Peter G. Mantle

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.

Claviceps africana sp. nov.; the distinctive ergot pathogen of sorghum in Africa

Stromata arising from ergot sclerotia from sorghum in Zimbabwe were different in colour and texture from those of Claviceps sorghi from sorghum in India, compounding other differences in the dynamics of the early stages of parasitism, the sugar composition of honeydew, the quantitative expression of secondary conidiation and the morphology of sclerotia. The distinctive sexual stage forms the basis of describing the African material as a new Claviceps species which also uniquely elaborates a group of dihydrogenated ergot alkaloids that are biosynthetic intermediates leading to the principal product dihydroergosine.

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.

Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A.

The potency of ochratoxin A (OTA) as a renal carcinogen in the rat in response to lifetime administration by oral gavage is a basis of current concern about possible human risk from dietary exposure to the mycotoxin. In this study, dietary delivery of OTA was chosen as the mode of administration, since this mimics human intake of OTA-contaminated food more accurately than gastric intubation. Young male Fischer rats were given approximately 300 µg OTA/kg body weight (bwt) daily until they reached 333 g; thereafter their daily intake was held at about 100 µg. Renal tumours, mostly unilateral carcinomas, were first discovered at week 75 and total incidence reached 25%. Statistical comparison of total carcinoma incidence (20%) in this study with that of the classic US NTP study suggested that OTA was significantly less carcinogenic when administered in feed than when given by oral gavage. The finding may moderate perceptions of a putative risk of trace amounts of OTA in some foodstuffs to human health.

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.

Nephrotoxic fungi in foods from nephropathy households in Bulgaria

The Penicillium spp. and Aspergillus spp. associated with 83 samples of foods from Bulgarian households, most of which had a history of Balkan endemic nephropathy, have been defined. Penicillium aurantiogriseum was the most common species of this genus and all isolates tested were acutely nephrotoxic in the rat. P. verrucosum , a species normally associated with ochratoxin A biosynthesis in Europe, was rare and all isolates failed to elaborate ochratoxin A in pure culture. Several isolates of P. citrinum produced abundant citrinin when moulding moist shredded wheat, but such had no acute effect on rat kidneys. One apparently unique Penicillium isolate, best assigned to P. solitum , produced ochratoxin A in pure culture. Aspergilli of the flavus and glaucus groups were common, but A. ochraceus was relatively uncommon and no isolate produced more than a trace of ochratoxin A in pure culture. Beans, maize and other cereals generally contained no detectable ochratoxin A ( −1 ). One apparently sound maize sample containing 153 g ochratoxin A kg −1 did not contain A. ochraceus but two ochratoxinogenic fungi were isolated, which best conformed to P. viridicatum and P. griseofulvum . Seeds containing these fungi accounted for most of the ochratoxin A in the whole sample. The natural occurrence of ochratoxin A in food in the region is ascribed mainly to fungi which do not fit the current concept of ochratoxinogenic penicillia, but the low general natural incidence both of ochratoxinogenic fungi and also of ochratoxin A in the present study hardly supports the popular putative epidemiological role of ochratoxin A in the highly mosaic distribution of Balkan endemic nephropathy.

Studies on some feed additives giving partial protection against ochratoxin A toxicity in chicks.

Significant protective effects of the feed additives: water extract of artichoke, sesame seed, Roxazyme-G and L-beta phenylalanine against the growth inhibitory effect of ochratoxin A (OTA) and associated pathomorphological changes were seen. Similarly, there was less OTA-induced decrease in serum total protein and increase of serum creatinine and urea in the chicks. Whereas OTA induced strong degenerative changes and an increase in weight of kidneys and liver as well as a decrease of the weight of lymphoid organs the additives variously gave protection against these changes. The protection of Roxazyme-G and sesame seed was better expressed in kidneys and liver, whereas the phenylalanine better protected the weight changes in gizzard, heart and the changes in differential WBC count. Notably, sesame seed gave strong protection against 5 ppm OTA-induced suppression of humoral immune response, for which artichoke also had some beneficial effect, whereas phenylalanine had hardly any effect.

Sesquiterpenoid metabolites from Stereum complicatum

Abstract A new sesquiterpene antibiotic, complicatic acid, isolated from cultures of Stereum complicatum (Fr.)Fr. has been shown to be dehydrohirsutic acid C . Hirsutic acid C was also isolated from the same fungus. [2- 14 C]-MVA was incorporated into both metabolites and complicatic acid has been shown to be formed from hirsutic acid C both in vivo and in vitro .

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.

Ochratoxin A: Potential epigenetic mechanisms of toxicity and carcinogenicity

Assessment of the significance to human health of ochratoxin A (OTA) in food is limited by a lack of human toxicity data. Therefore, OTA risk evaluation relies mainly on the use of animal data, with renal carcinogenicity in rat being considered as the pivotal effect. The elucidation of the mechanism of action would improve the use of the carcinogenicity data for risk assessment. Direct genotoxicity versus epigenetic mechanisms appears to be a key question. In this presentation, new biochemical and toxicogenomic results obtained in a recent European project (EU-Grant # QLK1-CT-2001-011614) will be summarized in the context of previously reported mechanisms of action including inhibition of protein synthesis, production of oxidative stress and alteration of cell signalling. Amongst others, the new data indicate that chronic administration of a carcinogenic dose of OTA affected cell-signalling pathways resulting in a significantly reduced renal antioxidant defence and increased oxidative DNA damage. These data confirm previous hypotheses involving oxidative stress as a possible key epigenetic mechanism of OTA toxicity and carcinogenicity.