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Opiates such as morphine and codeine are substances often misused to improve the performance of racing horses during competitions and are therefore on the International Federation for Equestrian Sports' list of banned substances. However, a positive antidoping test may be due to the consumption of feed (mainly alfalfa or oats) contaminated by poppy seeds containing the alkaloids morphine and codeine. In order to determine whether a positive antidoping test is the result of an intentional abuse of opiates or ingestion of feed contaminated by poppy seeds, we optimised conditions for the extraction of morphine and codeine from dehydrated alfalfa and developed and validated a gas chromatographic-mass spectrometric method for the simultaneous determination of both analytes. The most efficient extraction of morphine and codeine from dehydrated alfalfa was achieved using a citrate buffer pH4 followed by solid phase extraction. The method showed good linearity (R2>0.9980) in the tested concentration range (50-1600 ng/g), as well as good precision (RSD

The use of cannabinoid-based preparations by cancer patients raises concern whether delta-9-tetrahydrocannabinol (THC) can modulate or even compromise the effectiveness of concurrently administered anticancer drugs. Irinotecan (IRI) is a type of drug that causes various severe adverse effects such as diarrhoea, gastrointestinal toxicity and myelosuppression. Newer reports point to hepatotoxicity being an underestimated but important IRI side effect as well. This study focused on the evaluation of potentially detrimental interactions of IRI and THC in the liver of Wistar rats. Male rats were concomitantly exposed to IRI (at 100 mg/kg b.w., administered once i.p.) and THC (administered repeatedly for 1, 3 and 7 days per os at 7 mg/kg b.w.). Single IRI, THC and control groups were studied in parallel. We estimated changes in body and liver weight caused by the treatments, evaluated the level of primary DNA damage in hepatocytes using the alkaline comet assay, and evaluated the level of lipid peroxidation and catalase (CAT) activity. Changes in body weights and liver weights observed after the treatments suggest that all of the compounds at the tested doses and the applied experimental schedule produced acute toxicity and diminished the overall fitness of the exposed compared to control rats. It was most pronounced in rats administered a combined treatment. DNA damage was the most pronounced after 3-day treatment, and its highest level was observed in hepatocytes of single IRI-treated rats, followed by those given combined treatment. In contrast to 3-day, 7-day treatment with single THC slightly impaired hepatocyte DNA integrity. Rats given combined treatment demonstrated increased lipid peroxidation and higher CAT levels than those administered single IRI, at both time points, which may indicate that combined treatment induced more intense oxidative stress. Our findings provide evidence regarding a significant synergic enhancement of IRI toxicity caused by THC intake, which was confirmed using all of the applied biomarkers. Nevertheless, since we tested only one IRI and THC dose, further studies are required to further clarify their mutual interactions.

Organophosphate compounds are among the most frequently used pesticides worldwide. In this study, we evaluated the in vivo effects of the insecticide chlorpyrifos, which was orally administered to male Wistar rats at 0.160 mg/kg, 0.015 mg/kg, and 0.010 mg/kg for 28 days. Following treatment, the level of lipid peroxidation and acetylcholinesterase (AChE) activity was estimated in plasma and brain. To establish the potential DNA damaging effects of the exposure, we applied an alkaline comet assay on the white blood cells and brain tissue of the exposed and control animals. We found that 28-day exposure resulted in dose-dependent changes in AChE activity, which was significantly more depressed in the brain. Lipid peroxidation, presented as TBARS concentration, was elevated both in plasma and in the brain. All of the tested doses of chlorpyrifos were slightly genotoxic, both to the white blood cells and brain tissue. Taken together, our findings confirmed the AChE-inhibiting potency of chlorpyrifos and indicated that its toxicity was mediated through free radicals, which contributed to DNA instability. Considering that our study focused on very low doses of chlorpyrifos within toxicology reference values our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Organophosphorus (OP) compound poisoning is an important worldwide clinical and public health problem. Some nerve warfare agents have relatively recently been used in terrorist attacks, while pesticide poisonings represent the leading cause of poisonings reported globally. OPs act primarily as neurotoxins due to the irreversible inhibition of acetylcholinesterase (AChE), but there is increasing evidence for secondary mechanisms of their toxicity. Oximes act as an essential part of antidotal therapy by ensuring the recovery of inhibited AChE. Our recent in vitro and in vivo studies have drawn attention to oxime 1- (4-hydroxyiminomethylpyridinium)-4-(4- carbamoylpyridinium)-butane dibromide (K048) as a very potent reactivator of the nerve agent tabun- and pesticide-inhibited AChE. This study evaluated OP toxicity at molecular level and its potential alleviation by the use of an oxime. For this purpose, as it is one of the most potent AChE-inhibiting compounds available, we selected paraoxon as a model OP and estimated its ability to induce primary DNA damage in two human cell lines known for non- neuromuscular AChE expression. By applying the K048 oxime, we tested its ability to extenuate the effects of paraoxon. Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line HaCaT were cultivated in RPMI growth medium supplemented with glutamine, heat-inactivated fetal bovine serum, and antibiotics. Prior to treatment, cells were seeded in 6-well plates (3x105 cells/mL). The tested concentrations of paraoxon (1 µM), and K048 oxime (0.02 mM) were determined in a preliminary experiment on human erythrocytes. For the genotoxicity testing, we applied the same study design in both cell lines: (1) control group (untreated cells), (2) cells treated with paraoxon (for 10 min), (3) cells treated with K048 (for 30 min), (4) cells treated 10 min with paraoxon followed by a 30- min treatment with K048 oxime. After the treatments, aliquots of cells were used for the preparation of agarose microgels according to standard protocol for the alkaline comet assay. Slides were analyzed under fluorescent microscope using the Comet Assay IV analysis system (Perceptive Instruments Ltd., UK). The level of DNA damage was evaluated based on comet tail length, tail intensity, and tail moment. AChE activity measurements were performed by using the standardized spectrophotometric Ellman method. At the concentration tested, the K048 oxime did not induce a significant increase of primary DNA damage as compared to the negative control. In contrast, treatment with paraoxon, in spite of the relatively short exposure time, resulted in measurable genotoxicity both on A549 and HaCaT cells. At the same concentration, paraoxon caused a 93% erythrocyte AChE inhibition in our preliminary experiment. The most important observation was the lowering of primary DNA damage in cells treated with both compounds. In both cell lines, when applied after paraoxon, K048 caused a significant decrease of all comet assay parameters as compared to single paraoxon treatment. Furthermore, K048 was shown to reactivate about 87% of paraoxon-inhibited erythrocyte AChE. Apart from its high reactivation potency at the concentration tested, the K048 oxime also possesses antioxidative efficacy, which is an important property for a compound intended for use as an antidote.

