Book of Abstracts MYCOTOXINS and PHYCOTOXINS

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22-25 JULY 1985

SIXTH INTERNATIONAL SYMPOSIUM ON MYCOTOXINS AND PHYCOTOXINS

CSIR CONFERENCE CENTRE PRETORIA REPUBLIC OF SOUTH AFRICA SIXTH INTERNATIONAL SYMPOSIUM on MYCOTOXINS AND PHYCOTOXINS

ABSTRACTS

22-25 JULY 1985

CSIR CONFERENCE CENTRE PRETORIA REPUBLIC OF SOUTH AFRICA

under the auspices of

INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY

SOUTH AFRICAN COUNCIL FOR SCIENTIFIC AND INDUSTRIAL RESEARCH

and the SA Association for Food Science and Technology SA Biochemical Society SA Chemical Institute SA Society for Animal Production SA Society for Plant Pathology SA Veterinary Association

with the assistance of the CSIR Symposium Secretariat

S.3S1 PLENARY PRESENTATIONS

Synthetic studies on fungal metabolites and other natural products G Biichi PL1 Regulation of secondary metabolism in fungi A L Demain PL 2 Biomimetic synthesis of aromatic polyketide metabolites T M Harris and C M Harris PL 3 Global significance of mycotoxins C W Hessertine PL 4 Biology of toxigenic anamorphs B Kendrick PL 5 Biochemical mechanism of action of mycotoxins K-H Kiessling PL 6 Toxins from cyanophytes belonging to the Scytonemaceae R E Moore PL 7 Sampling, sample preparation, and sampling plans for foodstuffs for mycotoxin analysis D L Park, A D Campbell, T B Whitaker, A E Pohland and J W Dickens Abstract not received Pathology of mycotoxicoses: possibilities and limits of diagnosis HBSchieffer PL 8 Toxigenesis and biosynthesis of saxitoxin analogs YShimizu PL 9 Recent structural studies on mould metabolites CHTamm PL 10 Progress towards a biosynthetic rationale of the aflatoxin pathway CATownsend PL 11 Toxicology of naturally occurring microbial toxins YUeno PL 12 Determination of mycotoxins H van Egmond and W E Paulsch PL 13

-J. •• Biosynthetic studies on some polyene mycotoxins RVteggaar PL 14 SECTION LECTURES

Page Secondary metabolites produced by some Fusarium species J W ApSimon, B Blackwell, R Greenhalgh, R R King, R-M Meier, D Miller, J R J Pare and A Taylor S 1 The genetic basis of mycotoxin biosynthesis JW Bennett S2 Cyanoginosins - isolation, structure and biology D P Botes S 3 Mechanism of action for peptide toxins of freshwater cyanobacteria (blue-green algae) WWCarmichael S4 Recent studies on the immunochemical analysis of mycotoxins F S Chu S 5 A comparative study of the pathology of the ovine hepatogenous photosensitivity diseases, facial eczema and geeldikkop (tribulosis ovis), with special reference to their pathogenesis JAW Coetzer, T S Kellerman, W Sadler and G F Bath S 6 The role of tremorgenic mycotoxins in animal diseases R J Cole S 7 Lupinosis. The chemistry and biochemistry of the phomopsins J A Edgar S 8 Mycotoxin biosynthesis from di- to nonaketide precursors B Franck S 9 Mass spectrometry and n.m.r. spectroscopy of fungal extracts of some Fusarium species. R Greenhalgh Abstract not received Chemical studies relevant to the synthesis of some mycotoxins: the cyclopiazonic acids and viridamine CWHolzapfel S10 The role of aflatoxin in human cancer DPHHsieh S11 Analysis and dynamics of ochratoxin A in biological systems K Hurt S12 Synthetic studies on marine toxic polyethers - total synthesis of okadaic acid Mlsobe S13 3 Page

Mycotoxicoses of livestock in South Africa TSKellerman S14 Anti-cyanoginosin-LA monoclonal antibody: preparation and characterization R Kfir, E Johannsen and D P Botes S 15 Chronic pathological effects of some fusarial toxins N P J Kriek, W F O Marasas, S J van Rensburg, B Yagen and AZJoffe S16 The analysis and occurrence of patulin in apple juices SJKubacki S17 The natural contamination of cereals by toxic trichothecenes: risk to human health H Kurata and M Ichinoe S18 Factors affecting toxin production by fungi JLacey S19 Mechanism of action of the territrems, tremorgenic mycotoxins isolated from Aspergillus terreus K H Ling, H H Liou, T C Fu, L Kuo, M C Tsai and M Y Lin S 20 Hepatic metabolism and elimination of tremorgenic mycotoxins P G Mantle S 21 Fusarium moniliforme: A mycotoxicological miasma W F O Marasas S 22 Metabolism and residues of trichothecenes in different animals and tissues CJMirocha S23 Chemistry of some cytoreactive mold metabolites - A epitetrathiodioxopiperazine and two new cytochalasans S Natori S 24 Hepatocarcinogenicity of corn screenings naturally contaminated with Fusarium moniliforme P E Nelson and T M Wilson S 25 A rationale for the control of aflatoxin in feeds D L Park, A E Pohland and L Stoloff S 26 Aflatoxic suppression of cell mediated immune responses and interaction with T-2 toxin A C Pier, M J Varman and E L Belden S 27

4 Page Mass spectrometry/mass spectrometry as a tool for the identification and quantitation of mycotoxins R D Plattner S 28

Conformation of cyclic peptides: rhizonin and phomopsin A M Potgieter S 29

Commercial detoxification of aflatoxin-contaminated peanut meal A Prevot S 30

Important lesser known toxigenic fungi C J Rabie S 31 Analysis of toxins of Fusarium moniliforme P M Scott S 32 Physiological and morphological interactions in the toxic blue-green alga (Cyanobacterium) Microcystis aeruginosa W E Scott S 33 Biosynthetic studies on microbial metabolites containing a carbon- phosphorus bond H Seto S 34 Structural and biosynthetic studies on polyketide mycotoxins and fungal metabolites T J Simpson S 35 Molecular aspects of aflatoxin B, mutagenesis and carcinogenesis A-A Stark S36 Total synthesis of corynetoxins and tunicamycins T Suami S 37 Analytical techniques for dinoflagellate toxins J J Sullivan S 38 HPLC determination of aflatoxins and mammalian aflatoxin metabolites * PGThiel S39 Sequence determination of cyclic peptides by mass spectrometry • A A Tuinman S 40 < Role of mycotoxins in endemic liver and oesophageal cancer | S J van Rensburg S 41 Biosynthetic studies on mycotoxins using multiple stable isotope labelling and n.m.r. spectroscopy J C Vederas S 42 5 Page Conformations of mycotoxins utilizing X-ray diffraction and molecular mechanics techniques W H Watson S 43 Kinetic pulse labelling in the biosynthesis of mycotoxins L O Zamir S 44 POSTER PRESENTATIONS

