in corresponding unexposed subjects. Differences between mean tail intensity (0.67 vs. 1.22) and moment (0.08 vs. 0.16) as comet assay parameters and micronucleus test parameters (micronuclei, nucleoplasmic bridges and nuclear buds) were statistically significant between the two examined groups, suggesting that cytogenetic alterations occurred after microwave exposure. Concentrations of glutathione and malondialdehyde were measured spectrophotometrically and using high performance liquid chromatography. The glutathione concentration in exposed group was significantly lower than in controls (1.24 vs. 0.53) whereas the concentration of malondialdehyde was significantly higher (1.74 vs. 3.17), indicating oxidative stress. Results suggests that pulsed microwaves from working environment can be the cause of genetic and cell alterations and that oxidative stress can be one of the possible mechanisms of DNA and cell damage.

The involvement of mycotoxins in the development of endemic nephropathy

ZusammenfassungDie endemische Nephropathie (EN) ist eine Nierenerkrankung, die noch immer nicht wissenschaftlich erklärt werden kann. Die EN wird von einer hohen Prävalenz von urothelialen Tumoren bei der ländlichen Bevölkerung der endemischen Region begleitet. Man vermutet daher, dass eine natürlich vorkommende nephrotoxische und karzinogene Substanz in die Ätiologie involviert ist. Am meisten wird das Mykotoxin Ochratoxin A (OTA) als schuldige Substanz verdächtigt, da dieses eine gesicherte nephrotoxische und karzinogene Wirkung hat. Die vorliegende Arbeit bringt eine Übersicht über alle relevanten Studien über OTA in der Nahrung beziehungsweise im Blut und im Harn der Bewohner der betroffenen Endemie-Gebiete. Es werden auch Daten über das gleichzeitige Vorkommen von OTA mit anderen Mykotoxinen, wie Zitronin und Fumonisin B1 (FB1) in der Nahrung vorgestellt. Leider existieren keine Studien über das gemeinsame Vorkommen von OTA und anderen Mykotoxinen beim Menschen, und es gibt nur eine Studie über die Exposition mit FB1 in endemischen Gebieten. Diese Übersicht berichtet auch über experimentelle Daten, die in Zellkulturen und Labortieren erhoben wurden, die mit OTA und anderen nephrotoxischen Mykotoxinen (da die meisten Mykotoxin-Kombinationen synergistische Wirkung zeigten) behandelt worden sind. Es wird auch das Vorkommen von OTA- und Aristolochia-Säure-bedingten DNA-Veränderungen diskutiert.SummaryEndemic nephropathy is a human kidney disease that still escapes scientific explanation. It is accompanied by a high incidence of urothelial tumors in rural populations in endemic areas, which suggests that a natural nephrotoxic and carcinogenic compound may be involved in the etiology. The most imputed causative agent of endemic nephropathy is the mycotoxin ochratoxin A (OTA), because of its confirmed nephrotoxic and carcinogenic action. This paper presents a review of studies of OTA in food collected in the endemic areas and in blood and urine of their residents. Data on the co-occurrence of OTA and other nephrotoxic and carcinogenic mycotoxins such as citrinin and fumonisin B1 in food are also presented. Unfortunately, there is no study on the co-occurrence of OTA and other mycotoxins in humans and there is only one study on fumonisin B1 exposure in endemic areas. The paper also presents experimental data on cultured cells and laboratory animals treated with combinations of OTA and other nephrotoxic mycotoxins, because most such combinations show a synergistic effect. The occurrence of OTA- and aristolochic acid-DNA adducts is also presented.