Aim: Tabun is a highly toxic chemical warfare agent which causes fatal intoxication due to inhibition of acetylcholinesterase (AChE). Recent knowledge confirms the use of pyridinium oximes as causal antidotes for the protection/reactivation of AChE inhibited by organophosphorus compounds. K048 oxime showed an acceptable genotoxic profile both in vitro and in vivo models. Here we studied its potential neuroprotective effects in vivo using the alkaline comet assay in brain cells of rats which were given K048 oxime to relieve symptoms of tabun-poisoning. Methods: Altogether sixty male adult Wistar rats were used in the experiment. They were divided in five groups according to the treatment: (1) tabun-poisoned (75% LD50, s.c.), (2) K048-treated (25% LD50 i.p.), (3) atropine-treated (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), (4) concomitant treatment with K048 (25% LD50, i.p.) and atropine (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), and (5) negative controls given saline only (2 mL kg-1, i.p.). Rats were sacrificed 1, 6 and 24 h after the treatment, and comet assay on brain cells was performed. Results: We found that tabun itself caused a most notable increase of primary DNA damage in rat brain cells 24 h after its administration. K048 oxime had an acceptable genotoxicity profile at each time- point studied. Minor differences in the genotoxicity observed between three time-points could be explained by the metabolism of the compound. Concomitant administration of K048 with atropine changed its genotoxicity profile in the brain cells, especially 6 h and 24 after treatment, which has to be further studied. Conclusions: Taken together, K048 effectively counteracted the poisoning of rats by tabun, both alone and in combination with atropine. Observed neuroprotective ability speaks in favour of K048 as a promising molecule for therapeutic treatment against intoxication with organophosphorus compounds.

Aim: Oximes play an important role in the elimination of toxic effects caused by chemical warfare agents, especially in the cases resistant to the atropine treatment alone. K048 oxime as one of the promising antidote in humans recently showed an acceptable genotoxic profile. Present study is focused on potential hepatoprotective effects of K048 effects in vivo. Methods: We selected a model of tabun- poisoned rats, which were given K048 oxime to relieve symptoms of tabun-poisoning. The levels of primary DNA damage in liver cells were studied using the alkaline comet assay 1, 6 and 24 h after the treatments. Sixty male adult Wistar rats were divided in five groups according to the treatment: (1) tabun-poisoned (75% LD50, s.c.), (2) K048-treated (25% LD50 i.p.), (3) atropine-treated (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), (4) concomitant treatment with K048 (25% LD50, i.p.) and atropine (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), and (5) negative controls given saline only (2 mL kg-1, i.p.). Results: Tabun itself caused the highest level of primary DNA damage in hepatocytes 24 h after its administration. K048 oxime had an acceptable genotoxicity profile at each time point. Its hepatoprotective effects were obvious at each time point studied, and in all cases the levels of primary DNA damage measured in oxime-treated groups were lower or similar as compared to negative control. Minor differences in the genotoxicity observed between the time-points studied could be explained by the metabolism of the compound. Concomitant administration of K048 with atropine changed its genotoxicity profile in the hepatocytes, especially 6 h after treatment. Conclusions: K048 effectively counteracted the poisoning of rats by tabun, both alone and in combination with atropine. Hepatoprotective effects speak in favour of K048 as a promising molecule for therapeutic treatment against fatal poisoning with chemical warfare agents.

The work examines the level of metals in various tissues of wild caught Muraena Helena from Adriatic sea in the year 2010 by the atomic absorption spectrometry (Perkin-Elmer Analyst 600 Zeeman equipped with a THGA-600 graphite furnac). The analysis shows that Hg and As had highest levels in muscle, gills and especially liver while Pb and Cd had highest levels in skin.

Organophosphorus (OP) compounds cause fatal intoxication based on inhibition of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. There is an obvious need for appropriate therapeutic treatment against them due to their application in agriculture or their misuse in terrorist or war attack. Pyridinium oximes are causal antidotes employed for the protection/reactivation of AChE inhibited by OPs. Although their pharmacological profile is well known, possible cyto- and genotoxic effects have not been studied in detail so far. In present in vivo study we wanted to elucidate the toxicity profile of the standard oxime HI-6 and its modification - oxime K048 by simultaneous use of biochemical and genotoxicity assays. The catalytic activities of total cholinesterases (ChE) in plasma and of AChE in brain were determined 30 min, 60 min and 24 h after the rats were administered with single therapeutic dose of oximes (25% of their LD50). In addition, blood and brain tissue were sampled 24 h after treatment and used for the alkaline comet assay to study the levels of primary DNA damage in white blood cells (WBC) and brain cells. Experimental animals did not exhibit any clinical signs of poisoning following application of both oximes. Up to 25.3% decrease in plasma and up to 18.5% decrease in brain enzyme activities was observed in all experimental animals after application of HI-6. A decrease of enzyme activities after application of K048 was noticed only for time-point at 60 min in both plasma (11.9%) and brain (20%). These results indicate higher affinity of intact blood and tissue AChE for HI-6 but there were no significant differences compared to corresponding controls. Treatment with HI-6 and K048 did not induce a significant increase of DNA damage compared to the corresponding controls. Mean tail DNA% in the WBC of HI-6 treated rats was 0.27±0.03% (range: 0-3.90%), and in the brain cells 0.99±0.06% (range: 0-4.87%). Treatment with K048 induced slightly higher levels of primary DNA damage, compared to HI-6, both in the WBC (mean tail DNA% 0.56±0.07, range: 0-8.11%) and in the brain cells (mean tail DNA % 1.14±0.14, range 0-21.27%). Although both oximes had acceptable genotoxicity profiles, HI-6 induced lower level of DNA damage in the brain cells than K048 ; there were 6% of highly damaged comets with respect to the tail DNA% in HI-6 treated rats vs. 10% of highly damaged comets with respect to the tail DNA% in K048 treated rats. Taken together, results obtained by the enzyme assay and the alkaline comet assay, speak in favour of HI-6 as a promising molecule for therapeutic treatment against intoxication with OP compounds.

Oximes are pharmacologically important nucleophylic agents acting as antidotes against poisoning by organophosphorus compounds. In this study, effect of K048 oxime on cell viability and chromosome stability in vitro was evaluated in primary cell cultures of isolated human peripheral blood lymphocytes. K048 oxime at concentrations of 730, 200, and 7.3 nmol/dm3 was tested in 30 minutes treatment. Cytotoxic effect was tested by using differential viability staining with a mixture of acridine orange and ethydium bromide, while in evaluation of genotoxic potential hOGG1 modified comet assay, cytokinesis-blocked micronucleus assay, and chromosome aberration analysis were used. Viability staining showed that oxime treatment does not induce apoptosis (8.0% highest concentration vs. 7.0% control) or necrosis indicating lack of its cytotoxic effect (9.6% highest concentration vs. 7.3% control). HOGG1 modified comet assay revealed dose dependant increase in the level of oxidative primary damage to DNA, significant at all doses tested. Expressed as % of DNA in comet tail, oxidative damage level in K048 treated lymphocytes ranged from 7.73-11.46% compared to 2.12% in control lymphocytes. However, oxime treatment did not affect micronucleus frequency nor induced significant increase in formation of structural chromosomal aberrations. Micronucleus frequency in oxime treated lymphocytes ranged from 13-19 per 3000 binucleated cells compared to 15 micronuclei per 3000 cells in control cultures. Chromatid breaks were detected in both, control and oxime treated lymphocytes with no difference in frequency, while formation of acentric fragments was observed only in lymphocytes treated with lowest K048 concentration (7.3 nmol/dm3). However, observed level of acentric formation (2 per 200 cells) was not statistically significant. In applied battery of cyto/gentoxicity assays, considering applied testing conditions K048 oxime showed acceptable biocompatibility at the level of cell viability and chromatin/chromosome integrity. Since no increase in secondary genome damage was detected, induced primary oxidative DNA damage might be result of cell stress induced by treatment and are rather repaired than fixed as chromosome damage.

