Effects of fasting and gender on ochratoxin A toxicokinetics in F344 rats.

Ochratoxin A (OTA) is a mycotoxin that causes renal tumors in rats, particularly in males. In previous kinetic studies performed in fed conditions (Vettorazzi et al., 2008), mature F344 male rats presented a significantly lower OTA bioavailability than females and young animals. The objective of the present study was to evaluate two factors which could explain this different kinetic profile: the presence of food and the male-specific protein alpha-2u-globulin. Therefore, a 24h kinetic study has been performed in rats under fasting conditions. Food ingestion has been controlled in both sexes during two months. The presence of alpha-2u-globulin in the urine has been analyzed with SDS-gradient mini-gel electrophoresis. Fasting tends to increase the maximum OTA plasma concentrations and the rate of absorption. The relative bioavailability is significantly increased under fasting conditions only in males. Mature males consumed a higher amount of food but, as the OTA dose administered, it was proportional to body weight. The reason why the OTA bioavailability is more affected in presence of food only in males is unclear. Several possibilities, such as differences in gastric emptying, OTA-food interactions and the involvement of alpha-2u-globulin are discussed.

Clinicomorphological studies in chicks fed ochratoxin A while simultaneously developing coccidiosis.

The progression of coccidiosis and the resultant mortality were followed in chicks fed a OTA-contaminated diet. More complex and rapid progress of coccidiosis occurred in OTA-treated chicks than in chicks fed a OTA-free diet. The concentration of total protein in the serum was significantly decreased in the chicks in the OTA-treated group, whereas this was significantly increased in chicks infected with Eimeria tenella, irrespective of additional treatment with OTA. The serum glucose concentration was significantly increased in all the chicks exposed to OTA and/or suffering from coccidiosis, as was serum retention of uric acid in all groups, most notably in those consuming OTA. OTA induced degenerative changes in, and an increase in the weight of the kidneys, liver, heart and ventriculum; there was depletion of lymphoid tissue and a decrease in the lymphoid organs' weight and body weight. Coccidiosis induced only a slight growth depression and a slight increase in the relative weight of the kidneys and liver. The intensity of the clinical signs, the impairment of kidney function, macroscopic and histopathological changes, deviations in the weight of some organs and general depression in growth were greater when chicks infected with E. tenella were also given OTA.

Experimental one year ochratoxin A toxicosis in pigs.

Mild mycotoxic nephropathy was induced in 6 pigs by a diet containing ochratoxin A at 800 ppb, several times higher than that naturally encountered in some feed for pig production in Bulgaria. The nephropathy was expressed only as slightly hypertrophied kidneys with a faintly mottled surface, discernible at the end of the experiment to a skilled observer but probably not recognisable in routine slaughterhouse processing. Histological examination showed two types of changes: degenerative - affecting epithelial cells in some proximal tubules of pigs after 6 months, and proliferative changes in the interstitium which predominated after 1 year of exposure to ochratoxin A. Telangiectasis and lymph stasis were rarely seen. The renal lesions were similar to those described for classical mycotoxic porcine nephropathy formerly encountered in Denmark, but they were rather different from the porcine nephropathy which occurs spontaneously in Bulgaria. Measurement of ochratoxin A in serum provided analytical values complementary to feed intake and with similar concentration values. It also showed both accumulation with time, from 3 months to 6 months (approximately 1 ppm), and a 2-fold range of values within a group eating from a common feed source, as in commercial pig production. Mild symptomatology in this long, single-mycotoxin experiment serves to lessen somewhat the current perception of the direct renal toxicity of ochratoxin A alone, though a role in multi-toxin contexts is unquestioned.

Biosynthesis of ochratoxins by Aspergillus ochraceus.

Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin beta was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin alpha was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A.

Scorpinone: a new natural azaanthraquinone produced by a Bispora-like tropical fungus.