Page B BASIC ASPECTS OF SECONDARY METABOLISM IN RELA- TION TO TOXIN PRODUCTION

B1 Effect of water activity (Aw) on the production of asteltoxin by Emericella variecolor (Berk, et Br.) A E de Jesus, R M Horak, E M Lawson, and M Patel P1 B2 Effect of different antioxidants and free radical scavengers on aflatoxin production C Fanelli, A A Fabbri, S Pieretti, E Finotti, and S Passi P 2 B3 Microsomal and mitochondrial involvement in the production of aflatoxins induced by carbon tetrachloride in cultures of Asper- gillus parasiticus S Passi, M Nazzaro-Porro, E Finotti, G Panfili, A A Fabbri, and C Fanelli P3 B4 The effect of heat and radiation on germination and ochratoxin production of sclerotia of Aspergillus ochraceus N Paster, R Barkai-Golan, and R Padova P 4 B5 Production of fusarin C in liquid culture J M Farber, G W Sanders, and P M Scott P 5 B6 Zearalenone production by Fusarium species isolated from soybeans G Vaamonde, N B Bonera, and G T Scarmato P 6 B7 Effect of culture conditions on the toxicity and chemical composition of the toxin of Microcystis aeruginosa (UV 006) A J van der Westhuizen, J N Eloff, and G H J Kruger P 7

C BIOSYNTHESIS OF MYCOTOXINS AND PHYCOTOXINS

C1 O-Methyltransferases and aflatoxin biosynthesis R K Berry and M F Dutton P 8

C2 Metabolism of aflatoxin B2. by Aspergillus flavus M F Dutton P 9 C3 The effect of ethanol on the metabolite production of Aspergil- lus ustus A E de Jesus, R M Horak, P S Steyn, and R Vleggaar P10 Page

C4 Isolation, purification, and characterization of EC oxidase that transforms eremofortin C to PR toxin ' -' Y H Wei, S C Chang, and R D Wei P11 l; C5 A 13C n.m.r. study of the biosynthesis of secondary metabolites of Fusarium culmorum ; B A Blackwell, J D Miller, R Greenhalgh, and L Zamir P12 h C6 Biosynthesis of 3-acetyldeoxynivalenol and other metabolites '>• by Fusarium culmorum in a stirred jar fermentor J D Miller and B A Blackwell P13

E STRUCTURE AND CHEMICAL PROPERTIES OF MYCOTOX- INS AND PHYCOTOXINS E1Carbon-13 and proton resonance assignments in leucinostatin A C G Casinovi, L Radios, C Rossi, and L Tuttobello P14 E2 Structure/activity relationships of the fusarins W C A Gelderblom, P G Thiel, and K J van der Merwe P15 E3 Structure elucidation of the first naturally occurring trichothecene glycoside, a metabolite of Fusarium sulphureum C P Gorst-Allman, C J Rabie, P S Steyn, and R Vleggaar P16 E4 Unique insecticidal mycotoxin from Aspergillus melleus Yukawa isolated from cadaver of silkworm, Bombyx mori L T Ohtomo, H Sato, and K Yoshida P 17 E5 Quantum chemical studies of aflatoxin Bi, sterigmatocystin and versicolorin A and a comparison with their mutagenic activity R Pachter and P S Steyn P18 E6 Unexpected cyclizations upon oxidation of diplodiatoxin M Potgieter and P S Steyn P19 E7 Chemical reactions of the penitrems F R van Heerden, P S Steyn, and R Vleggaar P 20 E8 Trichothecenes from Fusarium compactum C M Maes, R M Horak, W F O Marasas, C J Rabie, P S Steyn, and R Vleggaar P 21 E9 Some secondary metabolites of Fusarium culmorum and F. roseum R Greenhalgh, R-M Meier, D Levandier, B A Blackwell, J D Miller, A Taylor, and J W ApSimon P 22 Page

E10 Reduction in levels of deoxynivalenol and its fate in contaminated soft wheat after chemical treatment J C Young, L Subryan, and D Potts P 23

E11 Some mycotoxin structures determined by X-ray crystallography at the NCRL J L M Dillen and P H van Rooyen P 24

G ANALYSIS OF MYCOTOXINS AND PHYCOTOXINS

G1 Rapid method for the determination of macrocyclic trichothecene toxins in field samples A Bata and A Vanyi P 25 G2 A monoclonal antibody based enzyme immunoassay for aflatoxin B, A A G Candlish, W H Stimson, and J E Smith P 26 G3 Thin layer immunoassays for mycotoxins WVDashek, J E Mayfield, NTLappas, CEO'Rear, and G C Llewellyn P 27 G4 Studies on aflatoxin analysis using liquid chromatography W R Day and Y Haroon P 28 G5 Immunoassay of the mycotoxin zearalenone (F2) S N Dixon and K Clarke P 29 G6 The international mycotoxin check sample programme M D Friesen P 30 G7 HPLC and GC-MS analysis of moniliformin in maize J Gilbert, M J Shepherd, J R Startin, and I Parker P 31 G8 Bioassay of cytochalasin E and other eleven mycotoxins using skin of one-day-old mouse T Glinsukon, S Sinlapanapaporn, and S Sriurairatana P 32 G9 Problems encountered in the routine analysis of foodstuffs for aflatoxins HKrohm P33 G10 Aflatoxin metabolites in human urine and liver in Zambia J S N Kaggwa, L A Oil, A C Bayley, and C E A Lovelace P 34 G11 Detoxication of aflatoxins by ETOXs-gasing J Leibetsedcr, J Bdhm, and C H Noonpugdee P 35

9 G12 The investigation of some aspects of a HPLC aflatoxin analysis method for groundnuts S A F Meyer, L H W Verhoef, and P J Grobler P 36 G13 Decontamination of groundnut meal containing aflatoxin Bi by treatment with calcium hydroxide and paraformaldehyde and consequences on aflatoxin M, level in the milk G Piva, A Pietri, and E Carini P 37 G14 Effects of four trichothecenes to larvae of brine shrimps {Arte- mia salina L) and of mosquitoes (Aedes vexans Meigen) H R Schmidt P 38 G15 Rapid thin layer chromatographic determination of aflatoxin Mi in milk M L Serralheiro and M L Quinta P 39 G17 Characterization and analysis of trichothecene mycotoxins by mass spectrometry J R J Pare, R Greenhalgh, P Lafontaine, and J W ApSimon P 40 G18 The role of technological processes in decreasing patulin con- tatnination of apple juices and apple wines T Lipowska and H Goszcz P 41

H BIOCHEMICAL MECHANISM OF ACTION OF MYCOTOXINS AND PHYCOTOXINS

H1 The pathological effects of immunosuppression of Plasmodium \ berghei - infected rats, with particular reference to survival, he- j patocarcinogenesis after aflatoxin Bt treatment ' S Angsubhakom, P Sathiropas, N Bhamarapravati, S Saha- phong, and P Thuvasethakul P 42

H2 Comparative study on the metabolism of diacetoxyscirpenol and zearalenone in pigs J Bauer, M Gareis, G Enders, and B Gedek P 43 - H4 Anatoxin-a(S): A neurotoxin produced by the freshwater cyano- j bacterium Anabaena flos-aquae NRC 525-17 I NAMahmoodandWWCarmichael P44 f

H5 Inhibition of cholinesterase by anatoxin-a(S) -i1 NAMahmoodandWWCarmichael P45 \