Since the mycotoxin theory of endemic nephropathy (NE) was put forward, the research on EN in Croatia and Bulgaria were focused on the nephrotoxic mycotoxin ochratoxin A (OTA) (1, 2), while the presence of other nephrotoxic mycotoxins such as fumonisin B1 (FB1) were tested in biological materials of inhabitants in EN areas only sporadically (3). It was proved that higher concentrations of OTA (4) and FB1 (5) might be found in cereals collected in EN area in Croatia than in control areas. Cereals in our country are very often contaminated with two or more mycotoxins (6). Very often finding of FB1 in our previous study and in study of Jurjević et al. indicated that the research on the simultaneous exposure to both mycotoxins would be interesting. In this study 45 human urine samples were collected in 2000 and 2005 from the same persons in the endemic village Kaniža in Croatia and 18 controls. In all samples the concentration of OTA and concentrations of sphinganine (Sa) and sphingosine (So) were measured using HPLC with fluorescence detector (7, 8). The ratio of Sa and So (Sa/So) was found to be good biomarker of exposure to FB1 in experimental animals because it increases significantly after exposure to this mycotoxin. In 2000 and 2005 the frequency of OTA- positive samples was higher in Kaniža (43% and 18 %, respectively) than in the control village (28% and 6%, respectively). The OTA concentration in samples collected in Kaniža in 2000 was higher than in 2005 (p

Fasciloides magna an etiological agent causing liver fasciloidiasis in deer has been recognized and described in Europe more than 130 years ago.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic and carcinogenic potential. Various animal models have demonstrated that OTA mainly impairs proximal tubule (PT) function and causes glucosuria, enzymuria, and diminished secretion of organic anion p-aminohippurate (PAH). The last indicates that OTA toxicity may target renal organic anion transporters (Oats ; subfamily of Slc22a drug transporters). Oat1 (Slc22a6) and Oat3 (Slc22a8) proteins reside in the basolateral membrane (BLM) of PT epithelial cells and mediate both OTA and PAH transport in a noncompetitive mode. Previous studies have shown that subchronic intoxication of rats with OTA leads to a dramatic reduction of PAH clearance/excretion, indicating that Oat1 and Oat3 may be involved in OTA nephrotoxicity, possibly via inhibiting their transport activity and/or expression in the cell membrane. However, these possibilities have not been clarified. In order to study effects of OTA on cell/tissue morphology and on cellular expression of renal Oat1 and Oat3, especially in view of male-dominant expression of these transporters in renal proximal tubules in rats and mice, we used an established subchronic in vivo model of OTA intoxication in adult male (M) and female (F) Wistar rats. The animals of both sexes were treated with various doses of OTA (0, 50, 125, 250 & 500 μ g/kg b.w., p.o., every 2nd day for 10 days). Using peptide-specific polyclonal antibodies, Oats were studied by Western blotting (WB) in total cell membranes (TCM) isolated from the cortex (C) and outer stripe (OS) tissues, and by immunofluorescence cytochemistry (IC) in tissue cryosections, whereas the expression of their mRNA was analyzed by RT-PCR using RNA from the renal C and OS. The study of tissue morphology revealed that OTA treatment resulted in damaged PT S3 segments in medullary rays, manifested by the loss of regular tubular structure and cell membranes, cell desquamation and other necrotic events. These phenomena were more severe at higher OTA doses, and were much more pronounced in M. As found in WB experiments, in spite of different levels of the Oat1 and Oat3 protein expression in isolated membranes (M>F), their expression in OTA-treated rats in both sexes followed a similar and dual, dose-dependent pattern. At low OTA doses (50-125 μ g/kg b.w.) the expression of Oat1 and Oat3 protein increased ~50% and ~300%, respectively, whereas high OTA doses (250-500 μ g/kg b.w.) decreased the expression of Oat1 (~70%) but not of Oat3 in respect to controls (vehicle-treated rats). By IC, the staining intensity of both transporters in proximal tubules completely matched the WB data. However, the RT-PCR data showed that the Oat1 mRNA expression was unaffected by low OTA doses, whereas high OTA doses downregulated it, and the expression of Oat3 mRNA was unaffected by any OTA dose. Also the expression of housekeeping gene GAPDH mRNA remained unchanged in all OTA-treated animals. Our data indicate that in rats: 1) the OTA treatment causes dose-related and M-dominant damage to cell morphology in proximal tubule S3 segments, 2) Oat1 and Oat3 have a similar pattern of protein expression for both sexes, with upregulation of both transporters at low but downregulation of only Oat1 at high OTA doses, 3) the upregulation of Oat1 and Oat3 proteins seems to be mRNA-unrelated and post-transcriptional, 4) previously observed inhibition of the renal PAH transport/secretion in rodents treated with high OTA doses may be explained by the loss of cell plasma membrane (transporting surface) and Oat1 protein in this membrane, and 5) upregulated Oat1 and Oat3 proteins at low OTA doses may possibly enhance OTA uptake and accelerate the development of nephrotoxicity.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

OTA is an environmental mycotoxin that can impair human and animal health. In the kidney, OTA primarily targets epithelial cells in the proximal tubule (PT). PT is the major site of organic anion (OA) secretion via basolateral transporters Oat1 (Slc22a6) and Oat3 (Slc22a8). Earlier studies have shown that OTA inhibits the renal secretion of OA p-aminohyppurate (PAH), possibly by targeting the expression of Oat1 and Oat3, but this possibility has not been verified. To see if OTA affects the expression of these two Oats, we treated adult male Wistar rats with various doses of OTA (0, 50, 125, 250 & 500 μ g/kg b.w., p.o.) every 2nd day for 10 days, and determined: a) the expression of Oat1 and Oat3 proteins by Western blotting (WB) of total cell membranes isolated from the kidney and by immunocytochemistry (IC) in tissue cryosections, and b) the tissue expression of Oat1 and Oat2 mRNA by end-point PCR. The WB study of protein expression revealed a dual, dose-dependent effect of OTA ; low doses (50-125 μ g/kg b.w.) increased the expression of Oat1 (~50%) and Oat3 (~300%), whereas high doses (250-500 μ g/kg b.w.) decreased the expression of Oat1 (~70%) but not Oat3 in respect to control. The IC data on staining intensity of both transporters in the PT completely matched the WB data. At the mRNA level the expression of Oat1 was unaffected by low OTA doses and downregulated by high OTA doses, whereas the expression of Oat3 was unaffected by any OTA dose. The mRNA expression of housekeeping gene GAPDH remained unchanged in all OTA-treated animals. We conclude that: a) low doses of OTA upregulate the expression of Oat1 and Oat3 proteins in the rat kidney, while high doses downregulate only the expression of Oat1, b) upregulation of Oat1 and Oat3 proteins is mRNA-unrelated, and seems to be post-transcriptional, c) previously observed inhibition of the renal PAH secretion in rodents may be related to the loss of Oat1 protein ; and d) at lower OAT doses, upregulated Oat1 and Oat3 proteins may enhance OTA uptake and accelerate the development of nephrotoxicity.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

