Optimised Fermentation Production of Radiolabelled Ochratoxin A by Aspergillus ochraceus with Maximum 14C in the Pentaketide Moiety for Exploring Its Rat Renal Toxicology.

In the context of the mysterious Balkan endemic nephropathy of the 1900s, and the discovery in the 1960s of the potent mycotoxin ochratoxin A, experimental research projects sought to explore any inter-relationship. Experimental lifetime administration of the toxin to male rats had revealed renal DNA adducts with the toxin, correlated with renal tumours, confirmation of which required molecular evidence. Consequently, production of 14C-ochratoxin A of a high specific radioactivity was required, practical biosynthetic detail of which had not previously been published. A fermentation study of Aspergillus ochraceous was carried out during 2002 for a European project, to select for the production of high-quality 14C-ochratoxin A, necessarily exploring for the maximum diversion of 14C-sodium acetate into the pentaketide portion of mycotoxin. Experimentation necessarily had to optimise the competitive context of fungal growth dynamics and addition of the biosynthetic precursor in the early days of shaken-flask fermentation before adding the radiolabelled precursor. From optimal fermentation, 50 mg of the 14C ochratoxin A was supplied within a European project for DNA adduct experimentation, but that proved negative as subsequently published. Experimental description of the radiolabelled ochratoxin A production was later made in a doctoral thesis, but is first publicised here. Further review of the literature reveals an explanation for the published failure to confirm rat DNA/ochratoxin A adduct formation, for which further experimentation is now recommended.

Rat Tumour Histopathology Associated with Experimental Chronic Dietary Exposure to Ochratoxin A in Prediction of the Mycotoxin's Risk for Human Cancers.

Mammalian animal toxicity of ochratoxin A (OTA) has focused largely in the past half-century on pigs because of initial recognition of it as a principal cause of intermittent growth suppression and renal disease caused by mouldy feed. Subsequent classical toxicology has used laboratory rodents because renal pathology in pigs raised questions concerning possible involvement in the human idiopathic bilateral renal atrophy of Balkan endemic nephropathy for which OTA was a focus of attention for human nephropathy through 1980s and into 2000s. Emphasis on human nephropathy has more recently concerned the plant metabolite aristolochic acid. Recognition that agricultural management can often minimise food and feed-stuff spoilage by OTA-producing Aspergilli and Penicillia has moderated some of the risks for animals. Legislation for human food safety combined with sophisticated analysis generally provides safety in the developed world. Chronic experimental exposure of male rats, in the absence of clinical dis-ease, specifically causes renal cancer. The possibility of this as a unique model for the human has generated considerable experimental evidence which may be more directly relevant for carcinogenesis in the complex kidney than that obtained from biochemical toxicities in vitro. Nevertheless, there does not appear to be any case of human renal or urinary tract cancer for which there is verified etiological proof for causation by OTA, contrary to much claim in the literature. To contribute to such debate, histopathology review of OTA/rat renal cancers, augmented where appropriate by immune profiles, has been completed for all remaining tumours in our research archive. Overall consistency of positivity for vimentin, is matched with occasional positives either for CD10 or the cytokeratin MNF 116. The current situation is discussed. Suggestion that OTA could cause human testicular cancer has also been challenged as unsupported by any experimental findings in rats, where the Leydig cell tumour immune profile does not match that of human germ cell neoplasms.

Immunohistochemical Review of Leydig Cell Lesions in Ochratoxin A-Treated Fischer Rats and Controls.

Ochratoxin A is best known as a potent renal carcinogen in male rats and mice after necessarily protracted ingestion, although valid extrapolation to any human disease has not been verified. The hypothesis that the toxin is a cause of human testicular cancer was proposed a decade ago and has proliferated since, partly through incomplete study of the scientific literature. Archived tumorous rat testes were available from Fischer F344 rats exposed to continuous dietary exposure for half of or the whole life in London in the 2000s. Renal cancer occurred in some of these cases and testicular tumours were observed frequently, as expected, in both treated and untreated animals. Application of clinical immunohistochemistry has for the first time consistently diagnosed the testicular hypertrophy in toxin-treated rats as Leydig cell tumours. Comparison is made with similar analysis of tumorous testes from control (untreated) rats from U.S. National Toxicology Program studies, both of ochratoxin A (1989) and the more recent one on Ginkgo biloba. All have been found to have identical pathology as being of sex cord-stromal origin. Such are rare in humans, most being of germinal cell origin. The absence of experimental evidence of any specific rat testicular cellular pathology attributable to long-term dietary ochratoxin A exposure discredits any experimental animal evidence of testicular tumorigenicity. Thus, no epidemiological connection between ochratoxin A and the incidence of human testicular cancer can be justified scientifically.

