Ochratoxin A in airborne dust and fungal conidia.

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCl, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9-1.0 microg/kg) was 74% (quantitation limit 0.150 microg/kg). OTA was found in 6 out of 14 settled dust samples (0.2-70 microg/kg). The total concentration of airborne conidia ranged from < 1.1 x 10(4) to 3.9 x 15(5) per m3, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m3. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4-0.7 and 0.02-0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments.

Ochratoxin A: a naturally occurring mycotoxin found in human milk samples from Norway.

The presence of ochratoxin A (OA) in human milk samples from different regions in Norway was investigated to determine the level of infant exposure to OA from human milk. OA was found in 38 (33%) of 115 human milk samples (range 10-130 ng l(-1)). It was found that 2-26% of the samples contained more than 40 ng l(-1) OA, which will cause a daily intake of OA from human milk exceeding the suggested tolerable dose of 5 ng kg(-1) body weight. Significant regional differences were found.

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum.

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4-4.1 pg per spore or 8-24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography.

[Homeopathy--time for official authorization?]. / Homøopati--tid for offentlig godkjenning?

The Norwegian government has recently appointed a committee to scrutinise alternative therapies and distinguish between serious and nonserious practitioners in preparation for future authorisation. Homoeopathy seems to be the most popular of alternative therapies in Norway, and counts Prime Minister Thorbjørn Jagland among contented patients. For this reason we have taken a closer look at the principles of homoeopathy, and the documentation. Just as in a recent report on documentation and the effect of selected alternative therapies, we too were unable to find studies of reasonable quality that were confirmed by others. Homoeopathists use theoretical physics to explain how water "remembers" the information from molecules no longer existing in the solution, when the liquid is shaken in a special way between every dilution. It does not matter whether the homoeopathist is serious or not as long as the remedy consists only of pure water. We conclude, therefore, that homoeopathy should not be authorised as a serious medical treatment in the Norwegian Health Service.

Citrinin, ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy.

Experiments with Neisseria meningitidis have shown that Fe3+ to some extent can reverse the toxicity of ochratoxin A and citrinin, as measured by inhibition zones around impregnated paper discs. Similar phenomena were observed with the less toxic ochratoxin B. Zearalenone also inhibited growth, but its effect was not counteracted by iron. The mycotoxins aflatoxin B1 and deoxynivalenol did not inhibit bacterial growth at all. Desferal (deferoxamine) also inhibited growth of meningococci, but iron totally abolished this inhibition. The results indicate that ochratoxin A and citrinin interfere with iron metabolism in this organism but that other additional toxic mechanisms are involved as well since a marked growth inhibition by both toxins was also observed in the presence of iron. One function of ochratoxin A and citrinin in nature could consequently be to affect the iron uptake of other competing microorganisms. Since both toxins interfere with iron and both cause nephropathy, a possible connection between these properties and lipid peroxidation is also briefly discussed.

Effects of ochratoxin A upon early and late events in human T-cell proliferation.

We have investigated the effect of ochratoxin A (OA) on both early and late events during activation of human T lymphocytes. As early activation parameters we chose to measure both changes in intracellular Ca2+ levels and the activity of protein kinase C following the triggering process. Our results demonstrate that concentrations of OA that inhibit overall activation had no effect neither on the absolute levels nor on the duration of the Ca2+ response. Furthermore, PKC activity as measured by phosphorylation of two specific cytosolic substrate substrate proteins, was also unaffected. However, when the cells were prestimulated for 48 h to measure effects on late events in the activation cascade, addition of graded concentrations of ochratoxin A down to 6.4 microns completely inhibited the DNA synthesis. This shows that ochratoxin A is able to block DNA synthesis efficiently even if the activation process has been running for 48 h in advance. To investigate whether the inhibitory effect on DNA synthesis could be ascribed to inhibition of protein synthesis, we carried out experiments to measure protein synthesis both in resting and activated T-cells. In resting T-cells protein synthesis was nearly abrogated by 12.5 microM ochratoxin A, but only minor effects were observed in stimulated cells. The low impact of ochratoxin A on protein synthesis in activated cells may indicate that other mechanisms than just a general inhibition of protein synthesis are operating.

Glycyrrhizic acid in liquorice--evaluation of health hazard.