Ochratoxin A (OTA) and fumonisin B1 (FB1) are nephrotoxic mycotoxins that contaminate various commodities, and humans are constantly exposed to low levels of these mycotoxins. Aim of this study was to find out whether very low doses of OTA and FB1, given separately or combined, induce oxidative stress in rat liver and kidney, and whether their effect is synergistic. The concentration of malondialdehyde (MDA) (1) and protein carbonyls (PCs) (2) and the activity of catalase (3) and superoxide dismutase (SOD) (4) were measured in the liver and kidney of rats orally treated with OTA (5 ng/kg b.w. and 50 ug/kg b.w.) and FB1 (200 ng/kg b.w. and 50 ug/kg b.w.), or their combinations. The lowest doses in this study correspondents to the estimated daily intake for OTA and FB1 for humans in Europe. The concentration of MDA and PCs was higher in the liver and kidney when 50 ug OTA/kg b.w. was applied (P

Literature data on carbofuran genotoxicity in vitro and in vivo are very scarce. There are few papers indicating that occupational exposure to this AChE inhibiting insecticide might be connected to increased risk of developing non-Hodgkin’ s lymphoma and lung cancer. Other authors showed its genotoxicity in vitro. We used comet and CBMN micronucleus assay combined with centromere probes to evaluate genome damage in lymphocytes of workers employed in carbofuran production. Also, the level of AChE activity in blood and plasma was measured. Only few workers exhibited AChE activity below 85%. Comet assay parameters were slightly but significantly elevated compared to control subjects, especially the long-tailed nuclei ratio. We found poor correlation between AChE activity and comet assay parameters, but significant effect of smoking and alcohol intake on the latest. In binucleated lymphocytes of workers significantly increased number of micronuclei, nuclear buds, and nucleoplasmatic bridges was detected. Proportion of micronuclei with centromere, DAPI signal positive micronuclei was also elevated. Micronucleus assay parameters also appeared to be significantly influenced by duration of exposure to carbofuran. Together with published data on carbofuran’ s effect on health our results might indicate the need for further evaluations of its genotoxicity using a range of different cytogenetic techniques.

Organophosphorus compounds are widely used as pesticides and unfortunately as nerve agents in chemical warfare. They are known inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) an enzyme that hydrolizes the neurotransmitter acetylcholine in the nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper three new bis-pyridinium compounds: K033 [1, 4-bis(2-hydroxyiminomethylpyridinim)butane dibromide], K027 [1, 4-hydroksyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as potential antidotes in tabun poisoned mice. Their antidotal effect was compared with TMB-4 [1, 3-bis(4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. In all experiments, oxime K033 in doses of 1/4 or 5% of its LD50 was used for the pre-treatment 15 minutes before tabun-intoxication. Also, one or 5 minutes after tabun application experimental animals received oxime K027, K033 or K048 (5% or 1/4 of its LD50) plus atropine sulphate as therapy. The antidotal efficacy of tested compounds was expressed as therapeutic factor (TF) and therapeutic dose (TD). Under same experimental conditions, our experiment selected compound K048 as the most reactivator of tabun inhibited AChE. Namely, this study has shown that the therapeutic regimen consisting of K033 in dose 5% of its LD50 as preatretment and &frac14 ; of LD50 of K048 plus atropine as treatment had the highest TF and TD. The TF was 13.3 LD50 of tabun, TD was 10 LD50 of tabun and insurance survival of all tested animals. In conclusion, treatment with these new bis-pyridinium oximes seems to be a very good alternative for current treatment in tabun poisoning. For this reason, these and other similar compounds require further investigation.

Mycotoxin ochratoxin A (OTA) is produced by Penicillium and Aspergillus spp. that frequently contaminate cereals and other commodities not only in tropical countries but also in countries with mild climate. OTA has hepatotoxic, genotoxic and carcinogenic effect in all tested animal species, and due to its pronounced nephrotoxicity it was supposed to be involved in the development of endemic nephropathy. Mechanism of its toxicity and genotoxicity is not understood. The proposed mechanisms of OTA toxicity involve inhibition of protein synthesis, disturbance of gluconeogenesis, and production of reactive oxygen species. In this study the importance of oxidative stress in the mechanism of OTA toxicity in rats was tested. Adult male Wistar rats were treated orally with multiple daily doses of OTA for 15 days and sacrificed 24 hours after last treatment. The doses of OTA were: 5 ng/kg b.w., 0.05 mg/kg b.w. and 0.5 mg/kg b.w., respectively. The lowest dose used in this study is the estimated daily intake for OTA for humans in Europe. In order to determine the effect of OTA on lipid peroxidation and oxidative damage of proteins, concentrations of malondialdehyde (MDA) and protein carbonyls were measured in liver and kidney homogenate. In liver two higher doses of OTA significantly increased concentration of MDA and protein carbonyls (p

In the early seventies of the last century, a hypothesis was put forward that mycotoxin ochratoxin A (OTA) was involved in the etiology of endemic nephropathy (EN). EN is a human interstitial bilateral noninflammatory kidney disease with fatal outcome with the highest prevalence in early seventies. Ten years after this peak, a high incidence of otherwise rare urothelial tumors was first noticed in the endemic area of Bulgaria, and then in other countries. The fact that the appearance of EN and these tumors in farmers is geographically limited suggests there may be a unique, and most probably natural cause of both diseases. Although OTA was found in the dust of grain and coffee in food production facilities, the main source of human exposure is food, because it contaminates food of vegetable and animal origin worldwide. Large follow-up studies were performed in Croatia and Bulgaria whose aim was to see whether the population of the EN regions was exposed to higher OTA concentrations than other populations. In both countries, mean blood OTA concentrations in the endemic populations were higher than in control populations. However, OTA was also found in food, feed and human blood in countries where EN has not been detected. Regional differences in OTA blood concentrations in healthy population have been established in Canada, Croatia, France, Sweden, Switzerland, and Tunisia. In Croatia, blood samples obtained from blood banks showed seasonal variations in OTA concentrations, and the highest number of OTA positive samples, as well as the highest mean OTA concentration were found in samples collected in the summer period. OTA was also found in breast milk (Italy, Norway, and Sweden). In the Czech Republic, Italy, Spain, and Turkey dialysis patients had higher OTA concentrations in blood than patients with other renal diseases or healthy persons. Several attempts have been made to link exposure to OTA in patients suffering from the end-stage kidney disease in North Africa with nephropathy.