Scorpinone (1), 3-methyl-6,8-methoxy-2-aza-9,10-anthraquinone, has been isolated from the mycelium of a cultured sterile fungus of Caribbean origin. The structure was elucidated by X-ray crystallography, and 2D NMR spectral data have been assigned. The compound is one of very few known fungal azaanthraquinones.

Biosynthesis of diaporthin and orthosporin by Aspergillus ochraceus.

Diaporthin and orthosporin were characterised from the fungus Aspergillus ochraceus D2306. Diaporthin was identified by high-resolution electron impact mass spectrometry and 1H and 13C NMR spectroscopy, from which new spectroscopic assignments were made. Orthosporin was also identified by mass spectrometry and both fungal metabolites are reported for the first time as co-metabolites and also as products of A. ochraceus. The methylation inhibitor ethionine affected production of both diaporthin and orthosporin in spite of no obvious methylation step in the biosynthesis of orthosporin, implying that extracellular orthosporin may arise by de-O-methylation of diaporthin. The biosynthetic origin of diaporthin was demonstrated by incorporation of [1-14C]acetate and [methyl-14C]methionine administered in early idiophase.

Differentiating the biosynthesis of pseudomonic acids A and B.

Pseudomonic acid A (1) has been the dominant commercial pseudomonate antibiotic produced by Pseudomonas fluorescens. In specific shaken flask conditions initial fermentation accumulation of 1 is followed by preferential accumulation of the 8-hydroxy derivative, pseudomonic acid B (2). Biosynthetic probing with a pulse of [1-14C] acetate or L-[methyl-14C] methionine at early, mid and late stages of the fermentation gave relative patterns of radioactivity in 1 and 2 that are inconsistent with an assumption that 2 arises by oxidation of 1, or that 1 is formed by reduction of 2. Since [methyl-14C] methionine only labels carbons in the 12-carbon part of the pseudomonate molecule that is thought to be an early biosynthetic moiety, the evidence from radiolabelling experiments implies that preferential early oxidation of this biosynthetic intermediate causes the pathway diversion to accumulate 2 instead of 1.

Caffeine: also a fungal metabolite.

Caffeine has been found to occur as a fungal metabolite and to be the principal alkaloid in sclerotia of Claviceps sorghicola, a Japanese ergot pathogen of Sorghum spp.

Susceptibility to secondary bacterial infections in growing pigs as an early response in ochratoxicosis.

Mycotoxic nephropathy was induced in twelve 14 kg pigs fed a dietary component, moulded by Aspergillus ochraceus and contributing ochratoxin A at 1 or 3 ppm for up to 3 weeks. Concurrently, salmonellosis arose spontaneously in all six animals treated at 3 ppm and all died between days 15 and 17. Two of the six pigs in the 1 ppm group died similarly but the rest, and all of six control animals, were unaffected. Clinical biochemistry and histology revealed changes typical of renal ochratoxicosis in all ochratoxin-treated pigs. Clinical and pathomorphological changes typical of salmonellosis were evident in all those that died and Salmonella choleraesuis was consistently isolated from their faeces and liver. In a further experiment at 1 ppm ochratoxin A in animals immunised against S. choleraesuis haemorrhagic diarrhoea resulted instead, associated with Serpulina hyodysenteriae and Campylobacter coli. There was concomitant evidence of immunosuppression and delayed response to immunization. For the first time, susceptibility to natural infectious disease has been demonstrated in pigs exposed to the immunotoxicity of ochratoxin A. Differentiation of biochemical and histological changes attributable to ochratoxicosis or to secondary disease may require reinterpretation of a classical description of experimental porcine ochratoxicosis.

Ochratoxin formation in Aspergillus ochraceus with particular reference to spoilage of coffee.