10 ; H6 The interaction of aflatoxin B, with rat plasma albumin in vivo and in vitro HWDirr.andJCSchabort P46 H7 The mechanism of inhibition of pyruvate dehydrogenase complex by the mycotoxin moniliformin P S Gathercole, P G Thiel, and J H S Hofmeyr P 47 H8 Mutagenic activity of secondary metabolites of Aspergillus ustus E Johannsen, R Kf ir, and R Vleggaar P 48 H10 Action of patulin on the glutathione pool in a yeast P Thonart, J Bechet, S Sene, and Z L Sumbu P 49 H11 Phomopsin A, the causative agent of lupinosis, interacts with microtubules in vivo and in vitro E M Tonsing, D J J Potgieter, I W Simson, P S Steyn, M Os- born, and K Weber P 50 H12 Influence of phomopsin and invalin on steroid hormone binding and MCF-7 cell proliferation C H van Aswegen, W M Lewko, J J van der Watt, H C Pot- gieter, NMJ Vermeulen, D J J Potgieter, and J L Wittliff P 51 H13 The influence of aflatoxin Bi in protein phosphorylation in rat liver J Viviers and J C Schabort P 52 H14 Trichothecene degradation by pure cultures of rumen bacteria K Westlake, M F Dutton, and R I Mackie P 53 Hi 5 Protective effect of vitamins against trichothecenes toxicity toward Sacharomyces cerevisiae B Yagen and S Halevy P 54 H16 Differential inhibition by T-2 toxin of total protein, DNA and isoprenoid synthesis in the cultured macrophage cell line J 774 R N Melmed and B Yagen P 55 H17 Study of the metabolization of trichothecene toxins A Bata and A Vanyi P 56

I PATHOLOGY OF MYCOTOXINS AND PHYCOTOXINS I1 Toxicity of Alternaria to chickens W L Bryden, K D Barrow amd D AI Suter P 60 11 13 Comparative performance of boars and gilts fed deoxynivalenol-contaminated diets D W Friend, H L Trenholm, B K Thompson, P S Fiser, and K E- Hartin P 61 14 Toxicity studies on toxin BE-4 from the blue-green alga, Micro- cystis aeruginosa P G Thiel, K Jaskiewicz, J E Fincham, and 0 P Botes P 62 15 Chronic experimental aflatoxicosis (Bi) in dogs T W Naude, P Bland van den Berg, F Reyers, and R C Tustin P 63 16 An experimental mycotoxicosis in sheep and goats caused by Drechslera campanulata, a fungal pathogen of green oats D J Schneider, W F O Marasas, M Collett, G C A van der Westhuizen, P S Steyn, and R M Horak P 64

J NATURALLY OCCURRING TOXICANTS IN HUMAN AND ANIMAL HEALTH J1 Aflatoxin examination of imported foods in Japan from 30 countries between 1972 and 1984 K Aibara Abstract not received J2 Occurrence of Fusarium mycotoxins in cereals in Italy A Bottalico, A Logrieco, A Visconti, and M Solfrizzo P 65 J3 Natural occurrence of trichothecenes and zearalenone in feedstuffs in the Federal Republic of Germany M Gareis, J Bauer, and B Gedek P 66 J4 Toxicity of Fusarium species associated with seeds of annual Medicago species in South Africa S C Lamprecht, W F O Marasas, P G Thiel, and D J Schneider P 67 J5 Natural occurrence of mycotoxins in Austria J Leibetseder, J Bohm, A M Abdelhamid, and C H Noonpug- dee P68 J6 A study on milk contamination by aflatoxin Mi in a restricted area in central Italy A Boccia, C Micco, M Miraglia, and M Scioli P 69 J7 Long term administration of low doses of mycotoxins in poultry. Residues of aflatoxin Bi and its metabolites in broilers and laying hens. C Micco, M Miraglia, L Benelli, A loppolo, Al Mantovani, and D Stasolla P 70 12 J8 Aflatoxin contamination in Zambian foods and feeds: preliminary studies H Njapau, E Walubita, A C Bayley, and F L C Kapumpe P 71 J9 Variation in the levels of aflatoxin in cow milk consumed in the city of Sao Paulo, Brazil M Sabino, A Purchio, and M A P Zorzetto P 72 J10 A rationale for the control of aflatoxin in human foods L Stoloff P73 J11 Co-existence of nivalenol, deoxynivalenol and zearalenone in cereal grains Y Ueno, U S Lee, T Tanaka, and A Hasegawa P 74 J12 Aflatoxin quality control on South African groundnuts L H W Verhoef, P J Grobler, and D de Jager P 75 J13 Identification of various T-2 toxin metabolites in chicken excreta and tissues A Visconti and C J Mirocha P 76 J14 Survey of milk for aflatoxin Mi A Visconti, A Bottalico, and M Solfrizzo P 77 J15 Studies on the toxicity of substances isolated from fungi growing on plant material H Bresch, H K Frank and R Miinzner P 78 J16 A study of storage problems under local conditions with or without the addition of sodium carbonate specifically of rice and maize in relation to mould growth and aflatoxin formation K Topsy, G K Chukowry, A Reesaul and M Kassee P 79

13 AMENDMENTS TO PROGRAMME

The following abstracts, as listed in the programme, were not available at the time of going to press or were withdrawn:

G16 H3 H9 - to be presented as a section lecture 12 - to be presented as a section lecture 17

Abstract received too late for inclusion: J17 - Toxicity of Monadenium lugardiae to albino rats G M Gundidza

14 SYNTHETIC STUDIES ON FUNGAL METABOLITES AND OTHER NATURAL PRODUCTS

G. H. BUCHI CAMILLE DREYFUS PROFESSOR Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 USA

A summary of current synthetic work and bacillosporins, naphthocyclinones and methoxatin.

PL1 REGULATION OF SECONDARY METABOLISM IN FUNGI

ARNOLD L. DEMAIN Department of Applied Biological Sciences M.I.T., Cambridge, MA 02139 U.S.A. Secondary metabolites (idiolites) are structurally diverse and unusual, generally are produced in mixtures with other members of the same chemical family, and usually are formed at low specific growth rates. In batch cultures, processes leading to the production of idiolites are often sequential; the cultures exhibit a distinct growth phase (trophophase) followed by a production phase (idiophase). However, trophophase and idiophase may overlap. Timing depends on the nutritional environment presented to the culture. Specific mechanisms regulating the onset of idiolite synthesis include repression by sources of carbon, nitrogen and phosphate and enzyme induction. Cessation of idiolite biosynthesis occurs via decay of idiolite synthetases as well as feedback inhibition and repression of these enzymes. With regard to control of mycotoxin biosynthesis, most studies have been done on ergot alkaloid, aflatoxin and patulin formation. Ergot alkaloid formation is mainly controlled by carbon source regulation (glucose), phosphate repression, induction (tryptophan), growth rate and feedback inhibition (agroclavine and elymo- clavine). Aflatoxin biosynthesis is controlled by nitrogen source repression (nitrate) and induction (glucose); zinc stimulates production in an unknown manner. Patulin production is regulated by nitrogen source repression, induction (6-methylsalicylic acid), growth rate and synthetase decay.