Ochratoxin A (OTA) is nephrotoxic and carcinogen product of moulds. In the early seventies the hypothesis of its involvement in the etiology of endemic nephropathy (EN) was put forward. EN is human interstitial bilateral noninflammatory kidney disease with fatal outcome that occurs in farmers’ households in geographically limited rural areas of Bosnia and Herzegovina, Bulgaria, Croatia, Rumania, Serbia and Montenegro. The prevalence of EN in Bulgaria, Serbia and Montenegro, and Croatia after the peak at early seventies has declining trend. Ten years after this peak, a high incidence of otherwise rare urothelial tumors was first noticed in the endemic area of Bulgaria, and then of other countries. These tumors are often multiple, affect mostly upper urothelial tract and show clinical picture different from tumors with the same location in patients out of the EN region. Geographically restricted area of the appearance of EN and these tumors in farmers suggests the possibility of the etiological factor for both diseases is unique, most probably of natural origin. Although OTA was found in dust of grain and coffee in food production facilities, the main source of human exposure is food, because it contaminates food of vegetable and animal origin worldwide. In Bulgaria and Croatia special attention was paid to OTA contamination of various commodities in areas with EN. Thus, in EN area of Bulgaria the OTA contamination of beans and maize showed a high variability in different years, but the mean OTA concentration and the frequency of positive samples was higher than in control areas. In EN region in Croatia, OTA was found in maize, wheat, barley, and beans and in some studies the mean OTA concentration in maize and wheat was reported to be higher in samples collected in EN area than in controls. Large follow-up studies were performed in Croatia and Bulgaria aimed to check whether the inhabitants of EN regions are exposed to higher OTA concentrations than other population. In both countries, albeit the variability of blood OTA concentration from year to year, the mean OTA concentration in inhabitants of endemic villages as well as higher number of OTA-positive samples was seen as compared to other regions. However, OTA was found in food, feed and human blood in countries where EN has not been detected. In Canada, Croatia, France, Sweden, Switzerland, and Tunisia, regional differences of OTA blood concentrations in healthy population have been found. In Croatia blood samples obtained from blood banks showed a seasonal variations in OTA concentrations, and the highest number of OTA positive samples, as well as the highest mean OTA concentration was in samples collected in summer period. OTA was also found in breast milk (Italy, Norway, and Sweden), and in urine (Bulgaria and Croatia) of healthy persons. In Czech Republic, Italy, Spain, and Turkey it was noticed that patients at hemodialysis have higher OTA concentration in blood than patients with other renal diseases or healthy persons. There are several attempts to link exposure to OTA in patients suffering of end stage kidney disease in North Africa with nephropathy.

Enzim acetilkolinesteraza (AChE) je izravni biomarker toksičnog djelovanja antikolinesteraznih spojeva kojima pripadaju brojni pesticidi po kemijskoj strukturi organofosforni spojevi i karbamati. Određivanje aktivnosti AChE u krvi radnika profesionalno izloženih antikolinesteraznim şpojevima jedina je mjera koja ukazuje na izloženost radnika tim spojevima, i način na koji se može uočiti potencijalna opasnost te spriječiti daljnja izloženost. Kroz petogodišnje razdoblje obrađeno u ovoj studiji radnici su kontrolirani jednom godišnje što svakako nije dovoljno s obzirom na nalaze koje smo našli kod određenog broja radnika. U njih su uz karakteristične simptome otrovanja, aktivnosti AChE pune krvi bile inhibirane 30% ili više. Međutim, pritužbe radnika nisu uvijek bile u skladu s inhibicijom enzima. Naime, u nekoliko osoba u kojih su bile prisutne subjektivne smetnje, inhibicija AChE nije bila značajno smanjena. Uvidom u ranija mjerenja i praćenja izloženosti radnika antikolinesteraznim spojevima u proizvodnji pesticida uočeno je da se kontrolom mjerenja aktivnosti AChE više puta unutar jedne godine može smanjiti broj otrovanih radnika i stupanj otrovanja. Također, upotrebom propisanih zaštitnih sredstava pri radu, i pridržavanjem higijenskih i sanitarnih odredaba moglo bi se pridonijeti još boljem stupnju zaštite radnika u proizvodnji pesticida.

This paper describes effect of glucocorticoide dexamethasone (DM) given intraperitoneally on the catalytic activity of buthyrylcholinesterase (BuChE) measured in plasma, liver and white adipose tissue of rats of both sexes. The effects of DM on the concentration of plasma lipids and lipoproteins were also tested. The rats were given multiple (2 and 4) pharmacological doses (0.4 and 3.0 mg kg-1 body mass) of DM. All animals were sacrificed 48 hours after the last dosing. The administration of DM significantly decreased the catalytic activity of BuChE in plasma and liver of all treated groups regardless of the sex. BuChE catalytic activity in white adipose tissue differed depending on dose and frequency of administration. In contrast to liver where both doses caused significant BuChE inhibition, in adipose tissue the lower DM dose did not inhibit BuChE activity, and the inhibition achieved by the higher dose was not as strong as in liver. This result corroborates an earlier hypothesis that BuChE is also synthesized in the adipose tissue. DM significantly increased plasma concentrations of triglycerides, total cholesterol and high-density lipoprotein (HDL) cholesterol and decreased low-density lipoprotein (LDL) cholesterol concentration. There was neither positive correlation between BuChE and triglycerides nor negative correlation between BuChE and HDL. Changes in lipid profile during DM treatment were not sex- and time-dependent.

Acethylcholinesterase (AChE) is an extremely active enzyme. Irreversible inhibition of AChE by organophosphorus compounds (OPs) results in the accumulation of endogenous acetylcholine in synaptic cleft and paralysis of nerve impulse transmission in the central and peripheral nervous system. In order to find the best treatment of organophosphate poisoning new compounds have been synthesized and tested. The aim of this study was to evalute the pretreatment efficacy of adamantyl tenocyclidine derivative TAMORF which antagonize the NMDA receptors and might protect AChE. Its antidotal effect was compared with physostigmine which also has a good prophylactic efficacy in soman poisoning. The therapeutic efficacy of TAMORF and physostigmine was tested on rats poisoned with two different sub-lethal dose of soman (&frac14 ; ; and &frac12 ; ; of LD50). Our results indicate that TAMORF when administered 5 min before intoxication clearly stopped soman-induced seizures and markedly improved respiration of animals. In contrast to TAMORF, physostigmine seems to be slightly effective in the elimination of soman-induced toxicity in rats. Catalytic activities of AChE in brain, especially after administration of higher dose of soman (&frac12 ; ; of LD50) were additionally lower throughout of the experiment, except after 24 h. Similar results with physostigmine in contrast to TAMORF were obtained in plasma also. In conclusion, treatment with TAMORF seems to be a good alternative for current pretreatment in soman intoxication.

