Fumonisin B1 causes multiple lesions in common carp (Cyprinus carpio).

Year-1 carp were fed ratios containing 100mg/kg and 10 mg/kg of added fumonisin B1 for 42 days. The experimental and control fish were examined clinically during the experiment and at the end all fish were necropsied and histological changes recorded. Blood vessels, liver, exocrine and endocrine pancreas, excretory and haematopoietic kidney, heart and brain were sensitive both to 100 and 10mg/kg of FB1 in the diet and the rodlet cell (RC) frequency was considerably increased in and around damaged tissues. Many damaged blood vessels contained stacks of RCs above the endothelium. Other changes subsequent to fumonisin exposure that have not been previously reported include scattered lesions in the exocrine and endocrine pancreas, and interrenal tissue, probably due to ischemia and/or increased endothelial permeability. Presented findings indicate the need for more intensive studies of fumonisin-induced toxicity in cultured fish.

The ability of fungal isolates from human lung aspergilloma to produce mycotoxins.

This study included 11 adult patients (seven men and four women) who had been surgically treated for pulmonary aspergilloma in the Republic of Croatia within two years. Mycological analysis was positive for Aspergillus genus in five samples of surgically removed tissue. A. fumigatus was isolated in three and A. versicolor in two samples. Their mycotoxigenic potency was determined by thin layer chromatography. A. fumigatus strains were found to produce aflatoxin B1 (AFB), and two of them aflatoxin G1. A. versicolor strains produced AFB1 and sterigmatocystin. Neither isolated Aspergillus strain produced aflatoxin G2 or ochratoxin A. Fungal growth and production of mycotoxins are the consequences of interaction of fungus, host and environment. One has also to take into consideration that the production of mycotoxins in vitro does not reflect what these fungi may produce in human organisms.

Studies of ochratoxin A-induced inhibition of phenylalanine hydroxylase and its reversal by phenylalanine.

Ochratoxin A (OTA) is a nephrotoxic, hepatotoxic, and teratogenic mycotoxin produced by storage molds on a variety of foodstuffs. Its chemical structure is composed of an isocumarin part linked to l-phenylalanine. Inhibition of phenylalanine hydroxylase and other enzymes that use phenylalanine as substrate is based on this structural homology. We have examined the effects of low doses of ochratoxin A on the activity of phenylalanine hydroxylase in kidney and in liver of experimental animals. Daily administration of ochratoxin A (50 microg/kg body wt, for 10 and 35 days, respectively) caused a significant reduction in the phenylalanine hydroxylase activity. Inhibition was more pronounced in liver than in kidney, although actual ochratoxin A concentration was higher in the kidney tissue. We observed an apparent increase in the affinity of phenylalanine hydroxylase for substrate following OTA administration to animals. However, simple competitive inhibition was observed for both tissues in vitro (K(i liver) = 0.0119 +/- 0.002 mM and K(i kidney) = 0.13 +/- 0.026 mM). Simultaneous application of ochratoxin A with phenylalanine could reduce inhibition of phenylalanine hydroxylase, in particular in liver. Enzyme activity was almost completely preserved after 35 days of combined treatment. The results obtained suggest that daily administration of ochratoxin A in low doses produced an inhibitory effect that could be diminished by competitive action of l-phenylalanine.

Production of cyclopiazonic acid by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

96 strains of Aspergillus flavus isolated from samples of stored grain and smoke-dried meat products were examined for ability to produce cyclopiazonic acid and aflatoxins, grown on mycological broth medium and malt extract agar. Five strains produced cyclopiazonic acid in the range of 0.5-30 mg/kg and 9 produced aflatoxin B1 (0.1-14.8 mg/kg) but none of them produced both cyclopiazonic acid and aflatoxins.

Influence of ochratoxin A treatment on the activity of membrane bound enzymes in rat brain regions.