PL2 BIOMIMETIC SYNTHESES OF AROMATIC POLYKETIDE METABOLITES

T. M. Harris and C. i-l. Harris Center in Molecular Toxicology & Department of Chemistry Vanderbilt University, Nashville, TN 37235, USA

The polyketide pathway is prominent among the biochemical routes leading to microbial metabolites including many of the mycotoxins. Investigations of the biosynthesis of polyketide metabolites, carried out in many laboratories throughout the world, have demonstrated the involvement of acetate and malonate as the basic building blocks. Oligoketo acids have been implicated as intermediates in the pathways; however these labile polycarbonyl species have eluded isolation or detection in biological systems. Methodology developed in the authors' laboratory has made it possible to synthesize many of the polycarbonyl compounds. This lecture will review these studies and subsequent studies which have been made of the chemistry of polyketo acids. Particular emphasis will be placed upon cyclizations which lead to aromatic natural products by routes and, in some cases, conditions which mimic those present in the biochemical systems. These studies involve examples of aromatic metabolites containing benzene, naphthalene, anthracene, napthacene and a number of pyran ring systems.

PL3 GLOBAL SIGNIFICANCE OF MYCOTOXINS

C. W. HESSELTINE

Ph.D., Mycologist Northern Regional Research Center, Agricultural Research Service U.S. Department of Agriculture, Peoria, Illinois 61604 This is about the 25th anniversary of the discovery of aflatoxin in peanut meal in Great Britain. Recently, the author conducted a worldwide survey in over 30 countries on what people considered the most important mycotoxins, areas of most needed research and least needed research, estimation of economic losses, commodities with mycotoxin problems, and where does the problem occur—storage, field, or both. Part of the lecture will be a summary of this survey. The second part will be a look into the future for the global needs of research and development in mycotoxins rather than a look backward. The literature published in the last 25 years has been well reviewed in books and review articles. Areas of needed research can be subdivided into analytical methodology, field studies, interaction of myco- toxic fungi with other microorganisms, characterization of newly discovered toxic compounds, metabolic pathways to toxin formation, prevention of mycotoxin development, diagnosis of poisoning in animals and man, effects of toxins on animal physiology, and interaction of mycotoxins with growing plants.

PL 4 BIOLOGY OF TOXIGENIC ANAMORPHS

B KENDRICK

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada NCL 3G1

The competition for food between toxigenic fungi and small mammals, and the interactions that result, are explored as paradigms for the biclogical and evolutionary implications of mycotoxin production. The life cycles, relationships and substrate preferences of the principal toxigenic mold genera are discussed, and some evidence, largely drawn from fungus-invertebrate interactions, is presented in support of the hypothesis that toxin production nay be much more widespread among ascomycetous anamorphs than has generally been thought.

PL5 BIOCHEMICAL MECHANI5?1 OF ACTION OF MYCOTOXINS

K.-H. KIESSLING Professor Department of Animal Nutrition and Management Swedish University of Agricultural Sciences, Sweden Symptoms of mycotoxicosis are a result of interactions of mycotoxins with functional molecules and subcellular organelles in the animal cell. The biological effects vary mainly according to the diversity in chemical structures of the mycotoxins, but also because of biological, nutritional and environmental factors.

For a few mycotoxins, especially aflatoxin B •], so many effects on biochemical systems have been described that only a fraction could be discussed here. Many of these effects are probably secondary to one and the same primary mechanism of action, others occur at such high concentrations that they will never happen in nature.

It is striking that so many mycotoxins act on the DNIA-RNA level. In most cases this gives rise to consequences on many levels and with pathological pictures caused by many co-operating factors. Less frequently, the effects are as specific as the inhibition of phosphoenolpyruvate carboxykinase by ochratoxin A.

Thus no generalized mechanism can apply to all mycotoxins - not even for one and the same mycotoxin in different circumstances. The mechanism of action for a mycotoxin in mammalian cell:? may not be applicable to plants and microorganisms if metabolic activation is involved.

Despite the many data accumulated so far, some of which have been accounted for in this review, the specific lesions responsible for the acute toxicity of many mycotoxins have not yet been identified. Much work therefore remains to be done in order to fully understand their mechanism of action.

PL 6 TOXINS FROM CYANOPHYTES BELONGING TO THE SCYTONEMACEAE

R. E. MOORE

Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822, U.S.A.

Powerful toxins are found in some blue-green algae. The most well-known are the water-soluble toxins associated with certain freshwater species, viz. anatoxin-a in Anabaena flos-aquae, saxitoxin-type compounds in Aphanizomenon flos-aquae, and cyclic peptides such as cyanoginosin-LA in Microcystis aeruginosa, and the lipophilic toxins present in a few species of marine Oscillatoriaceae, in particular aplysiatoxin and lyngbyatoxin A in Lyngbya majuscula.

Recently we have isolated two novel lipophilic toxins from a terrestrial species of Scytonemaceae. Their gross structures have been determined and will be presented at this symposium.

PL7 PATHOLOGY OF MYCOTOXICOSES: POSSIBILITIES AND LIMITS OF DIAGNOSIS

H.B. SCHIEFER

Dr.med.vet., Dr. habil, Professor of Veterinary Pathology Toxicology Research Centre, University of Saskatchewan Saskatoon, Saskatchewan S7N 0W0 Canada The goal of necropsy is to gain a complete understanding of the organism dissected, and the standard method for necropsies is essentially a reductive organological analysis of the pathological material. Toxic properties of chemicals manifest themselves as functional, biochemical or structural changes, and a documented functional change may give important clues as to where a structural change might be found. However, there is only a limited number of ways in which damage can appear. The pathologist is placed in the position of an intermediary between the basic sciences and the clinical sciences of medicine, and this demands from the pathologist to synthesize and to apply the totality of available scientific information to each case. No other discipline is charged with this responsibility, and it is the duty of the pathologist to present his final diagnosis at the level of organization where the investigation began: the total organism. The effects of various mycotoxins on various organs and systems of the body are to be considered first, and the specificity or non-specificity of a lesion with respect to a given or suspected mycotoxin has to be assessed. Unfortunately, many mycotoxins produce symptoms or lesions which do not allow for a diagnosis on the basis of singular observation, but when the lesions are considered in a holistic manner, i.e., judged from the total organism in relation to all available clinical history and epidemiological findings, many mycotoxins can be diagnosed accurately. Limitations are the non-specific nature of primary injury, which may even be masked by secondary effects (e.g., bacterial infection in instances of immunosuppressive mycotoxins), and late-appearing effects (e.g., cancer) which may be attributed to other carcinogens. Further complications arise due to the interaction of two or more mycotoxins and/or toxicants, and species variability with respect to type and site of response is another problem. Finally, while modern analytical techniques may allow for determination of traces of metabolites in the body, the causal link may be difficult to establish due to the fact that the primary source of the mycotoxin is no longer accessible for investigation. The most important limitation in pathology, however, is not having even a suspicion, which prevents even an attempt to make a diagnosis, because many hold the view that mycotoxins are agents in search of a disease. Judicious use of all the knowledge that is available nowadays, however, should permit a much higher rate of diagnoses than is generally thought to be possible.

