Ana Lucić

Ochratoxin A in human sera in the area with endemic nephropathy in croatia

Ochratoxin A (OA) is nephrotoxic fungal metabolite (mycotoxin) occurring in foodstuffs. The compound is causally associated with mycotoxin porcine nephropathy, a disease comparable with a human kidney disease called endemic nephropathy (EN). In this paper we presented results obtained over a 10-year period in the hyperendemic village Kaniza, and in control villages where no clinical cases of nephropathy had been found. In the hyperendemic village Kaniza and non-endemic villages the incidence of OA in human blood was up to 4.5% (range 2-50 ng/ml) and up to 2.4% (range 2-10 ng/ml), respectively. Almost all samples of food and feed, collected randomly in the hyperendemic village were found to contain OA. Considering marked exposure to OA in Kaniza, it was assumed that incidence of EN in this population could be related to OA contamination of food and feed.

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.

Quinuclidinium-imidazolium compounds: synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice

Abstract Four compounds were prepared: 3-oxo-1- methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH2)3- (III) and -CH2-O-CH2- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD50) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD50 dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD50 doses of Soman.

Antidotal efficacy of quinuclidinium oximes against soman poisoning

The efficiency of newly synthesized oxime derivatives of quinuclidinium were tested in vitro on soman inhibited acetylcholinesterase (AChE) of human erythrocytes and in vivo using soman poisoned mice. For this purpose, the inhibitory power of oximes (IC50), acute toxicity (LD50) as well as reactivating and protective capacities with respect to soman-inhibited AChE were determined for each of the oximes. All oximes tested were ineffective in vitro but protected mice very efficiently (BM-1 protects against 4LD50 of soman). The results indicate that the in vivo effectiveness of quinuclidinium oximes against soman poisoning may not be related to reactivation or protection of AChE but rather to some other mechanism of the cholinergic system.

226 Screening for fumonisins B1 and B2 in corn collected in Republic of Croatia

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.

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.

Imidazolium and quinuclidinium oximes as antidotes in soman poisoning

New synthesized oximes derivatives of imidazole and quinuclidines were tested in vitro using human erythrocyte acetylcholinesterase (AChE) inhibited by soman and in vivo using soman poisoned mice. The inhibitory power of oximes (IC 50), acute toxicity (LD 50) as well as reactivating and protecitve capacities with respect to soman-inhibted AChE were tested for each of the synthesized oximes. Derivatives of imidazoles demonstrated mostly weak reactivating and protective characteristics, both in vitro and in vivo. Only BMR-3 oxime was powerful reactivator of soman inhibte human AChe (55 %). BMR-4 oxime given together with atropine sulfate, provided a good in vivo protection against 1.8 and 2.2 x LD 50 of soman. On the contrary, all tested quinuclidne compounds are protective agents against soman in vivo (PP-1, PP-2, PP-3, PP-4 protect against 2-2.5 x LD 50 of soman). Bm-1 oxime has the best protection against soman inhibition of the AChE of all tested compounds (protect against 4 LD 50 of soman). Quinuclidines in vitro activity as reactivators of soman inhibited AChE, and their protective power against soman inhibition of AChE in vitro are negligible. The results indicate that in vivo effectiveness of quinuclidine oximes against soman poisoning is not related to their reactivatin or protective potentials for AChE; their good protective effect is more likely to be related to other mechanisms of the cholinergic system.