Monic acid A: a biomarker in clinical intra-nasal mupirocin medication for MRSA decolonisation.

CONTEXT: Mupirocin (BactrobanR) is widely prescribed for intra-nasal decolonisation of MRSA for in-patients awaiting surgery or self-medicated for out-patients although adherence for the latter is not monitored. Non-adherence is a widespread pharmaceutical problem but could encourage selection of antibiotic resistance. Mupirocin is only a topical antibiotic because it decomposes in stomach acidity to monic acid A, but this has not previously been exploited as a biomarker for clinical intra-nasal medication. MATERIALS AND METHODS: Urine from three catheterised patients in two London hospitals during and after mupirocin medication, was passed through Waters Oasis cartridges to isolate organic acids. Sensitive LC-MS-MS analysis for monic acid A in methanolic eluate has been developed to identify ∼10 pg. RESULTS: Monic acid A was quantified in all samples from one patient, translating into 6-46 ng from 12 mg mupirocin, assuming 1 L daily urine output. However, no urinary monic acid A was detected for two other patients. DISCUSSION AND CONCLUSIONS: Consistent occurrence of monic acid A in urine of one mupirocin patient shows for the first time that antibiotic distribution across nasal mucous membranes had generally been maintained during medication. In contrast, consistent absence in the two other patients requires wider study in hospital.

Immunohistochemical Analysis of Rat Renal Tumours Caused by Ochratoxin A.

Experimental renal cancer caused by ochratoxin A (OTA) in rats was first defined in the US National Toxicology Program (1989) and raised questions about any aetiological role in human urinary tract tumours. A review of histopathology in several rat kidney tumours from dietary OTA in recently described London studies, augmented by clinical immunohistochemistry for the first time for this mycotoxin, establishes their renal tubular cell origin. It had been assumed that the toxin might cause the human urothelial tumours associated with Balkan endemic nephropathy, but the present study could not support this. Comparison with a similar review of a metastasising renal tumour from a female rat of the NTP study consistently shows the kidney as the primary carcinogenic site for OTA. Morphological heterogeneity of these kidney tumours as epithelioid and/or sarcomatoid is revealed. Leiomyosarcoma was also diagnosed, and rhabdomyosarcoma differentiation was observed in the exceptionally aggressive NTP female tumour. The present pilot study involving immunohistochemistry indicates need for wider review of archived tumours for experimental evidence before formulating any epidemiological basis from a rat model for OTA's relevance to idiopathic human renal cell carcinoma. Although the NTP study concluded that females are less sensitive to OTA than males, some female tumours still had heterogeneous morphology.

Rat Kidney Cancers Determined by Dietary Ochratoxin A in the First Year of Life.

An experiment to explore renal carcinogenic efficacy of male rat exposure to dietary ochratoxin A (OTA) only in the first year of life has been made in comparison to lifetime exposure. Ten months exposure to OTA at 300 µg/kg b.w. was sufficient to cause high incidence of tumours which became apparent clinically after a latency of up to a year. As a putative model for human kidney cancer, the study shows a silent organ-specific carcinogenic effect through protracted exposure up to middle age and focused probably on very few nephrons. So far, tumourigenesis has not been recognised until in the last quarter of natural rat life, but for OTA, rat renal carcinogenesis requires both long exposure and only during the first year of normal longevity. The present findings offer an experimental framework within which systematic histopathology during tumourigenesis might show whether findings of mechanistic studies in key focal neoplasms can reasonably be applied to OTA as a putative renal carcinogen for idiopathic kidney cancer in humans. Already, the rat tumours mimic those occurring spontaneously in the Eker rat, and there is disparity between the large necessary OTA exposure in the rat and the trace amounts of OTA consumed by humans. In all such complex considerations it is important to adhere rigorously to established principles of disease epidemiology.

Contribution of organ vasculature in rat renal analysis for ochratoxin a: relevance to toxicology of nephrotoxins.