Literature on case reports, clinical studies and biochemical mechanisms of the sweet-tasting compound glycyrrhizic acid in liquorice was critically reviewed to provide a safety assessment of its presence in liquorice sweets. A high intake of liquorice can cause hypermineralocorticoidism with sodium retention and potassium loss, oedema, increased blood pressure and depression of the renin-angiotensin-aldosterone system. As a consequence, a number of other clinical symptoms have also been observed. Glycyrrhizic acid is hydrolysed in the intestine to the pharmacologically active compound glycyrrhetic acid, which inhibits the enzyme 11 beta-hydroxysteroid dehydrogenase (in the direction of cortisol to cortisone) as well as some other enzymes involved in the metabolism of corticosteroids. Inhibition of 11 beta-hydroxysteroid dehydrogenase leads to increased cortisol levels in the kidneys and in other mineralocorticoid-selective tissues. Since cortisol, which occurs in much larger amounts than aldosterone, binds with the same affinity as aldosterone to the mineralocorticoid receptor, the result is a hypermineralocorticoid effect of cortisol. The inhibitory effect on 11 beta-hydroxysteroid dehydrogenase is reversible; however, the compensatory physiological mechanisms following hypermineralocorticoidism (e.g. depression of the renin-angiotensin system) may last several months. It is not possible, on the basis of existing data, to determine precisely the minimum level of glycyrrhizic acid required to produce the described symptoms. There is apparently a great individual variation in the susceptibility to glycyrrhizic acid. In the most sensitive individuals a regular daily intake of no more than about 100 mg glycyrrhizic acid, which corresponds to 50 g liquorice sweets (assuming a content of 0.2% glycyrrhizic acid), seems to be enough to produce adverse effects. Most individuals who consume 400 mg glycyrrhizic acid daily experience adverse effects. Considering that a regular intake of 100 mg glycyrrhizic acid/day is the lowest-observed-adverse-effect level and using a safety factor of 10, a daily intake of 10 mg glycyrrhizic acid would represent a safe dose for most healthy adults. A daily intake of 1-10 mg glycyrrhizic acid/person has been estimated for several countries. However, an uneven consumption pattern suggests that a considerable number of individuals who consume large amounts of liquorice sweets are exposed to the risk of developing adverse effects.

[Significance of mycotoxins for health--present and past]. / Muggsoppgiftenes betydning for helsen--før og nå.

Investigations from several European countries indicate that, in former days, there was a connection between mycotoxins, mortality and reduced birth rate. In addition to being acutely toxic, many mycotoxins can interfere with the immune system, even in small concentrations. While tuberculosis was declining in Norway as a whole around year 1900, it culminated in Finnmark county. In addition, infant mortality was high. These people in the North ate rye flour imported from Russia, and harvested, transported and stored under conditions which may have favoured growth of toxic fungi. The mycotoxin ochratoxin A has been surveyed in several countries. The level in human sera seems to indicate continuous, widespread exposure of humans to ochratoxin A. Mycotoxin contamination of food and feeds may be a health problem of unknown dimensions.

Effects of ochratoxins A and B on prechondrogenic mesenchymal cells from chick embryo limb buds.

Prechondrogenic mesenchymal cells from the limb buds of 4-day chick embryos were cultured together with either ochratoxin A (OA) or ochratoxin B (OB) for 6 days. Both toxins inhibited the accumulation of cartilage proteoglycans and general protein synthesis in a concentration-related manner. The IC50 was 1.9 microM for OA and 6.2 microM for OB. Incubating the micromass with OA for periods ranging from 2 h up to 6 days did not produce any metabolites, indicating that OA was the proximal toxic compound in these studies. Neither preincubation with OB nor simultaneous exposure to non-toxic concentrations of OB together with various concentrations of OA influenced the toxicity of OA. The data indicate that interference of OA with general protein synthesis, both in vivo and in vitro, is an important mechanism underlying OA-induced embryotoxicity.

Mechanism of ochratoxin A-induced immunosuppression.

Ochratoxin A (OA) has been reported to affect immune function both at the level of antibody synthesis and natural killer (NK) cell activity. In the present study we demonstrate that exposure of purified human lymphocyte populations and subpopulations to the toxin will abrogate the cells' ability to respond to activating stimuli in vitro. Thus, both IL-2 production and IL-2 receptor expression of activated T lymphocytes are severely impaired. When the cells are preincubated with the analogue ochratoxin B (OB) prior to OA exposure, the inhibitory effect of OA is reversed. Furthermore, the inhibitory effect of OA on antibody production is not only due to blocking of T helper cell function. Highly purified B lymphocytes will not respond to polyclonal activators in vitro after a brief pulse with OA. The results strongly suggest that the toxin causes its immunosuppression through interference with essential processes in cell metabolism irrespective of lymphocyte population or subpopulation.

Fluorometric detection of 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and their N-acetylated metabolites excreted by the rat.