Production of ochratoxin on media by eight isolates of Aspergillus ochraceus from coffee or its processing environment in India, Indonesia, Kenya, and Brazil, and seven Brazilian isolates from other commodities, has been compared with yields in shaken fermentation on shredded wheat and coffee (Coffea arabica). Shredded wheat most consistently allowed expression of biosynthesis of ochratoxins A and B in yields up to 3.5% of the dry product. Culture on artificial media was an unreliable predictor of ochratoxin yield on both shredded wheat and coffee. Coffee was a relatively poor substrate for ochratoxin production particularly when sterilised. Notably, two Asian coffee isolates produced 400 mg kg(-1) ochratoxin A on unsterilised ground green coffee, showing this to be a preferred substrate for further experimentation. The study focused on isolates of A. ochraceus, which from evidence of culture on media would not be expected to be suitable fungi for future studies to establish both the fact of spoilage of coffee by A. ochraceus and the dynamics of ochratoxin formation by isolates of this species.

Influence of halogen salts on the production of the ochratoxins by Aspergillus ochraceus Wilh.

The first report of the biological production of bromo ochratoxin B by Aspergillus ochraceus Wilh. is presented as well as a study of the influence of potassium bromide, potassium iodide, potassium fluoride, and potassium chloride on the production of ochratoxin A and ochratoxin B. Potassium fluoride and potassium iodide inhibited the growth of the fungus, whereas potassium chloride substantially stimulated the production of ochratoxin A in shaken solid substrate fermentation on whole wheat or shredded wheat, generally giving a high yield of ochratoxins. Increasing levels of potassium bromide led to a decline in ochratoxin A production and an increase in bromo-ochratoxin B, ochratoxin B, and 4-hydroxy ochratoxin B. Nevertheless, A. ochraceus was much less versatile in the bromo analogues than other fungi, which produce metabolites containing chlorine. Analysis included aminopropyl solid-phase extraction column cleanup followed by quantitative analysis on reversed-phase HPLC using fluorescence detection and employing N-(5-chloro-2-hydroxybenzoyl)phenylalanine as an internal standard.

Uptake of radiolabelled ochratoxin A from soil by coffee plants.

[3H, 14C] Ochratoxin A, prepared biosynthetically, was applied in dilute NaHCO3 solution to the soil in which coffee plants had grown to four pairs of leaves. Three weeks later the compound, isolated from dilute NaHCO3 extract of leaves by immunoaffinity chromatography, was detected by scintillation counting as a 1-2 ppm component of leaf dry weight, greatly exceeding the trace (ppb) occurrence of ochratoxin A in some green coffees, which therefore might arise in the field directly from fungal activity in soil rather than from fungal infection of cherries or processed green coffee.

Isolation and characterization of 3-methoxy-2(5H)-furanone as the principal nephrotoxin from Narthecium ossifragum (L.) Huds.

The principal substance in Narthecium ossifragum (L.) Huds, responsible for the nephrotoxic effects on cattle, moose, goats and other ruminants has been isolated and identified by X-ray crystallography as 3-methoxy-2(5H)-furanone. The Fourier-transform infra-red, 1H and 13C nuclear magnetic resonance, and mass spectra are also given. The concentration in four different batches of plant material varied from 113 to 344 microg g(-1) (wet weight). Extracts of N. ossifragum and fractions derived from them, including purified 3-methoxy-2(5H)-furanone, were each dosed intraruminally, to young goats. 3-Methoxy-2(5H)-furanone of 99.9% purity (15 mg kg(-1) live weight) caused increased concentration of creatinine in serum within 2-3 days, typical of kidney damage caused by N. ossifragum, while toxic effect was obtained down to 4 mg kg(-1) live weight with less purified material (> or = 95%). Toxic effect was also obtained with synthesized 3-methoxy-2(5H)-furanone (30 mg kg(-1) live weight). The isomer 4-methoxy-2(5H)-furanone, detected in some of the batches of the plant material, was not toxic when dosed at 60 mg kg(-1) live weight.

Claviceps africana Discovered in India.