Ochratoxin A (OTA) is a mycotoxin produced by several Aspergillus and Penicillium species. It is known as a worldwide common food contaminant. This toxin has received considerable attention because of its deleterious effects on human and animal health. It has immunotoxic, hepatotoxic, teratogenic, neurotoxic and carcinogenic properties in different experimental animals, with the kidney being its main target organ. The role of OTA as a possible causative agent involved in the etiology of human Endemic nephropathy has still not been ruled out. To study adverse effects of OTA on cellular level, three renal cell lines (CV-1, Hek293, LLC-PK1) were cultivated and exposed to the different OTA concentrations, ranging from 0.1 to 50 μ M, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT) over 72 hours. Remaining cell viability after treatment with 20 μ M OTA amounted to 18.2 % for CV-1 and 64.9 % for LLC-PK1. The same OTA concentration induced cell death of almost 43 % Hek293 cell population. Induction of apoptotic and necrotic cell death was measured by anexin V cell labeling and flow cytometry analysis after being exposed to treatment in the same OTA concentration range over 24 hours. Results suggest to apoptosis and necrosis induction in OTA dose dependent manner. Ochratoxin A (OTA) is a mycotoxin produced by several Aspergillus and Penicillium species. It is known as a worldwide common food contaminant. This toxin has received considerable attention because of its deleterious effects on human and animal health. It has immunotoxic, hepatotoxic, teratogenic, neurotoxic and carcinogenic properties in different experimental animals, with the kidney being its main target organ. The role of OTA as a possible causative agent involved in the etiology of human Endemic nephropathy has still not been ruled out. To study adverse effects of OTA on cellular level, three renal cell lines (CV-1, Hek293, LLC-PK1) were cultivated and exposed to the different OTA concentrations, ranging from 0.1 to 50 μ M, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT) over 72 hours. Remaining cell viability after treatment with 20 μ M OTA amounted to 18.2 % for CV-1 and 64.9 % for LLC-PK1. The same OTA concentration induced cell death of almost 43 % Hek293 cell population. Induction of apoptotic and necrotic cell death was measured by anexin V cell labeling and flow cytometry analysis after being exposed to treatment in the same OTA concentration range over 24 hours. Results suggest to apoptosis and necrosis induction in OTA dose dependent manner.

U Zborniku radova seminara DDD i ZUPP iz 2004. godine detaljnije smo predstavili Direktivu o biocidnim pripravcima (98/8/EC) kojom se u Europskoj Uniji uređuje proces registracije i stavljanja u promet biocidnih pripravaka. Za aktivne tvari u biocidnim pripravcima koje su već bile na tržištu prije stupanja na snagu Direktive o biocidima (14. 5. 2000.) predviđeno je da se na nivou EU provede proces procjene sigurnosti/djelotvornosti prije nego se uvrste na popise dozvoljenih aktivnih tvari, poznate kao Annex I, IA i IB. Nakon dovršene prve faze programa revizije, 2003. je u drugoj Uredbi o reviziji objavljen popis aktivnih tvari koje su obuhvaćene procesom identifikacije i notifikacijete kao takove ostaju na tržištu u desetogodišnjem periodu revizije, kao i popis aktivnih tvari koje se u određenim rokovima povlače s tržišta EU. Europska komisija također je objavila i Prvi zajednički izvještaj o provođenju odredbi direktive u 15 zemalja članica za razdoblje od 2000.d0 2003. Hrvatska, kao zemlja kandidat za članstvo u Uniji, u prijelaznom periodudo potpunog prihvaćanja zakonodavstva EU, može kao najjednostavnije rješenje zadržati postojeći sustavstavljanja biocida u promet, odnosno zadržati Zakon o otrovima. Ujedno je potrebno da se, čim se donese novi hrvatski Zakon o biocidima (i zakon o opasnim tvarima) počnu postupno primjenjivati i neke njegove odredbe, uz istovremenu prilagodbu i razvoj sustava koji će omogućiti njihovo potpuno provođenje.

Endemic nephropathy (EN) is human fatal renal disease of unknown origin that occurs in geographically limited area of Croatia and some other countries. The most plausible theory of the aetiology of EN links it with the exposure to nephrotoxic mycotoxins through ingestion of contaminated food. In this study the concentration of mycotoxin ochratoxin A (OTA) and sphingosine/sphinganine (Sa/So) ratio, the biomarker of fumonisins exposure, were checked in human urine collected in endemic village (Kaniža) in Croatia and in a control village. The mean OTA concentration in urine of the group of subjects at risk (relatives of patients with EN, N=31), group of suspected (with some signs of EN, N=19), and in healthy persons from EN village, (N=21) were 4.3, 1.8, and 0.9 ng/ml, respectively. The mean concentration in control group (N=33) was 2.1 ng OTA/ml, and was not statistically different to other groups. The mean Sa/So ratio in urine of subjects at risk, healthy subjects and in suspected was 0.82, 0.80, 0.51, respectively. Although this ratio was the lowest in control group, (0.16) there was no statistical difference when compared to other groups. There was no correlation of OTA concentration and Sa/So ratio in urine.

Although the basic mechanisms of toxicity of the highly toxic nerve agents has been known for a long time, there are still major problems with the effective treatment of acute poisoning by them, especially by soman. The objective of this study was to evaluate the efficacy of tamorf (a new synthesized adamantyl derivative containing morpholine group) on rats poisoned with soman. In this study rats given subcutaneously &frac14 ; ; and &frac12 ; ; LD50 of soman were treated intraperitoneally with tamorf (2.5 mg/kg b.w.) before or after soman injection. Catalytic activity of acethylcholinesterase (AChE) was measured in plasma and brain at different time periods from 10 minutes up to 24 hours after application of soman. Tamorf was applied alone or together with atropine sulphate (10.0 mg/kg b.w.). The best effect of tamorf in rats given &frac14 ; ; LD 50 of soman was obtained when it was given as pre-treatment together with atropine. Treatment with tamorf increased the catalytic activity of AChE in plasma and brain of all soman poisoned rats, except when given up to 10 minutes after &frac12 ; ; LD 50 of soman. Our results suggest that the treatment with tamorf together with atropine antagonise the acute toxicity of soman, and could be supplementary therapy in soman poisoning.