Assumptions surrounding the kidney as a target for accumulation of ochratoxin A (OTA) are addressed because the contribution of the toxin in blood seems invariably to have been ignored. Adult rats were maintained for several weeks on toxin-contaminated feed. Using standard perfusion techniques, animals were anaesthetised, a blood sample was taken, one kidney was ligated, and the other kidney perfused with physiological saline in situ under normal blood pressure. Comparative analysis of OTA in pairs of kidneys showed marked reduction in the perfused organ in the range 37%-98% (mean 75%), demonstrating the general efficiency of perfusion supported also by histology, and implying a major role of blood in the total OTA content of kidney. Translation of OTA values in plasma to whole blood, and its predicted contribution as a 25% vascular compartment in kidney gave values similar to those in non-perfused kidneys. Thus, apparent 'accumulation' of OTA in kidney is due to binding to plasma proteins and long half-life in plasma. Attention should be re-focused on whole animal pharmacokinetics during chronic OTA exposure. Similar principles may be applied to DNA-OTA adducts which are now recognised as occurring in blood; application could also extend to other nephrotoxins such as aristolochic acid. Thus, at least, quantitative reassessment in urological tissues seems necessary in attributing adducts specifically as markers of potentially-tumourigenic exposure.

Revealing a Pre-neoplastic Renal Tubular Lesion by p-S6 Protein Immunohistochemistry after Rat Exposure to Aristolochic Acid.

Aristolochic acid (AA) has, in the last decade, become widely promoted as the cause of the Balkan endemic nephropathy and associated renal or urothelial tumours, although without substantial focal evidence of the quantitative dietary exposure via bread in specific households in hyperendemic villages. Occasional ethnobotanical use of Aristolochia clematitis might be a source of AA, and Pliocene lignite contamination of well-water is also a putative health risk factor. The aim of this study was two-fold: to verify if extracts of A. clematitis and Pliocene, or AA by itself, could induce the development of renal or urothelial tumours, and to test the utility of the ribosomal protein p-S6 to identify preneoplastic transformation. Rats were given extracts of A. clematitis in drinking water or AA I, by gavage. After seven months, renal morphology was studied using conventional haematoxylin and eosin and immunohistochemistry for ribosomal p-S6 protein. Plant extracts (cumulative AA approximately 1.8 g/kg b.w.) were tolerated and caused no gross pathology or renal histopathological change, with only faint diffuse p-S6 protein (except in the papilla) as in controls. Cumulative AA I (150 mg/kg b.w. given over 3 days) was also tolerated for seven months by all recipients, without gross pathology or kidney tumours. However, p-S6 protein over-expression was consistent particularly within the renal papilla. In one case given AA I, intense p-S6 protein staining of a proximal tubule fragment crucially matched the pre-neoplastic histology in an adjacent kidney section. We briefly discuss these findings, which compound uncertainty concerning the cause of the renal or upper urinary tract tumours of the Balkan endemic nephropathy.

Changes in male rat urinary protein profile during puberty: a pilot study.

BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, α2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n = 3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease. RESULTS: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (α2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins. CONCLUSIONS: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models.

Comparative immunohistochemical analysis of ochratoxin A tumourigenesis in rats and urinary tract carcinoma in humans; mechanistic significance of p-S6 ribosomal protein expression.

Ochratoxin A (OTA) is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis.

H NMR spectroscopy-based metabolomic assessment of uremic toxicity, with toxicological outcomes, in male rats following an acute, mid-life insult from ochratoxin a.

Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. (1)H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by (1)H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic.

Lifetime, low-dose ochratoxin A dietary study on renal carcinogenesis in male Fischer rats.

Carcinoma arising from male rat renal parenchyma is an aspect of the nephrotoxicity of ochratoxin A (OTA) and is a factor in considering application of animal data to human health risk assessment. We present experimental data to complement already published and to complete dose-response findings for dietary OTA. From 34 rats, only four unilateral renal carcinomas (12%) developed during a 2-year exposure to dietary OTA, contaminated to give the same weekly overall dosage as in the 50 µg kg(-1) gavage-dosing regimen of an NTP study (30%). Statistical analysis included adjustment for premature leukaemia deaths, resulting in the carcinoma incidence of 35% (10-81%), and showed no significant difference from NTP (incidence of 43% (23-49%)) due to the smaller number of animals. However, absence of microscopic neoplastic renal lesions in premature decedents argues for minimal effect of the 47% leukaemia on carcinoma expression in the present experiment. This would fit with previously published findings showing significantly less carcinoma expression from a regimen administering an OTA dose in feed than was achieved by a lower dose by gavage as in the NTP study. It is concluded that chronic gavage administration of OTA to male rats may optimise carcinoma incidence for toxicological purposes, but that the dietary mode gives data more applicable to assessing putative health risk for humans.

Comments on "Ochratoxin A: In utero Exposure in Mice Induces Adducts in Testicular DNA. Toxins 2010, 2, 1428-1444"-Mis-Citation of Rat Literature to Justify a Hypothetical Role for Ochratoxin A in Testicular Cancer.