PL 8 TOXIGENESIS AND BIOSYNTHESIS OF SAXITOXIN ANALOGS

YUZURU SHIMIZU

Professor Department of Pharmacognosy and Environmental Health Sciences College of Pharmacy, University of Rhode Island, Kingston, R.I. 02881, U.S.A.

Saxitoxin (1) was first isolated as a paralytic shellfish poison (PSP) from Alaskan butter clam in 1957. Later the origin of the toxin was determined to be the dinoflagellate, Gonyaulax catenella. Since then, more than ten new related toxins have been isolated and their structures determined. The toxin producing organisms are curiously scattered from different genera of dino- flagellates to blue-green and macro red algae. An extensive survey of various strains of Gonyaulax tamarensis has also indicated that the toxigenicity varies tremendously even in an identical species. Although the chemistry of saxitoxin and its analogs has been a subject of vigorous investigation, the biosynthesis of these conspicuous molecules have been left unsolved mostly due to technical difficulties encountered in the feeding study. Our group has recently succeeded in determining the origins of all atoms in the toxin molecule. The details of the biosynthesis work and possible mechanisms of the toxigenicity will be discussed.

PL9 RECENT STRUCTURAL STUDIES ON MOULD METABOLITES

CH. TAMM

Institute of Organic Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056, Basel

Anguidine (diacetoxyscirpenol) is the major metabolite of Fusarium sambucinum (Fungi imperfecti). A series of minor metabolites has been isolated from cultures of this microorganism. Some of their structures have been elucidated. Their biogenetic relationship to the trichothecenes is discussed. Using anguidine as the starting material macrocyclic trichothecenes have been synthesized. Special attention is paid to the stereoselective synthesis of the building blocks of the macrocyclic segment. Methods for the asymmetric synthesis of a-hydroxy^esters have been developed. Also the enantioselectivity of the enzymes, pig liver esterase and a-chymo- trypsin, has been studied extensively in order to prepare chiral synthons from symmetrical starting materials. The former are suitable for the construction of a variety of optically active mycotoxins and other natural products, such as the cytochalasans.

PL10 PROGRESS TOWARDS A BIOSYNTHETIC RATIONALE OF THE AFLATOXIN PATHWAY

CRAIG A. TOWNSEND B.A., Ph.D. Department of Chemistry, The Johns Hopkins University Baltimore, Maryland 21218, U.S.A.

The biosynthesis of aflatoxin Bj (2) will be discussed from the perspective of advanced anthraquinone intermediates in the pathway. The roles played by averufin (1_, R=H) and nidurufin (1_, R=0H) in bisfuran formation will be evaluated through an array of synthetic and spectroscopic approaches in experiments using wild-type and mutant strains by Aspergillus parasiticus. Lastly, biomimetic and stereochemical experiments will 5i presented to test biogenetic hypotheses in in vitro chemistry.

PL11 TOXICOLOGY OF NATURALLY OCCURRING MICROBIAL TOXINS

Y UENO Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya Tokyo 162, Japan

After discovery of the aflatoxins in the early 1960's, research on fungal toxins and their role in human and animal diseases intensified greatly and also became more systematized. Aflatoxins, ochratoxins, zearalenone, trichothecenes and others are probable causative agents in important diseases in humans and animals.

Toxicologically, the mycotoxins are classified into; 1) inhibitor of energy production (citreoviridin etc.), 2) inhibitor of protein synthesis (trichothecenes etc.), 3) cytoskeleton modifier (cytochalasins etc.), 4) estrogens (zearalenone), 5) tremorgen (penitrem etc.), and 6) mutagen and carcinogen (aflatoxins etc.).

A number of unicellular microalgea, either planktonic or benthic, produce various kinds of phycotoxins such as saxitoxins, okadaic acid and its esters, dinophysistoxins, ciguato in, maitotoxin, brevetoxins, hemolysin-1 and others. Protogonaulax spp. and Pyrodiniutn bahamense var. compressa in paralytic shellfish poisonings, Dinophysis fortii in diarrhetic shellfish poisoning, Gambierdiscus toxins in ciguatera, and Ptychdiscus brevis in iteurotoxic shellfish poisonings are examples of toxic microalgea-origin. Mass growth of Prymmnesium parvum, which produces hemolysin-1, causes death of fishes.

Ciguatosin and maitotoxin induce an increase of Na -influx and saxitoxin blocks Na -channels. Aplysiatoxin and debromoaplysiatoxin of Lyngbya majuscula are a potent tumor promotor, and a hepatotoxic peptide of Microcystis auruginosa exhibits an interaction with actin.

These informations revealed that the mycotoxins as well as phycotoxins are expected as an excellent tool for toxicological and pharmacological researches.

PL 12 DETERMINATION OF MYCOTOXINS

H.P.VAN EGMOND AND W.E.PAULSCH

Agricultural Engineer (Food Technology) National Institute of Public Health and Environmental Hygiene P.O.Box 1, 3720 BA Bilthoven, the Netherlands

Since the early 1960's, the period of Turkey X disease, the availability of methods of analysis has played a keyrole in the development of mycotoxin survey and research programmes. Initially bioassay techniques have been used, but their limitations led chemists to develop more selective and reliable methods of analysis. Generally, the basic steps of chemical analytical methods for mycotoxins involve extraction with organic solvents, cleanup by column chromatography or liquid-liquid partitioning, separation of the toxin from matrix components, detection, quantitation and confirmation of identity. Various approaches exist to the separation, detection and quantitation, such as thin layer chromatography (TLC), a reliable and relatively simple technique with a broad scope of application, minicolumn chromatography (MCC), useful for screening purposes, high performance liquid chromatography (HPLC), a technique which offers the possibility of automating the ultimate separation and quantification step and gas liquid chromatography (GLC), which is mainly used in the analysis for trichothecenes. Procedures to confirm the identity of mycotoxins may be based on in situ derivatizations on TLC plates, pre-column derivatizations in HPLC systems, or on mass spectrometry.

The newest development in the determination of mycotoxins involves the application of immunochemical procedures, such as Enzyme Linked Immuno Sorbent Assay (ELISA) and Radio Immuno Assay (RIA). Although still in the development stage it is to be expected, that these techniques will play an important role in mycotoxin methodology in the near future.

PL 13 BIOSYNTHETIC STUDIES ON SOME POLYENE MYCOTOXINS

R. VLEGMAR

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa

Citreoviridin, asteltoxin and aurovertin B are members of a group of mycotoxins which act as inhibitors of oxidative phosphorylation in mitochondria. The biosynthetic origin of the carbon skeleton of these metabolites was studied by incorporation of C-labelled precursors: [l- C]-, [2- C]-, 13 13 2 13 2 13 [l,2- C2]-, [l- C,2- H3]-, and [2- C,2- H3]acetate, (25)-[methyl- Cl- methionine and [l-13C]- and [3-13C]propionate. The results show that aurovertin B and asteltoxin can be formed via two biosynthetic pathways. The first pathway involves the D-methylation of a Cjo-polyketide precursor at

C18, followed by loss of the chain-initiating acetate unit, C19~C2o- The second pathway involves a Cig-precursor formed from a propionate chain- initiating unit and eight malonate units. In addition the arrangement of intact acetate units in asteltoxin derived from [l,2- C2Jacetate proves that a 1,2-bond migration occurs during the formation of the 2,8-dioxabicyclo- [3.3.0]octane moiety. Citreoviridin is derived from a C^-polyketide formed from an acetate chain-initiating unit and eight malonate units.

