Experimental coccidiosis provoked by Eimeria acervulina in chicks simultaneously fed on ochratoxin A contaminated diet.

The progression of coccidiosis provoked by Eimeria acervulina was followed in chicks fed on OTA-contaminated as well as on OTA-free diets. More heavy progress of duodenal coccidiosis, including mortality, occurred in OTA-treated chicks as can be seen from the higher value of lesion (3.50) and oocyst (31.65) indices. A stronger decrease of serum total protein was found in OTA-treated chicks (22.80 g/l) than in chicks infected with E. acervulina(24.20 g/l), but that decrease was strongest in chicks treated with OTA and simultaneously infected with E. acervulina (19.71 g/l). The serum concentration of uric acid was significantly increased in all chicks exposed to OTA, most notably in those additionally infected with E. acervulina (1020.6 (micro mol/L), whereas the serum enzyme activity of AST was increased only in chicks infected with E. acervulina and highest in those fed OTA contaminated diet (122.2 U/L). OTA induced degenerative changes in kidneys, liver and heart as well as a depletion of lymphoid tissue in the lymphoid organs and a decrease of body weight. Coccidiosis induced only a slight growth depression and duodenal hemorrhages in addition to characteristic duodenal damages. The impairment of kidney function, histopathological changes and general growth depression were stronger when chicks infected with E. acervulina were also given OTA.

A toxicogenomics approach to identify new plausible epigenetic mechanisms of ochratoxin a carcinogenicity in rat.

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

Persistent karyomegaly caused by Penicillium nephrotoxins in the rat.

Continuous or intermittent consumption by rats of food moulded by Penicillium aurantiogriseum induced prominent and extensive histopathological changes within several weeks seen specifically at the renal cortico-medullary junction. Many cells of the P3 segment of proximal tubules contained either giant nuclei or multiple enlarged nuclei, described in this text as karyomegaly, but also included within a cytomegalic change. The changes contrasted with the tubular cell necrosis and concomitant mitosis elicited after only four days consumption of nephrotoxic mould. Unilateral nephrectomy enabled persistence of histopathological changes to be assessed directly after detailed histology at an earlier stage. After ten days consumption of food with a 100-fold excess of fungal extract containing the amphoteric nephrotoxins, the typical acute histopathology evolved, over a period of three weeks on normal diet, into the bizarre karyomegalic histopathology, implying a latent effect. Karyomegaly persisted for at least twelve months after nephrotoxin dosage ceased. P. aurantiogriseum karyomegaly was much more striking than that induced by a relatively high chronic dose of another Penicillium nephrotoxin, ochratoxin A. Although the study does not attempt to measure relative potencies, qualitatively similar ultrastructural changes (enlarged nuclei, proliferation of smooth endoplasmic reticulum and thickening of proximal tubule basement membranes) were induced by the two types of nephrotoxin. The broadly toxic ochratoxin A is the popular putative aetiological agent in the mysterious and insidious Balkan endemic nephropathy and associated urinary tract tumours. As the renal carcinogenicity of ochratoxin A in rats follows karyomegaly, the striking karyomegaly induced by P. aurantiogriseum in the proximal tubules of the kidney must be considered as a potential factor in human chronic renal disease.

Actions of a tremorgenic mycotoxin on amino acid transmitter release in vivo.

The tremorgenic mycotoxin verruculogen was administered directly into the brain of freely moving rats by the use of cannula systems that superfused either the cortical surface or the lateral ventricular space. The tremor produced by these CNS routes was compared with that produced by i.p. administration of the toxin or the dried mycelium of the fungus that synthesizes the verruculogen. The nature and degree of tremor produced by the central vs peripheral routes suggest that the site of action of verruculogen is not immediately adjacent to the cannula sites in the brain. Measures of the amino acids in the superfusates collected during the verruculogen-induced tremor showed an increase in the excitatory neurotransmitters, glutamate and aspartate in superfusates from the lateral ventricle but not in superfusates from the cortical surface. The differential effect on transmitter release suggests that a subcortical action of verruculogen is responsible for its tremorgenic activity.

Biosynthesis of glyantrypine by Aspergillus clavatus.

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.

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.

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.

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.

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.

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.

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.

Amino acid neurotransmitter release from cerebrocortical synaptosomes of sheep with severe ryegrass staggers in New Zealand.

Enhanced unstimulated release of the excitatory amino acid neurotransmitters aspartic acid and glutamic acid was measured in cerebrocortical synaptosomes prepared from sheep showing severe symptoms of ryegrass staggers. A similar pattern of deviation from neurologically normal animals was seen whether the sheep had contracted the disorder naturally or whether they had been deliberately caused to develop the disorder by grazing specific pastures rich in endophyte infected ryegrass. Deranged neurotransmitter release may reasonably be involved in the chemical pathology of ryegrass staggers and the findings are in accord with the putative synonymy of lolitrem toxicosis and ryegrass staggers.

The role of Penicillia in ryegrass staggers.

Tremorgenic strains of Penicillium verrucosum var cyclopium, P canescens, P janthinellum, P novaezeelandiae and P estinogenum were isolated from the faeces of 15 of 23 affected sheep and cattle in eight of nine field outbreaks of ryegrass staggers. One tremorgenic strain of P griseofulvum was isolated from the faeces of one of 25 sheep grazing in unaffected flocks. Tremorgenic strains of P verrucosum var cyclopium, P canescens, P janthinellum and P estinogenum were also isolated from the A horizon of New Zealand soils. Since a large proportion of experimentally dosed live P verrucosum var cyclopium died during passage through the gut, the faecal evidence from naturally staggering animals suggests that at least some outbreaks of ryegrass staggers are caused by tremorgenic Penicillia and that their source may be soil.