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of mouldy food. We examined the neuroactive potential of ochratoxin A by measuring the changes in the activities of several membrane bound, cytoplasmic and lysosomal enzymes in the brain of adult female rats, following subchronic application of ochratoxin A. The activities of both soluble and membrane bound fractions of ecto-5'nucleotidase, ecto-Ca2+/Mg2+ATPase, alanine aminopeptidase, gamma-glutamyl transferase, as well as activities of lactate dehydrogenase and of N-acetyl-beta-D-glucosaminidase were followed. Biochemical effects were examined in cerebral cortex, cerebellum and hippocampus. The results obtained showed physiologically significant alterations in the activity of enzymes tested. The changes were found to be time-dependent and regionally selective. Compared to controls, statistically significant increases in gamma-glutamyl transferase were observed in all three brain regions, while in the case of alanine aminopeptidase activities differed with regard to region, the highest increase being observed in hippocampus. Ecto-Ca2+/Mg2+ATPase and ecto-5'nucleotidase showed distinct changes lasting for 20 days of treatment, while increase in the activities of N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase were visible only at the beginning of the treatment. By the end of the trial the activities of almost all enzymes returned back to normal values.

Use of UV photography to identify aflatoxin-producing strains of Aspergillus flavus and A. parasiticus.

UV photography in glucose, yeast extract (GY) agar medium was tested as a simple and rapid method for the distinction of aflatoxin-positive from aflatoxin-negative strains of Aspergillus flavus and A. parasiticus. In the UV photographs aflatoxin-producing moulds were identified as grey or black colonies, whereas aflatoxin-nonproducing moulds appeared as white colonies. Of the aflatoxin-positive strains detected by the UV photographic method, 10% was confirmed by extraction of the GY agar medium and mould mycelium in chloroform, extracts which were analysed subsequently using thin-layer chromatography. Confirmation of aflatoxigenic strains was achieved by biosynthesis on liquid medium yeast extract sucrose (YES) broth.

Ochratoxin A impairs activity of the membrane bound enzymes in rat pancreas.

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of moldly food. Ochratoxin A has a number of toxic effects, some of which may be related to the changes in the cell membrane. We measured the activities of 5 pancreatic, membrane bound enzymes in female Fisher rats that were given low oral doses of ochratoxin A (120 micrograms/kg body weight per day) during 20-35 days. The amount of toxin corresponds to 1.5 mg/kg in the feed, daily. These doses are in the range of natural contamination found in feed. The enzymes studied were alanine aminopeptidase, alkaline phosphatase, ecto-Ca2+/Mg(2+)-ATPase, gamma-glutamyl transferase and ecto-5'-nucleotidase. Treatment lasting 20 days caused a strong decrease in the activity of alanine aminopeptidase, Ca2+/Mg(2+)-ATPase and alkaline phosphatase to 0.76 +/- 0.04, 0.53 +/- 0.03 and 0.30 +/- 0.02 of the control values, respectively (p < 0.05). No significant changes in the activity of gamma-glutamyl transferase and 5'-nucleotidase were observed. However, activity of alanine aminopeptidase returned to normal values after 35 days of treatment, suggesting an adaptation of the organism, or a substitution of a released enzyme. Activities of alkaline phosphatase and Ca2+/Mg(2+)-ATPase remained significantly reduced to 0.42 +/- 0.03 and 0.52 +/- 0.04, respectively (p < 0.01). We conclude that treatment of rats with low doses of ochratoxin A resulted in reduction of the activities of the membrane bound enzymes, most probably by inducing their release, as a result of the impairment of the functional integrity of cell membranes.

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat.

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17.8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium--APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat--SW). A. flavus strains produced mainly aflatoxin B1, and had various levels of toxigenicity (1.4-3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B1 B2 G1 G2, while the other ones produced AF B1 + G1 only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.

Effect of ochratoxin A on brush border enzymes of rat kidney.