Mycotoxin ochratoxine A (OTA)is a wellknown nephrotoxic compound. The aim of the study was to check the time-cours of representative kidney brush-border and cytosolic enzymes released in urine and concentration of malondyaldehide (MDA) in urine during and after OTA treatment. Adult rats were receiving i.p. doses of 500 ug/kg b.w. of OTA three times a week for four weeks, and their urine was collected after the 1th, 3th, 6th, 9th, and 12th dose, and 14 and 21 days after the 12th dose (follow up). 24-hour urine measurements included the catalytic activity of alkaline phpsphatase (AP), leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and isocitrate dehydrogenase (ICDH) and concentrations of OTA and MDA. OTA affects all parameters measured in urine. After the 6th dose, the concentration of MDA and the catalytic activity of AP, LAP, LDH, and ICDH reached their peak (278, 195, 680, 730, 346% respectively) and were significantly different from controls (p < 0.05). However, althought the rats received 6 more doses of OTA, all measured parameters showed a declining trend. The catalytic activity of brush-border enzymes LAP and AP were significantly higher for a longer period than of LDH and ICDH. The 21-day follow.up urine volume, concentration of creatinine and MDA, as wellas the activity of all measured enzymes did not differ from controls. The effect of OTA-induced release of brush-border enzymeis more pronounced than that of cytosolic enzymes. The catalytic activity of brush-border and cytosolic enzymes released to urine, as well as urinary MDA concentration reverted to control values 21 days after the end of treatment.

Prije dvije godine na istom skupu postavili smo pitanje potrebe osnivanja državne agencije koja bi na stručnim i znanstvenim osnovama obavljala cijeli niz poslova za potrebe državne uprave u području registracije pesticida. tada je rečeno da i da bi se naše nacionalno zakonodavstvo trebalo prilagoditi zahtjevimaEU s obzirom na strateški cilj RH u odnosu na EU. U ime tih nastojanja u proteklom razdoblju RH donijela je velik broj zakona koji su u Hrvatskom saboru izglasani po hitnom postupku. zakoni su nažalost u velikom broju slučajeva pisani i izglasani bez konzultiranja stručne i znanstvene javnosti što je rezultiralo donošenjem niza zakona koji su samo deklarativno prilagođeni propisima i direktivama EU, dok su u stvarnosti nelogični, neprimjenjivi, kontradiktorni i u određenim slučajevim čak i apsurdni. Očit primjer izrazito lošeg zakona čija je primjena u velikoj mjeri upitna je Zakon o kemikalijama izglasan na zadnjoj sjednici Hrvatskog sabora u prošlom sazivu po hitnom postupku.

The aim of our study was to find whether ochratoxin A (OTA) induces the apoptosis and/or necrosis of kidney tissue in rats. In the first experiment, the highest number of apoptotic cells was found in rats sacrificed one day after OTA administration (1.00 mg/kg b.w., i.p.). The number of apoptotic cells reduced gradually and they were not seen nine day after OTA administration. A possible dose-dependence of histological changes was checked in kidney tissue of rats given 0.25, 0.50 or 1.00 mg of OTA/kg b.w., i.p. three times a week for four weeks. The number of apoptotic cells showed a clear dose-dependence, but necrosis was absent even at the highest dose. The time-dependent appearance of lesions related to OTA administration was checked by administering 0.50 mgOTA/kg body weight to rats, and sacrificing them one day after 1, 3, 6, and 9 doses/administrations, or 6 and 21 day after 12 doses/administrations. Long-term administration is associated with continued and increased apoptosis without necrosis, suggestive of OTA's role in the pathogenesis of progressive renal atrophy.

Mycotoxins contamination of food and feed is an important health problem, both in humans and animals, almost allover the world. Ochratoxin A (OTA) is nephrotoxic fungal metabolite known to cause nephropathy in farming animals and it is probably involved in the aethiology of human endemic nephropathy (EN). The different protective methods are focused on the removal of mycotoxins from contaminated foodstuffs or to neutralize their toxic activity. Klioptiolits and especially zeolits have been successfully used in agricultural production as very potent absorbents. Commercially available product "Megamin pulver" a zeolite prepared by special tribomechanical procedure was tested in vitro as a possible OTA adsorbent. The OTA absorption by tribomechanically processed zeolit was tested under different pH values (pH 2, pH 4, pH 7.5), simulating conditions in certain parts of digestive system. The presence of OTA was determined by use of HPLC (with detection limit of 0.2 ng/ml). In all pH conditions absorptive possibilities of Megamin were observed, however the highest absorptive properties were noticed at pH 2, suggesting its best potential absorptive effect for OTA in the stomach.

Mycotoxins are a large group of naturally occurring toxins. Some of them are highly toxic substances, expressing their toxicity on various organs and cells in human and animal organisms. There have been some attempts to use mycotoxins for military purposes, alone or in combination with classical chemical agents. For nephrotoxic mycotoxin ochratoxin A (OTA), as well as for a number of other mycotoxins, the mode of their toxic activity is still not known. To study adverse effects of OTA on cellular level, Green monkey kidney cell line CV-1 were cultivated and exposed to OTA. Cell cultures were exposed to OTA dissolved in dimethyl sulfoxide, in concentrations range 0.004 - 400 μ M over 24, 48 and 72 hours, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT). In all treatment periods the lowest concentration of OTA which resulted in cell viability inhibition was found to be 4 μ M OTA. In treatment period of 24h at OTA concentration 20 μ M remaining cell metabolic activity was found to be 45.1 % in comparison with untreated control group (100 % metabolic activity). The same OTA concentration over longer time period resulted in higher inhibition of metabolic activity, leaving only 35.8 % and 18.2 % activity after 48 and 72h, respectively. Thus differences in activity may be explained by higher percentage of death cells after longer treatment period and consequently smaller metabolic activity that can be measured by use of MTT test. The IC50 value after treatment period of 24 hour was 18.7 μ M OTA. In OTA-positive samples of human sera obtained from people living in the area where fatal human kidney disease called Endemic Nephropathy in Croatia is present, OTA concentrations varied between 2 and 50 ng/ml (4.96 and 124 μ M) OTA (1). Toxicokinetics studies using monkey as an animal model, and the calculations based on available data from humans naturally exposed to OTA have shown that the toxin in those species is predominantly eliminated by renal filtration (2). Our in vitro results strongly suggest that concentrations found in the human blood by natural exposure to the OTA can cause severe damage of kidney cells in vivo. It is known that cells exposed to cytotoxic drugs are undergoing necrotic or apoptotic processes. Which of those processes will be expressed is very much related to mode of action of the cytotoxic substance as such. Based on our results and the data from the published literature, it seems that cytotoxicity of OTA is connected with induction of apoptosis rather then necroses.