Incorporation of [l-13C,18O2]acetate and O2 gas into citreoviridin, aurovertin B, and asteltoxin established the origin of the oxygen atoms and provided stereochemical and mechanistic information on the biosynthetic pathways of these metabolites.

The incorporation of (2S)-f methyl-x3 CIme thionine and [l,2-13C2]acetate into the fusarins, metabolites of Fusarium moniliforme, points to a biosynthetic pathway involving the condensation of a Cxv~polyketide and a Ci* intermediate, e.g. oxaloacetate, of the Krebs tricarboxylic acid cycle.

PL 14 SECONDARY METABOLITES PRODUCED BY SOME FUSARIUM SPECIES

J.W. ApSIMON1, B. Blackwell2, R. Greenhaigh2, R.R. King3, R.-M. Meier1, D. Miller2, J.R.J. Pare2 and A. Taylor".

Carleton University, Ottawa, Canada, K1S SB6

The Canadian Mycotoxin Program brings together a group of experts from several disciplines to study the Fusarium species responsible for mycotoxicoses in Canadian agricultural products. Large scale liquid cultures of Fusarium Roseum and Culmorum have led to our ability to isolate a variety of different secondary metabolites whose structures will be presented at this symposium.

Structural variations of the materials obtained allow us to make comments on the biosynthetic routes available in these and similar species.

^ttawarCarleton Chemistry Institute, Carleton University, Ottawa, Ontario, Canada, K1S 5B6. 2CBRI, Agriculture Canada, Ottawa, Ontario, Canada. ^Agriculture Canada, Research Station, Fredericton, New Brunswick, Canada. **Atlantic Regional Laboratory, National Research Council, Halifax, Nova Scotia, Canada.

S1 THE GENETIC BASIS OF MYCOTOXIN BIOSYNTHESIS

J. W. BENNETT

PROFESSOR OF BIOLOGY DEPARTMENT OF BIOLOGY TULANE UNIVERSITY NEW ORLEANS, LA 70118 U. S. A. Although fungal genetics is a sophisticated and well advanced discipline, the genetics of fungal secondary metabolism, including mycotoxins, is a neglected field. Industrially important fungi have been subjected to repeated rounds of. mutagenesis, screening, and selection in the search for strains yielding high antibiotic production. Other approaches to strain improvement via the parasexual cycle and recombinant DNA technology have been less successful. Never- theless, this research on antibiotic-producing fungi serves as a model system for similar genetic research on mycotoxin-producing species.

Mutational dissection of mycotoxin biosynthetic pathways is employed with patulin, ergot, and aflatoxin. Genetic studies with trichothecene-producing species are almost nonexistent, perhaps because of problems with strain degeneration and the lack of quick plate assays for trichothecene production. The best characterized systems for mycotoxin genetics are with the aflatoxin-producing species Aspergillus flavus and A. parasiticus. The parasexual cycle is elucidated in both species, and blocked aflatoxin mutants can be isolated using assays involving quick-screening plate techniques. Protoplast fusions are successful between auxotrophs of certain strains. The availability of a well-characterized transformation system in Aspergillus nidulans would suggest that transformation may become possible in A. flavus-parasiticus as well. This would open the doors for applying modern approaches of molecular biology to mycotoxin genetics.

Recent genetic studies in our laboratory have concentrated on aflatoxigenic strains of A. parasiticus. Three areas of research have been emphasized: 1) isolation of blocked aflatoxin mutants, 2) elucidation of optimal haploidization agents for analysis of parasexual crosses, and 3) analysis of segregants from toxigenic and non-toxigenic diploid strains. Four presumptive aflatoxin mutant have been isolated, including one high aflatoxin B2~producing strain. Benlate- treatment of diploid colonies (not spores) has been the most efficient means of isolating haploid segregants. Finally, intensive studies were directed toward one diploid strain synthesized between a brown-spored, norsolorinic acid-producing, lysine-requiring mutant and a white-spore, versicolorin A-producing, serine-requiring mutant. Non-aflatoxigenic variants were isolated by serial ay- celial maceration. Non-aflatoxigenic variants simultaneously lost the ability to produce norsolorinic acid and versicolorin A. Aflatoxigenic and non-aflatoxigenic diploids were treated with benlate and para-fluorophenylalanine. Mutant markers were scored in resultant segregants. Most spore color and »y- celial color segregants were prototrophic. Surprisingly, SOM non-aflatoxigenic . variants yielded segregants bearing color Markers for norsolorinic acid and versicolorin A. These segregants were highly unstable.

S2 CYANOGINOSINS - ISOLATION, STRUCTURE AND BIOLOGY

D.P. BOTES MOLECULAR BIOCHEMISTRY DIVISION, NCRL, CSIR, P 0 BOX 395, PRETORIA OOO1 Natural blooms of the cyanobacterium Microcystis aeruginosa have been responsible for livestock losses In South Africa, Australia, Canada and other countries with similar climatic conditions. Evidence of liver damage in humans as well as gastroenteritis, diarrhoea and dermatitis have been recorded where Microcystis had been incriminated as the cause. Both toxic and non-toxic strains can be recovered from the same bloom and environmental factors influence the dominance of a particular strain. Knowledge of the structure therefore became of considerable interest for devising preventive measures and for possible antidote and sensitive immuno- or bioassay developement. The extreme hydrophobic nature of these toxins renders them amenable to butanol extraction at acidic pH. DEAE-cellulose chromatography, utilizing volatile buffers, affords a good first step separation allowing reasonable amounts of material to be handled. Reverse-phase h.p.l.c. on a C-18 column suffices to clean the toxin variants from further impurities. Amino acid analysis suggested a penta-peptide or penta-peptide repeat structure. However, 'H-n.m.r. at 500 MHz indicated a considerably more complex structure. N-CHy^VIa and a novel p-amino acid 3-amino-9-methoxy- 10-phenyl-2,6,8-trimethyl-deca-4,6dienoic acid (Adda), both labile during acid hydrolysis and thus escaping detection by ami no acid analysis, are also constituent moieties of the structure. Seven toxin variants have been isolated and D-Ala, erythro-p-CHyD-iso-Asp, D-iso-Glu, N-CH3-Mla and Adda are common to all. In addition two variant L-Amino acids form part of the cyclic heptapeptide structure. N-CHrMla is readily reduced to the stable N-CHrAla. A conserved N-CHrAlanyl-Ala sequence is preferentially cleaved by 95% trifluoroacetic acid in all variants and the linearized derivatives of some variants are amenable to f.a.b.m.s., giving rise to recognizable sequence ions. *H-n.m.r. allowed the connectivity of Adda to be deduced. A monoclonal antibody against aeruginosin L,A crossreacted with all variants. This reactivity seems to correlate with high segmenta1 mobility as determined by relaxation time at 500 MHz. An ELISA procedure, utilizing this monoclonal antibody, 1s presently being developed In order to detect low levels of toxin In water to be purified for domestic use.