Ochratoxin A was given orally at 60 microgram/kg body weight in neutral olive oil to Fischer rats for 30 days, at which time they were killed. Clinical state, weights of animals and of their organs and urea and creatinine concentrations were not affected during the exposure period. Significant increases in the activity of enzymes in urine were found: 60% increase in alanine aminopeptidase, 45% increase in gamma-glutamyl-transferase and 90% increase in alkaline phosphatase. These changes indicate early pathological changes in the kidney. Relatively small amounts of the toxin thus affect kidney membrane cells.

Ochratoxinogenicity of Aspergillus ochraceus strains from nephropathic and non-nephropathic areas in Yugoslavia.

Mycological analyses of 855 samples of stored grains and dried meat collected in period 1980-1987 from individual households in the nephropathic and wider non-nephropathic area in SR Croatia in Yugoslavia showed 10% of samples to be contaminated with Aspergillus ochraceus. Ability to produce ochratoxin A (OA) was tested in 70 samples (27 from nephropathic areas and 43 from non-nephropathic areas). The detection was carried out under UV-light (365 nm) (light blue fluorescence) and 6 OA-producers were found. A biosynthetic procedure on liquid nutritional substrate with saccharose and yeast extract as well as a method using wet crushed wheat revealed that 37% of the samples from a nephropathic area, and 35% of the samples from a non-nephropathic area produce OA. In the nephropathic area 1/10 strains was a strong producer of OA (concentration crushed wheat 135 mg/kg, and 240 mg/l on YES liquid substrate), 1/10 strains was a moderate producer (concentration 16.6 mg/l and 0.07-7.0 mg/l and 0.1-10.4 mg/kg). Among the strains isolated from a wider non-nephropathic area no strong producers of OA were found, but 2/15 strains were moderate producers of OA (concentration of OA 20.4-27.0 mg/l and 15.0-33.7 mg/kg). The other strains, 8/10 on the crushed wheat and 13/15 on the liquid substrate, were weak producers of OA with concentrations of OA between 0.2-9.0 mg/l and 0.2-10.0 mg/kg with the two methods respectively.

The in vivo evaluation of poly(lactic acid) microcapsules of pilocarpine hydrochloride.

Poly(lactic acid) microcapsules of pilocarpine hydrochloride were prepared and evaluated by measuring a miotic effect in rabbits. The microcapsule suspension showed prolongation of miosis and an improved bioavailability when compared with a standard eye dosage form.

The mycotoxicological chain and contamination of food by ochratoxin A in the nephropathic and non-nephropathic areas in Yugoslavia.

Research was carried out on the distribution of moulds on cereals in vegetation and in storerooms in the period from 1974 to 1981 and on ochratoxin (OA) in stored maize and wheat as well as residues of OA in the organs of swine in the nephropathic and non-nephropathic areas in the SR of Croatia, Yugoslavia. It was shown that moulds belonging to toxogenic species contaminate cereals in vegetation to an approximately equal degree in both areas (Penicillium 6.6-20.0%, Aspergillus 2.5-6.6% and Fusarium 80-100%). Stored cereals were contaminated by species of Penicillium 75-82.8%, Aspergillus 2.5-27.1% and Fusarium 57.1-82.5%, with a somewhat higher degree of contamination in the nephropathic area. Ochratoxin A occurs on cereals on the whole territory of the SR of Croatia, but average concentrations are higher in the nephropathic area (45% of the positive findings of OA were over 2 mg/kg). Residues of OA in the kidneys (16-77 micrograms/kg), liver (0-21 micrograms/kg) and blood (36-77 micrograms/l) were detected in 38 organs taken from swine in the nephropathic area, but this toxin was not found in the 6 samples taken from the non-nephropathic area. In the same organs histopathological changes were found in the kidneys (interstitial nephritis with parenchymal degeneration of the distal parts of the tubular epithel) and liver (interstitial hepatitis with fatty degeneration of the liver).