The African ergot pathogen (Claviceps africana Frederickson, Mantle, & de Milliano) of sorghum (Sorghum bicolor (L.) Moench) was recently discovered in the Americas (4) and Australia, having previously only been recognized outside Africa in Thailand and Japan (3). The fungus provides a striking example of intercontinental epiphytotics of uncertain origins. Another fungus (C. sorghi Kulkarni, Seshadri & Hegde), the anamorph of which (Sphacelia sorghi McRae) is morphologically similar to that of C. africana and also causes ergot disease of sorghum, is considered to be the pathogen endemic to the Indian subcontinent (1,2). Five isolates of endemic ergot pathogen of sorghum from different locations in Southern India were provided as C. sorghi by ICRISAT, Hyderabad. The isolates were morphologically indistinguishable when cultivated on an asparagine-sucrose-salts agar, producing a white mycelium but no spores. Suspensions of hyphal fragments of each isolate were inoculated into gaping florets of a male-sterile sorghum grown at the Chelsea Physic Garden, London, in 1998. Infection of a few florets occurred with difficulty (<0.1% efficiency) by two of the isolates (from Andhra Pradesh and Madhya Pradesh states) to give a pathology typical of C. africana (2), especially with respect to the prominent young sphacelium forcing the glumes apart before first exudation of honeydew, the low concentration of honeydew oligosaccharides, and the white cascade of secondary sporulation on honeydew. This sphacelial fructification functioned as highly infective inoculum in other inflorescences, readily producing similar pathology leading to the formation of persistently small, roughly spherical "sclerotia" that were typical of C. africana in the recent American epiphytotics, but bearing none of the sclerotial characteristics of C. sorghi. Analysis of ergot tissue from near-mature inflorescences revealed dihydroergosine, an alkaloid that differentiates C. africana from C. sorghi, together with festuclavine, the identity of which was shown by GCMS (gas chromatography-mass spectrometry) (2). This evidence from pathogen isolates was complemented by analysis of a sample of small, spherical "sclerotia" from ICRISAT that also had a similar alkaloid composition. It is therefore clear that C. africana is now in India and this influences not only the interpretation of data on sorghum ergot disease published in recent years from that region, where the identity of the pathogen may not have been rigorously monitored, but also future phytopathological strategies for sorghum more widely in Asia. References: (1) R. Bandyopadhyay et al. Plant Dis. 82:356, 1998. (2) D. E. Frederickson et al. Mycol. Res. 95:1101, 1991. (3) P. G. Mantle and H. A.-G. Hassan. Int. Sorghum Millets Newsl. 35:97, 1994. (4) E. M. Reis et al. Plant Dis. 80:463, 1996.

Porcine nephropathy in Bulgaria: a progressive syndrome of complex or uncertain (mycotoxin) aetiology.

Macroscopic nephropathy was observed in 506 pigs at slaughter in Bulgaria in 1993/94. Histopathological changes were mainly degenerative and proliferative, and were linked with kidney hypertrophy similar to that of the classical Danish Syndrome. Retention cysts formed by dilated tubules, activation or proliferation of capillary and vascular endothelium, and the development of neoplastic tissue were also observed. The most advanced pathology took the form of extensive interstitial fibrosis. Traces of ochratoxin A were found in the kidneys of the majority of 96 cases examined, and in some feed samples taken retrospectively from farms or commercial sources. The dietary ochratoxin concentration (100 micrograms/kg), calculated from serum analyses, closely matched the average of individually analysed feeds. In other feeds no ochratoxin A was detected and the cosmopolitan mycobiota isolated did not include the ochratoxinogenic Penicillium verrucosum that caused the Danish syndrome. Aspergillus ochraceus was rare and the isolates did not synthesise ochratoxin in laboratory culture. The unconfirmed diagnosis of ochratoxicosis suggests a complex or multi-toxin aetiology for this rather common chronic disease in Bulgaria.

The effects of the tremorgenic mycotoxin penitrem A on the rat cerebellum.