S3 MECHANISM OF ACTION FOR PEPTIDE TOXINS OF FRESHWATER CYANOBACTERIA (BLUE-GREEN ALGAE)

W.W. CARMICHAEL Associate Professor Aquatic Biology/Toxicology Department of Biological Sciences Wright State University Dayton, Ohio, U.S.A. 45435 Peptide toxins of freshwater cyanobacteria are produced by strains of Anabaena flos-aquae, Microcystis aeruginosa and Oscillatoria agardhii. Only the toxic peptides of Microcystis have been chemically examined and they are referred to as microcystis toxins, microcystin or most recently, cyanogenosins. These toxins are small molecular weight linear or cyclic penta or heptapeptides with a molecular weight between 600 and 900 Daltons. These toxic peptides isolated from geographically different toxic strains, all show similar toxin- ology (LD50 i.p. mouse = 50 to 100 ug/kg) in laboratory animals. Upon gross necropsy, animals show enlarged hemorrhaged livers with other tissues appearing normal. Histological examination of the liver reveals necrosis with loss of architecture in the hepatic cords. TEM studies reveal that both hepatocytes and endothelial cells are damaged. The lungs are mildly congested with occasional patches of debre. This debre, referred to as atypical thrombi, has been suggested to be responsible for the death of the animal, via pulmonary congestion and right heart failure. This has led to two opposing views for the mechanism of action; a direct effect on hepatic and endothelial cells, with death due to cardiovascular shock or an indirect effect due to pulmonary congestion and right heart failure, which leads to the hemorrhaged and necrotic liver. To examine the two possibilities, we purified the peptide toxin of M. aeruginosa strain 7820 and found it,to be a cyclic hexapeptide with an LD5Q i.p. mouse of 50-100 ug/kg. Time course pathology studies indicated that liver damage occurred prior to the appearance of pulmonary thrombi and in no animal was the thrombi extensive enough to be life-threatening. Anesthetized rats cannulated to measure pressure in the jugular vein, hepatic portal vein and the femoral artery showed no pressure patterns that would indicate venous congestion. Venous congestion with a rise above 10 mmHg of pressure would be a necessary prerequisite tocause right heart failure. Concurrent measurements of lactic acid and femoral arterial pressure showed a rise in lactic acid levels paralleling the fall in systemic arterial pressure. Isolated liver perfusion experiments, with rats, showed that the toxin had an effect which paralleled and mimicked that seen in whole animal studies. Total hepatectomized rats were found to be resistant to the toxin. From this evidence, it is concluded that the toxin of M. aeruginosa 7820 has a direct effect on the liver and that the cause of death in acute toxicity is heroor- rhagic, hypovolemic shock.

S4 RECENT STUDIES ON IMMUNOCHEMICAL ANALYSIS OF MYCOTOXINS

F. S. CHU

Department of Foo^ Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706, U.S.A. Because of the potential hazards of mycotoxins to human and animal health, a search for a dependable, sensitive, specific and simple method for the detection of mycotoxins in foods and feeds, as well as biological fluids, has become highly desirable. Methods commonly used are either lacking in specificity or are limited by their sensitivity. To overcome some of the difficulties encountered with the chemical and biological methods for mycotoxin analysis, attempts have been made to elicit antibodies against mycotoxins for immunoassays. Specific polyclonal antibodies against aflatoxins (afla) Bl, Ml, B2a, Bl diol, Gl and Ql, ochratoxin A (OTA), T-2 toxin, diacetoxyscirpenol, deoxyvermcarol, sterigmatocystin and zearalenone as well as monoclonal antibodies against afla Bl, afla Ml and T-2 toxin have been obtained from several laboratories in recent years. Sensitive and specific radioimmunoassays (RIA), as well as enzyme 1 inked-immunosorbent assays (ELISA), of these toxins in foods and biological fluids have also been developed. The sensitivity of RIA is generally in the range of 0.1 to 5.0 ng per assay or 2-5.0 ppb of toxin in foods or body fluids; whereas the sensitivity varies from 0.01 ppb for afla Ml in milk and urine to 5 ppb of afla Bl in peanuts in the ELISA. As little as 2.5 pg of mycotoxin can be detected in ELISA tests. Specific antibodies against several important mycotoxin metabolites including afla DNA adducts and T-2 toxin have also been made available, thus allowing detection of mycotoxin metabolites in body fluids. Two immunohistochemical techniques for monitoring afla Bl, OTA and T-2 toxin have been developed. With the availability of specific antibodies against mycotoxins, immunoaffinity columns have been prepared and have also been used as a cleanup tool for mycotoxin analysis. Details on the recent progress of the immunoassay of mycotoxins, the advantages and disadvantages of different immunoassays as well as problems associated with immunochemical research on mycotoxins will be reviewed and discussed.

S5 A COMPABATIVE STUDY OF THE PATHOLOGY OF THE OVINE HEPATOGENOUS PHOTOSENSITIVI- TY DISEASES, FACIAL ECZEMA ANC GEELDIKKOP (TBIBDLOSIS OVIS), WITH SPECIAL REFERENCE TO THEIR PATHOGEHESIS J.A.W. COETZER, T.S. KELLEBMAN, V. SADLER and Q.F. BATH BVSc, BVSc (Hons), M Mcd Vet (Path) Section of Pathology, Veterinary Sesearch Institute, Onderstepoort 0110

The subject of this study was the pathological and scanning electron microscopical changes in the biliary systems of sheep suffering from facial eczema or geeldikkop (Tribuloeia ovis), or made photosensitive by ligation of the common bile duct. While an obliterative cholangitis is responsible for the retention of phylloerythrin in facial eczema, the occlusion of bile ducts with crystalloid material (microliths) appear to perform a similar function in geeldikkop. The similarities and differences between the 2 diseases are discussed in the light of their pathogenetit mechanisms.

S6 THE ROLE OF TREMORGENIC MYCOTOXINS IN ANIMAL DISEASES

R. J. COLE Research Mycotoxicologist, Ph.D. USDA, ARS, National Peanut Research Laboratory 1101 Forrester Drive, S. E., Dawson, Georgia 31742, USA Based on current knowledge, fungal tremorgens can be classified into four different chemical groups. The largest and presumably the most economically significant group consists of the paspalitrems, lolitrems, janthitrems, aflatrem, and penitrems. These metabolites all contain the basic indole/ terpene (derived from tryptophan and geranylgeraniol) nucleus originally discovered in paspaline, paspalicine, and paxilline. Most members have been strongly implicated in the etiology of natural intoxications. The verruculogen-fumitremorgen group chemically consists of tryptophan and proline or hydroxyproline united through a diketopiperazine functionality. Members of this group have also been implicated in natural intoxications. A third chemical group, the tryptoquivalines, have not been implicated in any natural intoxication and their ability to induce tremor in animals has not been con- clusively established. The territrems represent a chemical group that differs from all other tremorgenic fungal metabolites by the absence of indole as part of their chemical constitution. The latter also have not yet been implicated in any animal disease syndrome. The demonstrated and suggested roles of these groups of fungal tremorgens will be discussed in detail.