Excretion and transformation of [14C] penitrem mycotoxins in sheep bile.

[14C] penitrem A (1.1 mg) was given intravenously to a sheep under barbiturate anaesthesia. The radioactivity was almost completely excreted in the bile within four hours but the penitrem had been transformed to more polar products. [14C] penitrem E (4.5 mg) was eliminated similarly. Incubation of the penitrems with sheep or rat liver homogenate did not result in any metabolic products. However, in sheep bile, whether previously boiled or not, complete transformation to more polar products occurred spontaneously within seven hours at 37 degrees C, and apparently non-enzymic process which seems to be a novel attribute of bile.

Mycotoxic tremorgens of Claviceps paspali and Penicillium cyclopium: a comparative study of effects on sheep and cattle in relation to natural staggers syndromes.

Claviceps paspali sclerotia were dosed orally at various levels to sheep and cattle and the neurotoxic effects are described. Evidence is presented which indicates that the tremors are produced by a neutral tremorgen fraction. The ergot alkaloids, principally D-lysergic acid alpha-hydroxyethylamide and its isolysergic acid isomer, are not involved. The mixture of C paspali tremorgens has not been separated and characterised but its components are chemically related to other known tremorgens, such as penitrem A, produced by saprophytic moulds, notably Penicillium cyclopium. P cyclopium mycelium and C paspali sclerotia were dosed to produce comparable effects in sheep and in cattle. Similarly, effects of penitrem A and tremorgen concentrate of C paspali were compared after intravenous dosage to sheep. Observed enurotoxic effects of both moulds were identical in most respects. A low level of tremorgenic activity was detected at the honeydew stage of C paspali. These findings are discussed in relation to staggers syndromes occurring in grazing livestock.

Further studies concerning the toxicity of ingested ergot sclerotia (Claviceps purpurea) to young and growing pigs.

In three experiments pigs were offered diets contaminated with milled ergot sclerotia. In a metabolism study 0-5 per cent (w/w) of American wheat ergot decreased urinary nitrogen losses and improved the efficiency of nitrogen retention. When early weaned piglets ingested a diet contaminated with 2-5 per cent of the ergot, feed intake was reduced and growth rate decreased. Reduction in growth rate was also evident in growing pigs given a diet containing ergotised English wheat. These latter pigs had lesions in stomach, intestine and liver, consistent with a toxic insult. With respect to feed intake and growth these results agreed with those of an earlier study, although a toxic response was produced by a lower dosage rate and there was additional internal evidence of toxicity. Improved nitrogen retention was contrary to earlier evidence at higher dose rates but may merely reflect a tonic action which, at higher doses, becomes toxic.

Metabolism and excretion of [14C] verruculogen in a sheep.

[14C] Verruculogen (75 micrograms/kg) was given intravenously to a sheep under barbiturate anaesthesia to prevent the severe tremor and convulsions which would otherwise have occurred. Two hours later 28 per cent of the tremorgenic mycotoxin was detected in the liver, bile and small intestine. Approximately 0.5 per cent was excreted in the urine. Trace amounts of radiolabel were detected in the cortex and corpus striatum of the brain. Verruculogen was metabolised by the liver and converted completely to four more polar products, including two isomeric forms of desoxy-verruculogen and the weakly tremorgenic mycotoxin TR-2. The principal and most polar metabolite excreted is probably an isomer of TR-2.

A comparative study of sheep and pigs given the tremorgenic mycotoxins verruculogen and penitrem A.

The moulds Penicillium simplicissimum and P crustosum and the tremorgenic mycotoxins, verruculogen and penitrem A, isolated from them, were given to sheep and pigs to compare their potencies. Pigs were generally less susceptible and in both species penitrem A was less potent than verruculogen. Five-month-old lambs seemed more susceptible to mycelium containing verruculogen than were 15-month-old sheep given a similar oral dose relative to bodyweight. Repeated daily dosing of lambs and sheep for five days with P simplicissimum failed to enhance the effect, indicating that verruculogen toxicity was not cumulative. Long and short acting barbiturate anaesthesia blocked the effects of lethal doses of tremorgens. Sedation with diazepam diminished, but did not block, mycotoxin-induced tremors suggesting that there was no specific action of this anticonvulsant sedative on tremorgens.

Some consequences of the ingestion by young and growing pigs of feed contaminated with ergot.

Growing pigs were fed freshly milled ergot sclerotia (Claviceps purpurea Fr (Tul)) from North American wheat containing 0-31 per cent of total alkaloids of which 30 per cent was ergotoxine, 30 per cent ergosine, 20 per cent ergotamine and 20 per cent other ergot alkaloids. Pigs were induced to ingest a diet heavily contaminated with ergot (10 per cent w/w). The presence of ergot reduced feed intake and growth of the pigs, but there was no evidence of peripheral necrosis, of internal or external haemorrhage, or of changes in haemotological characteristics. Balance experiments in which the diet was contaminated with 4 per cent (w/w) of ergot showed that 90 per cent of the alkaloids were absorbed by the pigs, but none could be detected in tissues or urine. The ingestion of ergot did not affect the digestibility of the diet, but the urinary excretion of nitrogen was increased and the retention of nitrogen reduced. The presence of ricinoleic acid was shown to be a useful diagnostic feature of ergot contamination of diets, digesta and faeces; it was not found in body tissues.