Lipid peroxidation is essential for phospholipase C activity and the inositol-trisphosphate-related Ca2+ signal

ABSTRACT Reactive oxygen species (ROS) are produced in enzymatic and non-enzymatic reactions and have important roles in cell signalling but also detrimental effects. ROS-induced damage has been implicated in a number of neurological diseases; however, antioxidant therapies targeting brain diseases have been unsuccessful. Such failure might be related to inhibition of ROS-induced signalling in the brain. Using direct kinetic measures of lipid peroxidation in astrocytes and measurements of lipid peroxidation products in brain tissue, we here show that phospholipase C (PLC) preferentially cleaves oxidised lipids. Because of this, an increase in the rate of lipid peroxidation leads to increased Ca2+ release from endoplasmic reticulum (ER) stores in response to physiological activation of purinoreceptors with ATP. Both vitamin E and its water-soluble analogue Trolox, potent ROS scavengers, were able to suppress PLC activity, therefore dampening intracellular Ca2+ signalling. This implies that antioxidants can compromise intracellular Ca2+ signalling through inhibition of PLC, and that PLC plays a dual role – signalling and antioxidant defence.

Fumonisin B1: a neurotoxic mycotoxin

Abstract Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium spp. moulds that contaminate crop, predominantly maize, all around the world. More than 15 types of fumonisins have been indentified so far, but FB1 is the most abundant and toxicologically the most significant one. FB1 has a wide range of toxic effects, depending on animal species. In horses FB1 causes equine leukoencephalomalacia (ELEM), in pigs pulmonary oedema and in experimental rodents nephrotoxicity and hepatotoxicity. In humans exposure to FB1 is linked with higher incidence of primary liver cancer and oesophageal cancer, which are frequent in certain regions of the world (such as Transkei region in South Africa) where maize is staple food. The occurrence of neural tube defect in children in some countries of Central America (such as Mexico and Honduras) is connected with the consumption of FB1-contaminated maize-based food. However, possible involvement of FB1 in the development of human diseases is not clear. Nevertheless, the International Agency for Research on Cancer (IARC) has classified FB1 as a possible carcinogen to humans (group 2B). FB1 is a causative agent of ELEM, a brain disorder in equines, indicating that brain is a target organ of FB1 toxicity. Several studies on experimental animals or on cell cultures of neural origin have established that FB1 has a neurodegenerative potential, although the mechanism of its neurotoxicity is still vague. The aim of this article is to give an overview of available literature on FB1 neurotoxicity and involved mechanisms, and to offer a new perspective for future studies. Fumonizin B1 (FB1) jest mikotoksin koji proizvode plijesni roda Fusarium spp. koje nalazimo kao onečišćivače žitarica, ponajprije kukuruza diljem svijeta. Od svih do sada izoliranih fumonizina FB1 se najčešće može naći na kukuruzu, a i najtoksičniji je fumonizin. FB1 ima različite toksične učinke ovisno o životinjskoj vrsti. Tako u konja izaziva leukoencefalomalaciju kopitara (ELEM), u svinja plućni edem, a za eksperimentalne je glodavce nefrotoksičan i hepatotoksičan. U ljudi je izloženost FB1 povezana s razvojem primarnog karcinoma jetre i karcinoma jednjaka koji se učestalo pojavljuju u regijama svijeta (kao Transkeiska regija u Južnoj Africi) u kojima ljudi rabe kukuruz u dnevnoj prehrani. I pojavljivanje defekta neuralne cijevi u nekim je zemljama Srednje Amerike (kao Meksiko i Honduras) povezano s učestalom konzumacijom kukuruzne hrane kontaminirane s FB1. Ipak se sa sigurnošću ne može povezati razvoj navedenih bolesti u ljudi s izloženosti FB1. Međunarodna agencija za istraživanje raka (IARC) klasificirala je FB1 kao mogući karcinogen za ljude (grupa 2B). Kako je FB1 uzročnik ELEM-a, poremećaja središnjega živčanog sustava (SŽS) u konja, to upućuje na mogućnost da FB1 uzrokuje promjene u mozgu. Nekoliko studija na pokusnim životinjama i na staničnim kulturama stanica podrijetlom iz SŽS-a potvrdilo je da je FB1 neurotoksičan iako mehanizam neurotoksičnosti FB1, pa tako i mehanizam njegove toksičnosti, još nije razjašnjen. Stoga je cilj ovoga rada dati pregled dostupne literature o neurotoksičnosti i mehanizmu neurotoksičnosti FB1 kako bi se omogućilo bolje planiranje budućih istraživanja.