A manuscript in the journal recently cited experimental rat data from two manuscripts to support plausibility of a thesis that ochratoxin A might be a cause of human testicular cancer. I believe that there is no experimental evidence that ochratoxin A produces testicular cancer in rats or mice.

A pilot study of nuclear instability in archived renal and upper urinary tract tumours with putative ochratoxin aetiology.

DNA ploidy measurement has been applied uniquely to wax-embedded tissue of primary renal cell and metastatic tumours of a key experimental researcher on porcine ochratoxicosis, a control, and four transitional cell carcinomas from cases of Balkan endemic nephropathy. Primary renal tumour was diploid, and hyperdiploid metastasis was within the lower ploidy range for typical renal cell carcinoma. Three Balkan primary tumours showed extensive aneuploidy indicating marked nuclear instability, similar to model rat renal carcinoma caused by ochratoxin A. In contrast, much less nuclear instability in the putative occupational ochratoxicosis case fitted poorly with the ochratoxin A model.

Oncological outcomes in rats given nephrocarcinogenic exposure to dietary ochratoxin a, followed by the tumour promoter sodium barbital for life: a pilot study.

The potent experimental renal carcinogenesis of ochratoxin A (OTA) in male rats makes the dietary contaminant a potential factor in human oncology. We explored whether the tumour promoter sodium barbitate could shorten the otherwise long latency between exposure to toxin and tumourigenesis. Young rats, of a hybrid in which mononuclear leukaemia was rare, were given feed contaminated (5 ppm) with OTA for 36 weeks to initiate renal tumourigenesis. Some individuals were thereafter given sodium barbitate (500 ppm in drinking water) for life. Pathological outcomes were studied at or near the end of natural life. Renal tumours in males given barbitate became evident after latency of one year, but only slightly before those without barbitate. In contrast, female mammary tumourigenesis was advanced by at least 6 months synchronously in all rats given the OTA-barbitate regimen compared to tumourigenesis in controls. Diagnosis of malignant mammary angiosarcoma in a female given the OTA-barbitate regimen is a new finding in the rat. The long latency of OTA-induced renal tumourigenesis was not notably susceptible to accelerated promotion by barbitate, contrasting with an apparently marked effect of barbitate on development of mammary tumours.

Pathological outcomes in kidney and brain in male Fischer rats given dietary ochratoxin A, commencing at one year of age.

Malignant renal carcinoma, manifest in morbid ageing rats, is the striking component of an otherwise silent response after about nine months of exposure to ochratoxin A in the first year of life (daily intake ~100-250 µg/kg body weight). Reasons for the long latency are unclear, as is whether there would be a similar carcinogenic response if toxin exposure started at one year of age. Therefore, 24 male Fischer rats were given 100 µg ochratoxin A as a daily dietary contaminant for 35 weeks from age 50 weeks. Plasma ochratoxin A concentration reached a maximum value of ~8 µg/mL within one month of starting the toxin regimen. No renal carcinomas occurred. Four renal adenomas, two of which were only microscopic, were found among the six rats surviving for 110 weeks. The findings raise new questions about a difference between young adults and mature adults in sensitivity of male rats to the ochratoxin A-induced DNA damage necessary for renal carcinogenesis. A pilot histological study of perfuse-fixed brains of the toxin-treated rats showed no gross abnormalities, correlating with the consistent absence of behavioral or neurological disorders from chronic ochratoxin A exposure regimens in the range 100-250 µg/kg/day during the second half of life. Reasoned questioning concerning ochratoxin A as a neurotoxic mycotoxin is made.

Contrasting nephropathic responses to oral administration of extract of cultured Penicillium polonicum in rat and primate.

Liquid- or solid substrate-cultured Penicillium polonicum administered in feed to rats over several days evokes a histopathological response in kidney involving apoptosis and abnormal mitosis in proximal tubules. The amphoteric toxin is yet only partly characterized, but can be isolated from cultured sporulating biomass in a fraction that is soluble in water and ethanol, and exchangeable on either anion- or cation-exchange resins. After several weeks of treatment renal proximal tubule distortion became striking on account of karyocytomegaly, but even treatment for nearly two years remained asymptomatic. Extract from a batch of solid substrate fermentation of P. polonicum on shredded wheat was incorporated into feed for rats during four consecutive days, and also given as an aqueous solution by oral gavage to a vervet monkey daily for 10 days. Treatment was asymptomatic for both types of animal. Rat response was evident as the typical renal apoptosis and karyomegaly. In contrast there was no such response in the primate; and neither creatinine clearance nor any haematological characteristic or serum component concentration deviated from a control or from historical data for this primate. The contrast is discussed concerning other negative findings for P. polonicum in pigs and hamsters. Renal karyomegaly, as a common rat response to persistent exposure to ochratoxin A, is not known in humans suspected as being exposed to more than the usual trace amounts of dietary ochratoxin A. Therefore the present findings question assumptions that human response to ochratoxin A conforms to that in the rat.

