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Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of fungi. OTA induces a tubular-interstitial nephropathy in humans and in animals. It has been implicated as one of the aetiological agents involved in the development of endemic nephropathy. OTA-induced oxidative stress and apoptosis may play key roles in the development of chronic tubulointerstitial nephritis connected to the long-term exposure to this food contaminant. We studied the effects of low doses of OTA on kidney cells. Wistar rats were treated with 120 microg OTA/kg bodyweight daily, for 10, 30 or 60 days. Toxin concentration in kidney was proportional to the time of exposure, and amounted to 547.2, 752.5 and 930.3 ng OTA/g kidney tissue after 10, 30 and 60 days, respectively. OTA treatment caused an increased number of cells undergoing apoptosis in both proximal and distal epithelial kidney cells. The apoptotic cells were visualised using the TUNEL assay and staining with haematoxylin and eosin in situ. The number of apoptotic cells in rats treated for 10, 30 and 60 days increased by 5-, 6.4- and 12.7-fold, respectively, compared with the control cells. However, DNA electrophoresis did not show characteristic fragmentation (DNA laddering). The oxidative stress was evident via increased malondialdehyde formation. The concentration of lipid peroxides showed an increase (36%), but the activity of superoxide dismutase decreased (26%) in 60-day treated rats. In spite of the observed biochemical and morphological changes in the kidney cells, renal functional status was preserved to the end of experiment. This study demonstrates that a combination of morphologic and biochemical markers can be used to monitor early cell death in OTA-induced renal injury. We have shown that the exposure to the relatively low OTA concentrations has activated apoptotic processes and oxidative damage in kidney cells.

The objective of this study was to evaluate the efficacy of thienyl phencyclidine (tenocyclidine, TCP) and its newly synthesized adamantyl derivatives containing piperidine (TAPIP), pyrolidine (TAPIR) and morpholine (TAMORF) groups, which were tested with or without standard therapy in mice poisoned with organophosphates (OPs) and carbamates. These compounds with potential activity at the N-methyl- D-aspartate and muscarinic receptors showed low acute toxicity, having LD50 values varying from 106.00 mg/kg (TCP) to >504.00 mg/kg body weight (TAMORF). TCP and its adamantyl derivatives were administered intraperitoneally (2.5 mg/kg body weight) together with atropine (10.0 mg/kg body weight) and with or without 1/4 LD50 of the oxime HI-6. Each compound administered with atropine had a therapeutic effect against poisoning with carbamates propoxur, aldicarb and Ro 02-0683 (protective ratio of tenocyclidines was from 3.99 LD50 of aldicarb to >16.00 LD50 for propoxur). However, the efficacy of those compounds in combination with atropine was lower against poisoning with the OP insecticide dichlorvos (DDVP) and chemical warfare agents soman and tabun. In soman-poisoned mice, the best therapeutic effects were obtained with the combination of HI-6 plus atropine and test compounds, with protective ratios being from 5.40 to 7.12 LD50 of soman. The results suggest that TCP and adamantyl tenocyclidines could be used in combination with atropine as antidotes in carbamate poisoning and as adjuvant therapy to HI-6 and atropine in soman poisoning.

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.

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.