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.

Impact of fumonisin B1 on glutamate toxicity and low magnesium-induced seizure activity in neuronal primary culture.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium spp. mould that contaminates maize world-wide. Although its neurodegenerative potential is well established, mechanisms and acute effects of FB(1) on neurons are still not completely understood. Our previous study on astrocytes and neuroblastoma cells demonstrated that acute FB(1) exposure inhibits mitochondrial complex I and leads to mitochondrial membrane potential depolarization and calcium deregulation. To further explore the mechanisms of FB(1) neurotoxicity, we here investigated the effects of acute FB(1) co-exposure with glutamate and in the low magnesium model of epilepsy on neuronal calcium level, mitochondrial membrane potential, and cell death in glio-neuronal cultures. FB(1) increased the glutamate-induced calcium signal in neurons and changed neuronal calcium signals to more sustained intracellular calcium rises in the low magnesium model of epilepsy that coincided with mitochondrial membrane potential depolarization. FB(1) co-exposure increased the percentage of dead neurons in low magnesium conditions dose dependently when compared with low magnesium exposure only, whereas in FB(1) and glutamate co-exposure neuronal death remained unchanged when compared with glutamate treatment only. Our results show that FB(1) makes neurons more vulnerable to glutamate-induced toxicity and epileptiform conditions, indicating that FB(1) can enhance the detrimental effect of these conditions on neurons.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay.

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 +/- 0.53, 7.52 +/- 3.32, 7.85 +/- 2.24 microg/mL, and 0.87 +/- 0.09, 0.99 +/- 0.06, 1.09 +/- 0.15 microg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 æg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 æg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

Seed-borne fungi and ochratoxin A contamination of dry beans (Phaseolus vulgaris L.) in the Republic of Croatia.

The study was designed to identify seed-borne fungi on bean (Phaseolus vulgaris L.) crops grown in 13 counties of the Republic of Croatia and their association with ochratoxin A (OTA) production. Bean samples (N=45) were collected in Croatia in 2001 shortly after the harvest and were stored at -20 degrees C for mycological and mycotoxin analyses. The most common fungi isolated were Cladosporium spp. (98%) Alternaria spp. (75%), Aspergillus spp. (73%), Rhizopus spp. (73%), Penicillium spp. (69%), Fusarium spp. (38%), Botrytis spp. (27%), Trichothecium spp. (24%), and Chaetomium spp. (18%). OTA was found only in samples contaminated with Penicillium and Aspergillus spp. Using HPLC (detection limit 0.25 microg/kg), OTA was found in 17 out of 45 samples (38%), and the mean concentration in positive samples was 0.41+/-0.21 microg OTA/kg. Beans from south Croatia (Adriatic coast) were OTA-free and the least mould-infected, while the mean OTA concentration and mould infection of samples from other regions were similar. The OTA contamination of beans in our country is low. Although beans are not severely contaminated with OTA, their consumption may contribute to the exposure to OTA from other commodities.

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.

Changes in plasma lipids after a non-lethal dose of cycloheximide in rats.

This paper describes a study of the effect of a single intraperitoneal non-lethal dose of cycloheximide (CHM; 2.0 mg/kg body weight) on the concentration of plasma lipids and lipoproteins in male rats killed one, two, three, four and nine days after receiving the dose. The concentration of triglycerides, total cholesterol, high-density lipoproteins (HDL)-cholesterol and low-density lipoproteins (LDL)-cholesterol was measured in treated and control animals. The effect of CHM on the concentration of triglycerides, total cholesterol, HDL-cholesterol, and LDL-cholesterol was visible in rat plasma throughout the study. Total cholesterol and HDL-cholesterol concentrations showed the same pattern of changes, probably due to the reversible inhibition of apolipoprotein apo A-I synthesis by CHM. The concentration of triglycerides decreased after a lag period of three days when the reserves of apolipoprotein apo B, the main apolipoprotein of very low-density lipoproteins (VLDL)-cholesterols produced in the liver, were consumed.

Variations of ochratoxin A concentration in the blood of healthy populations in some Croatian cities.

The nephrotoxic mycotoxin ochratoxin A (OTA), a common contaminant of cereals, has been implicated in the etiology of endemic nephropathy. It was also frequently found in low concentrations in blood of healthy populations in countries where endemic nephropathy is not known. However, data on regional and seasonal differences in the frequency and concentration of OTA in human blood are scarce. In June, September and December 1997, and March 1998, about 50 human blood samples were collected randomly from blood donors for blood banks in the Coatian cities of Osijek, Rijeka, Split, VaraZdin and Zagreb. OTA was measured in the total of 983 samples using an HPLC technique with fluorescent detection. The daily intake of OTA was estimated from the mean concentration found in different cities and at different times of year. Samples containing OTA above the detection limit (0.2 ng/ml of plasma) were found in populations from all Croatian cities at all collecting periods. The highest frequency (59%) of samples containing OTA above the detection limit and the highest mean concentration (0.39 ng/ml) were found in June. Both the frequency and the mean concentration were lowest in all samples in December (36% and 0.19 ng OTA/ml, respectively). Osijek was the city with the highest frequency of OTA-positive samples (81%) and the highest mean OTA concentration (0.56 ng/ml). The total mean concentration of OTA in blood of healthy population in Croatia is lower (0.30 ng/ ml) than the mean concentration in European countries as a whole (0.90 ng/ml). The estimated daily intake, calculated from the mean concentration in all blood samples, is 0.40 ng OTA/kg body weight, which is much lower than that proposed by World Health Organization as the tolerable daily intake (16.0 ng/kg body weight). Healthy populations of Croatia are exposed to low, but seasonally and regionally variable amounts of OTA.

Contamination of food with mycotoxins and human health.

Mycotoxins are natural contaminants of cereals and other commodities throughout the world. They are produced by various strains of moulds, particularly in tropical countries. Due to significant trade of cereals, humans in temperate countries can also be exposed to mycotoxins. The most common route of exposure to mycotoxins is ingestion, but it may also involve dermal, respiratory, and parenteral routes, the last being associated with drug abuse. Apart from acute and chronic toxic effects on human health called mycotoxicosis, some mycotoxins are proved or suspected human carcinogens. This paper describes various human diseases caused by ergot, afflatoxin, ochratoxin A, 3-nitropropionic acid, trichothecene, zearalenone, and fumonisin. It also gives a quick review of human carcinogenicity evaluations of the international Agency for Research on Cancer and of regulatory limits of mycotoxin concentrations in various commodities.

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.

Ochratoxin A in blood of healthy population in Zagreb.

Healthy blood donors from the city of Zagreb were checked for the presence of a nephrotoxic mycotoxin ochratoxin A (OTA) in the plasma. Samples of blood were collected in June, September, and December 1997, and March 1998, totalling 200 or 50 in each round. The concentrations of OTA were measured using high pressure liquid chromatography (HPLC) method (detection limit 0.2 ng OTA/ml of plasma). The frequency of OTA-positive samples (> 0.2 ng/ml of plasma) showed significant seasonal variation (P < 0.001). The frequency of OTA-positive samples was the highest in March (65%) and it gradually decreased towards December (12%). The high frequency of positive samples coincided with seasons favouring growth of moulds and production of toxins. The daily intake of OTA by healthy persons in Zagreb was estimated from the mean concentration of OTA in samples collected during the whole year (0.19 ng OTA/ml plasma). The estimated daily intake was 0.26 ng/kg b.w., that is, substantially below the tolerable daily intake proposed by World Health Organization (16.0 ng/kg b.w.).