Structures of covalent adducts between DNA and ochratoxin a: a new factor in debate about genotoxicity and human risk assessment.

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after (32)P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined (32)P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3'dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in male Dark Agouti rats.

In rats fed dietary ochratoxin A (5 ppm for 3, 6 or 9 months) no renal tumours occurred throughout natural life of the group treated for 3 months, during which the ochratoxin dose was 3 times that in the high dose group of the NTP study. Bilateral renal carcinoma occurred in one rat in the 6 month group. Four rats treated for 9 months developed unilateral renal carcinoma. Overall latency between ceasing toxin exposure and discovering tumours was 35-97 weeks. Experimental verification of a 'no observable effect level' was made for feed containing 400 ppb, equivalent to approximately 7 microg ochratoxin A/day for Dark Agouti rats for up to 2 years, during which mean daily dose commenced at approximately 50 microg/kg, but later for adults was in the range 30-20 microg/kg. This data doubles the daily in vivo threshold dose from the NTP study ( approximately 15 microg/kg), and could influence human risk assessment. An at least 3 month threshold period for exposure to exceptionally high daily OTA intake (90 microg; 640-450 microg/kg) raises doubts over interpretation of experimental molecular data for OTA exposure at lower dose for up to 3 months in studies aimed at understanding carcinogenic mechanism.

Metabolic pathway of oligosaccharides in sorghum honeydew caused by Claviceps africana / Rota metabólica de oligossacarídeos em exsudato da "doença açucarada" do sorgo causado por Claviceps africana

The occurrence of the alditol oligosaccharides in the Claviceps afriana honeydew is partly as a rational expression of the pathogen's selective nutritive metabolism of the sucrose supplied by the host plant. The experiments were carried out in laboratory and when 14C-D-sucrose, 14C-D-fructose or 14C-D-mannitol radiolabelled saccharides were incorporated into: a) sorghum plant infected by C. africana, b) whole and macereted micelia tissue and c) cell-free honeydew of C. africana, it was observed that the glucose moiety of sucrose was not involved in oligosaccharides formation. Glucose was used by the pathogen as nutritional source. Part of the unused fructose moiety was reduced to mannitol by the pathogen's enzymes which was also excreted into honeydew where reductase activity accepted 14C mannitol. The mannitol was linked with fructose in a 2-position synthesizing the disaccharide 1-beta-D-fructofuranosyl-D-mannitol and then the process was repeated by the mannitol moiety of the disaccharide to yield the trisaccharide 1,6-di-beta-D-fructofuranosyl-D-mannitol, which became dominant. The direct formation of alditol saccharides from monosaccharides in this way seems to be unique to C. africana, contrasting with the fructosyl transfer from sucrose to sucrose which is usual in others ergot parasites.

A ocorrência de oligossacarídeos contendo alditol em exsudações da doença açucarada produzidos por Claviceps africana é em parte uma expressão racional do metabolismo seletivo de nutrição do patógeno na tansformação da sacarose fornecida pela planta. Quando açúcares radioativos marcados com 14C (D-sucrose, D-fructose e D-mannitol) foram incorporados em: a) planta do sorgo infectada por C. africana, b) tecido micelial inteiro e macerado isolado de C. africana e c) exsudato livre de células de C. africana, foi observado que a glicose da sacarose não estava envolvida na formação de oligossacarídeos, sendo usada pelo patógeno como fonte nutritiva. Parte da frutose não utilizada da sacarose foi reduzida a mannitol por enzimas do patógeno. As enzimas envolvidas também atuaram no exsudato da "doença açucarada", ativando a ação da redutase pela incorporação do 14C manitol. O manitol foi ligado na posição 2 da fructose, formando o dissacarídeo 1-beta-D-fructofuranosyl-D-mannitol e, posteriormente, adicionada mais uma fructose ao mannitol do dissacarídeo, formando o trisacarídeo 1,6-di-beta-D-fructofuranosyl-D-manitol, que se tornou predominate. A formação direta de oligossacarídeos contendo alditol a partir de monossacarídeos parece ser exclusiva em C. africana, contrastando com a ação enzimática da frutose transferase a partir de sucrose para sucrose, a qual é muito comum em outros patógenos tipo "ergot".