Zearalenone (ZEA) is natural product of some Fusarium moulds that grow on cereals in mild and tropical climatic zones. ZEA has very low acute toxicity, but the ZEA contamination may present a serious health problem due to its estrogenic activity in animals and humans. For some time in the past it was used as in the treatment of dismenorhoic disturbances. There is no data on its carcinogenicity in humans because of the lack of epidemiological studies. In our country data on the ZEA contamination of wheat are rather scarce. The only data, which can be found in open literature, are obtained by use of TLC methods, which are relatively insensitive. Therefore we have decided to introduce a sensitive and reliable HPLC method and to test it on the wheat infected artificially in the field with Fusarium moulds. Wheat was artificially infected in field with Fusarium moulds and harvested in Summer 2002. Samples (N=55) were collected and stored at -80 0C until analyzed. ZEA was extracted from wheat samples as described by Llorens A. et al. (Food Addit Contam 2002 ; 19, 272-281) and samples were cleaned-up using Bond Elut C-18 columns (Varian, Harbor City, CA, USA). The concentration of ZEA was determined using HPLC with fluorescent detector. Mobile phase consisted of methanol and water (80+20), and the flow rate was 0.5 ml/min. The separation of ZEN was performed on C-18 reverse phase analytical column (LiCroCART, Merck, Darmstadt, Germany) 250x4 mm, 5 mm particle size. For fluorescence detection wavelengths were set at lex 274 nm and lem440 nm. Calibration curve, calculated from standards prepared in methanol was linear (r2=0.9992). Reproducibility of the method (precision form day to day) calculated as relative standard deviation (RSD) was 5.6%. Detection limit of the method was 0.39 mg/kg and recovery was 108%. ZEA was found in all 55 samples of wheat artificially infected in field with Fusarium moulds. The concentration range was from 0.67 to 10.98 mg/kg and mean concentration was 3.47 mg/kg. These results indicate that the introduced HPLC method for ZEA determination in wheat may be used for naturally contaminated samples.

Ochratoxin A (OTA) is the nephrotoxic product of moulds that contaminate food and feed at all climatic conditions. It is supposed to be involved in the mechanism of endemic nephropathy, disease known also to occur in our country. The mechanism of OTA toxicity is not yet understood. The proposed mechanisms of OTA toxicity are the inhibition of t-RNA phenylalanine syntethase, inhibition of phosphoenolpyruvate carboxykinase and the enhancement of lipid peroxidation. Biological marker of lipid peroxidation is malondialdehyde (MDA), the final product of lipid peroxidation. Apoptosis is the type of active cell death caused by intrinsic factors. It occurs in physiological and pathological conditions. It was observed in some tumors as well as the consequence of exposure to some toxic compounds. The aim of the study was to check whether the single dose of OTA causes apoptosis in kidney tissue and changes of lipid peroxidation in kidney and urine. In female rats given single OTA dose (1.0 mg/kg.b.w., i.p.) and sacrificed 1, 2, 6 and 9 days afterwards, the number of apoptotic cells in kidney (mean&plusmn ; ; ; SD) was 19&plusmn ; ; ; 23, 13&plusmn ; ; ; 19, 0 and 0, respectively. The same was not seen in kidney tissue of control rats (given vehicle only). The OTA concentration in kidney, urine and plasma of treated animals was the highest at the first time point and afterwards decreased gradually. However, the concentration of MDA in kidney and urine of treated animals did not differ from that in control animals. The appearance of apoptotic cells in kidney of OTA-treated animals seems to be in correlation with OTA concentration. The lack of changes in MDA concentration in OTA-treated animals indicates that MDA is not involved in the induction of apoptotic cells in experimental animals.

Ochratoxin A (OTA) is a common contaminant of cereals and other commodities of animal and vegetable origin all round the world. Its nephrotoxic and carcinogenic properties are well established in experimental animals. Beans (Phaseolus vulgaris) are common food in our country, especially in winter period, but the data on OTA contamination of beans are rather scarce. The aim of this study was to analyze the OTA concentration in naturally contaminated beans with a more sensitive method than in previous studies. During fall 2001, bean samples (N=45) were collected in 13 counties of Croatia and stored (– 20 oC) until analyzed. Samples were cleaned up by means of immunoaffinity columns (OchraTest, Vicam) and OTA analyses were performed on HPLC (Varian, USA) with fluorescence detection. Standard curve prepared from OTA-spiked samples in the concentration range 0.25 to 5.0 mg/kg was linear (r2 = 0.9982). Detection limit of method was 0.25 mg/kg, reproducibility (day to day precision) expressed as RSD was 5.5 %. Such a low detection limit enabled the detection of OTA in 42 % of analyzed bean samples in the range of 0.25 to 0.92 mg/kg. The mean OTA concentration in positive samples was 0.39 &plusmn ; ; 0.21 mg/kg (mean &plusmn ; ; SD). Such a low concentration of OTA in beans does not represent a significant origin of human exposure to OTA, but due to the very long half-life of OTA in humans (35.55 days), it may contribute to the exposure to OTA from other commodities.

In this paper three new pyridinium compounds: 1-phenacyl-2-methylpiridinium chloride, 1-benzoylethylpyridinium chloride, and 1-benzoylethylpyridinium-4-aldoxime chloride have been synthesized, and tested in vitro using human erythrocyte acetylcholinesterase (AChE) inhibited by soman and in vivo in soman poisoned mice. The inhibitory power (IC50), as well as reactivating (% R) and protective capacity (P50) of each compound was tested on soman-inhibited human erythrocyte AChE. Their acute toxicity (LD50) was tested in mice and observed in 24-hour period. All tested compounds were found to be reversible inhibitors of AChE. Their AChE binding affinity was compared with their protective effect (PI) on soman phosphonylated enzyme. These compounds have shown weak protective effectiveness without reactivating potency either in vitro or in vivo. Our results show that tested compounds are relatively toxic (their LD50 was from 80.0 to 105.0 mg/kg body weight) and inadequate antidotes in soman poisoning. Their protective effect is more likely to be related to other mechanisms of the cholinergic system.

This paper describes two different procedures of ochratoxin A (OTA) extraction from urine samples. A procedure with acidic chloroform-methanol mixture, followed by solid-phase extraction (SPE) clean-up was compared with extraction on commercial Chem Elut columns with a chloroform-formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a RSD

Fumonisins are mycotoxins produced by some strains of Fusarium moulds. They cause equine leukoencephalomalacia, pulmonary edema in swine and hepatic and kidney lesions in various laboratory animals. Fumonisins are frequently found as contaminants of corn in temperate climatic zone, but the knowledge about their occurrence in our country is scarce. 49 corn samples were collected during fall 2002. from 14 counties of Republic of Croatia. Samples were purified by means of immunoaffinity clean-up procedure and the concentration of fumonisin B1 (FB1) and fumonisin B2 (FB2) were determined by HPLC method with fluorescent detection. Limit of detection for both mycotoxins was 10 mg/kg. Reproducibility of the method expressed as Relative Standard Deviation (RSD) was below 10 %. FB1 was found in all analyzed samples of corn in the range of 142-1378 mg/kg, and the mean concentration was 460 mg/kg. Most of samples (45 %) contained between 200 and 400 mg B1/kg. FB2 was found in 3 samples (6 %), and the concentrations were 68, 109 and 3084 mg/kg. Although the year when samples were collected was extremely wet and the frequency of finding, as well as the concentration of mycotoxins is certainly not common, our results indicate that fumonisins are very frequent contaminants of corn in our country.