Within 10 minutes of intraperitoneal injection of penitrem A (3 mg/kg), rats develop severe generalized tremors and ataxia that persist for up to 48 hours. These are accompanied by a three- to fourfold increase in cerebellar cortical blood flow. Mitochondrial swelling occurs in cerebellar stellate and basket cells within 30 minutes of dosing and persists for more than 12 hours without leading to cell death. From 2 hours, Purkinje cell dendrites show early cytoplasmic condensation accompanied by fine vacuolation of smooth endoplasmic reticulum and enlargement of perikaryal mitochondria. From 6 hours, many Purkinje cells develop intense cytoplasmic condensation with eosinophilia that resembles "ischemic cell change," and from 12 hours, many other Purkinje cells show marked watery swelling. Astrocytes begin to swell from 0.5 hours after injection and show hypertrophy of organelles from 6 hours. Also from 6 hours onward, discrete foci of necrosis appear in the granule cell layer, while permeability of overlying meningeal vessels to horseradish peroxidase becomes evident at 8 hours. All changes are more severe in vermis and paravermis. Despite widespread loss of Purkinje cells, the animals' behavior becomes almost normal within a week. While tremor occurs with doses of 1.5 and 0.5 mg/kg, cellular damage is minimal. The tremor mechanism differs from that of harmaline since destruction of inferior olivary nuclei abolishes neither the tremor response to penitrem A nor the cellular damage. No morphological changes are found in other brain regions. The affinities of penitrem A for high-conductance calcium-dependent potassium channels and for gamma-aminobutyric acid receptors with the probability of resultant excitotoxity are considered to be important underlying factors for these changes.

Nephrotoxicity in goats caused by dosing with a water extract from the stems of Narthecium ossifragum plants.

Seven goats were given a single dose of an aqueous extract derived from 30 g (wet weight) of Narthecium ossifragum per kg liveweight. Their serum creatinine and urea concentrations increased to day 5 but then fell to normal by day 10. Serum magnesium increased to day 4 and decreased to normal by day 9. Their serum calcium concentration was lower than normal on days 4, 5 and 6. Histopathological examination of the kidneys of goats killed or found dead 2, 4, 6, 8, 11 or 16 days after dosing revealed tubular epithelial cell degeneration and necrosis. Regeneration of the tubular epithelium and signs of interstitial fibroplast proliferation and fibrosis could be seen in animals killed on days 8, 11, 16 and 42. No signs of liver damage were observed in 3 goats dosed with the insoluble plant material from 40 g (wet weight) Narthecium ossifragum per kg liveweight. The total dose was divided into three doses, which were given intraruminally within 7 h. The activities of aspartate aminotransferase, gamma-glutamyltransferase and glutamate dehydrogenase remained within the normal range in all 10 goats after dosing.

Further studies on the presence, qualities and effects of the toxic principles from Narthecium ossifragum plants.

One calf was dosed during one day with an aqueous extract from 3.0 kg (wet weight) of Narthecium ossifragum and another was dosed on the same day with the insoluble plant residue. The concentrations of serum creatinine and magnesium increased only in the calf dosed with the aqueous extract, while the activity of glutamate dehydrogenase increased only in the serum of the calf dosed with the plant residue, so differentiating the nephrotoxic and hepatotoxic principles as water-soluble and water-insoluble compounds, respectively. One calf was dosed with 30 g (wet weight) N. ossifragum flower stems per kg live weight during one day and another was dosed with 30 g (wet weight) N. ossifragum leaves per kg live weight on the same day. The serum creatinine and urea concentrations and also the activities of glutamate dehydrogenase, aspartate aminotransferase and gamma-glutamyltransferase in the serum increased in the calf dosed with the flower stems, whereas there was only a slight temporary increase in the creatinine concentration in serum from the calf dosed with the leaves. However, histopathological examination of the kidneys of the calf dosed with the flower stems revealed severe tubular necrosis and degeneration. It therefore appears that both the toxic principles are present in the flower stems of N. ossifragum rather than in its leaves. The serum creatinine concentration was significantly increased in a non-ruminating calf dosed with an aqueous extract from 32 g (wet weight) N. ossifragum per kg liveweight during one day, showing the intrinsic nephrotoxicity of the plant.