A sensitive fluorometric method has been developed for the determination of carcinogenic amino imidazoazaarenes (AIA compounds) which are formed during the cooking of food. They are separated by t.l.c. followed by spraying with dimethylsulfoxide and visualization by long-wave u.v. light. Quantification is done by fluorescence scanning. This method was applied to the determination of the metabolites in urine and feces from rats given 5 mg/kg body wt i.p. of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). Metabolites extractable by organic solvents were identified as the parent compound and their N-acetylated derivatives (AcIQ, AcMeIQ). Between 0.1 and 1% of the administered dose was recovered as these metabolites in urine during 24 h. The amounts of MeIQ and AcMeIQ found in feces were approximately 1 and 3% respectively. AcIQ was not detected in feces while IQ, MeIQ and their N-acetylated derivatives were found in bile. The metabolites in urine were identified by m.s. and in feces by t.l.c. and their fluorescent properties. When IQ or MeIQ were incubated with rat liver cytosol in the presence of acetyl-CoA, N-acetylated derivatives were formed. The reverse reaction, deacetylation, also took place in the liver cytosol, with Km values of 67 and 32 microM for AcIQ and AcMeIQ respectively.

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats.

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.

Ochratoxin A-induced porcine nephropathy: enzyme and ultrastructure changes after short-term exposure.

Four pigs were treated with ochratoxin A (800 micrograms/kg) for five consecutive days. Subsequently, urine and bile were collected and kidneys were perfusion fixed unilaterally. Liver and kidney samples were examined for the distribution of ochratoxin A and metabolites in subcellular fractions and the effects of the toxin on protein synthesis and enzyme activities. Ochratoxin A and the hydrolytic product, ochratoxin alpha, were found in urine. Elevated levels of toxin accumulation in kidney (283 ng/g) compared with liver (189 ng/g) and toxin-mediated reductions in protein synthesis and enzyme activities in kidney identified it as a target organ of ochratoxin toxicity. Ultrastructural investigations of kidney in toxin-exposed animals identified a process of condensation of cellular material with disappearance of membranes and continuous desquamation in the lower part of the proximal convoluted tubules. In target cells peroxisomes appeared to have lost membrane integrity and the organelles were leaking materials into the cytosol. Reduction of structural integrity was associated with an increase in the presence of catalase and cyanide insensitive fatty acid oxidase activity in the soluble kidney fractions.

Effects of ochratoxin A metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and in vivo protein synthesis of hepatoma cells.

The ochratoxin A (OTA) metabolite (4R)-4-hydroxyochratoxin A [4R)-OTA) inhibits the aminoacylation of phenylalanine tRNA catalyzed by phenylalanyl-tRNA synthetase (PheRS) with a Ki-value of 0.9 mM as compared to 1.3 mM for OTA. It also inhibits protein synthesis and cell growth in the same manner as OTA. Ochratoxin alpha (OT alpha) does not affect either protein synthesis or cell growth.

Oxidation of two hydroxylated ochratoxin A metabolites by alcohol dehydrogenase.

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K(m) and turnover number for 10-hydroxyochratoxin A were 110 muM and 0.1 s(-1), respectively.

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.

Effects of two metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, on immune response in mice.

The metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, were investigated for immunosuppressive properties in BALB/c mice. The standard plaque-counting technique for the estimation of antibody-producing spleen lymphocytes was used. (4R)-4-hydroxyochratoxin A was found to be an immunosuppressor almost as highly effective as ochratoxin A. Doses of 1 microgram of (4R)-4-hydroxyochratoxin A per kg administered to mice caused an 80% reduction in the number of cells producing immunoglobulin M (90% with ochratoxin A) and a 93% reduction in cells synthesizing immunoglobulin G (92% with ochratoxin A). Ochratoxin alpha, however, was ineffective. A possible mode of action is discussed.

Metabolism of ochratoxin A by rats.

Albino rats were given ochratoxin A (6.6 mg/kg body weight) intraperitoneally or per os. Independent of route administration, 6% of a given dose was excreted as the toxin, 1 to 1.5% as (4R)-4-hydroxyochratoxin A, and 25 to 27% as ochratoxin alpha in the urine. The metabolite (4S)-4-hydroxyochratoxin A, which is formed by rat liver microsomes in the presence of NADPH, was not detected. Only traces of ochratoxins A and alpha were found in feces. Identical experiments were carried out with brown rats, since the Km value for the formation of the 4S epimer was considerably lower when brown rat microsomes were used. About the same ratios of metabolites and metabolite recoveries as those found for albino rats were found for brown rats. Brown rats were also given the two hydroxylated metabolites and ochratoxin alpha (0.66 mg/kg body weight) intraperitoneally. The three compounds were excreted in the urine; within 48 h, 90% recovery of ochratoxin alpha and 54 and 35%, respectively, of the 4R and 4S isomers were observed.

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.