S7 LUPINOSIS The chemistry and biochemistry of the phomopsins

J.A. EDGAR

B.Sc.(Hons) Ph.D. CSIRO, Division of Animal Health, Private Bag No. 1, P.O. Parkville, Victoria 3052, Australia. Lupinosis is a mycotoxicosis of domestic animals feeding on Lupinus species (lupins) infected with the fungus Phomopsis leptostvomifovmis. It is characterised by anorexia, listlessness, a severe fatty liver and signs of mitotic arrest in the liver cells. The principal toxin produced by P.lepto- stromifortnis, phomopsin A, is a chlorine-containing cyclic hexapeptide involving 2,3 and 3,4-didehydro and 3-hydroxy aminoacids and has proved to be an extremely effective inhibitor of microtubule formation. It's effect on tubulin and microtubules provides a basis for many of the symptoms associated with lupinosis.

S8 MYCOTOXIN BIOSYNTHESIS FROM DI - TO NONAKETIDE PRECURSORS

B. FRANCK

Organisch-Chemisches Institut, Universitat Munster, D-4400 Munster

The majority of mycotoxins is formed from polyketide precursors containing 2-10 acetate units. The tremendous diversity of these biogenetic pathways was established by feeding labeled acetates and determining the isotope positions in the mycotoxins thus obtained. However in contrast to other areas of biogenetic research it was .seldom tried to prove the postulated biogenetic sequences by incorporation experiments with advanced intermediates. The reasons for this are: 1) Difficulties in synthesizing the labeled intermediates 2) Assumption that the biosynthesis from acetate to the completed mycotoxin proceeds in a closed enzyme system, not accessible to intermediates 3) Doubts concerning the passage of advanced intermediates through cell membranes. A number of postulated and other possible intermediates of mycotoxin biosynthesis from di-, octa- and nonaketides were synthesized in labeled form and applied to mould cultures. Thereby it turned out that the arguments 2) and 3) were mostly unfounded. Thus it was possible to gain precise and novel information on mycotoxin biosynthesis from polyketides, which brings about mechanistic and metabolic understanding and the development of useful biomimetic syntheses.

Reference; 1) B. Franck, Angew. Chem. 9£ (1934) 462; Angew. Chem. Int. Ed. Engl. 21 (1984) 493.

S9 CHEMICAL STUDIES RELEVANT TO THE SYNTHESIS OF SOME MYCOTOXINS: THE CYCLOPIAZONIC ACIDS AND VIRIDAMINE

C.W. HOLZAPFEL M.SC., Ph.D. Rand Afrikaans University, Johannesburg 2000, South Africa

A number of structurally interesting mycotoxins such as the ergot alkaloids and the cyclopiazonic acids contain a common structural feature vz. an isoprenoid moiety joined, at least on one point, to the 4-position of an indole ring system. Much of the published work directed at the synthesis of these compounds or their biogenetic precursors involved the construction of suitable 4-substituted indoles. The routes to these synthons are generally inefficient and relatively expensive. An alternative and more direct approach to 4-isoprenyl substituted indole and related compounds, additionally functionalised in the side chain, involves the carboxolytic fission of suitably N-substituted 5-amino 1,4-dihydronaphthalenes. The method, based on the vinylnitrosonium ion chemistry developed by Eschenmoser, involves consecu- tive [2 + 4] cycloaddition - deprotonation - [21 + 4']retrocyclisation. This method was used for the stereospecific synthesis of (E)-4-(4'-indolyl)- 2-methylcrotonaldehyde. The conversion of this aldehyde into biogenetic precursors of the ergot alkaloids and cyclopiazonic acid, respectively, will be described. Alternative methods for the introduction of the •y.^-dimethyl- allyl side-chain into heterocyclic aromatic nuclei of some mycotoxins such as viridamine will also be discussed. The key step involves the reaction of a suitable imidazole precursor with bis-*-dimethylallylnickel(I) bromide. Methods for these synthesis or dedihydrodioxopiperazines and their application in the synthesis of viridamine will be discussed.

S10 nouc or AFUxoxm n BDHUI CATCKR

D. P. H. HSIEH Professor and Department Chairman Department of Environmental Toxicology, University of California, Davis, California 95616 U.S.A.

Aflatoxins are a family of carcinogenic mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus. The toxigenic strains of these fungi frequently infest grains, oil seeds, tree nuts, and other agricultural commodities used for food and ingredients of animal feed. The widespread occurrence of aflatoxin makes human exposure to this family of foodborne carcinogens difficult to avoid. Human exposure to aflatoxin is largely through ingestion of aflatoxin-contaminated foodstuffs and edible animal tissues which contain residues and metabolites of parent toxins ingested by domestic animals. Inhalation of aflatoxin-containing grain dusts by farmers and grain processors is also an occupational hazard of concern. Aflatoxin B-, the most abundant and the most potent member of the aflatoxin family, is one of the most carcinogenic compounds known for the rat and rainbow trout. The principal target organ is the liver, although tumorigenicity to the intestine, cc\on, kidney, and lung has been found. The major metabolite of aflatoxin B^ found in milk and other edible tissues of animals having ingested aflatoxin B^ is aflatoxin H^c This metabolite is about 2 to 10% as hepatocarcinogenic as aflatoxin B1 and is directly ingested by human populations as a contaminant of dairy and other animal products. Epidemiological studies of various regions in Asia and Africa have provided evidence for the involvement of aflatoxin in the etiology of human primary hepatocellular carcinoma. However, studies conducted in North America have not produced similar evidence. It is therefore widely accepted that other etiological factors, especially the hepatitis B virus, are also involved. At present, the incidence of liver cancer more strongly correlates with exposure to aflatoxin than with hepatitis B virus infection. The role of aflatoxin in the etiology of cancer *•> extrahepatic organs has received relatively little attention. However/ in view of its potency as a complete carcinogen and the prevalence of cancer in intestine, colon, kidney, and lung in human populations, aflatoxin should be examined more closely for its contribution to the etiology of cancer in these organs.

S11 ANALYSIS AND DYNAMICS OF OCHRATOXIN A IN BIOLOGICAL SYSTEMS

KARL HULT Ph.D Department of Biochemistry and Biotechnology The Royal Institute of Technology, S-100 44 Stockholm, Sweden The strong fluorescence of ochratoxin A has been used in a number of detection methods developed for the toxin. Most of these methods rely on TLC or HPLC in the determinative step. We have in my laboratory developed a spectrophotofluorometric method for the analysis of ochratoxin A in which the fluorescence of the toxin is measured in a buffer before and after enzymic hydrolysis of the molecule's amide bond. This procedure combines the quantitation of the toxin with the confirmation by using the differences in the spectra of the substrate and the product. The sampling process for the analysis of ochratoxin A, and any other analyte, is as important as the analytical determination used. Thus, the sampling of cereals for mycotoxin analysis is a big problem because of the heterogenous distribution of the toxins in the lot to be investigated. For ochfatoxin A this problem can be solved by using the pig as sampler and the blood of the pig as sample. By analysing the occurrence of ochratoxin A in pig blood it is possible to calculate the general occurrence of the toxin in the feed used. With this method it is possible to monitor variation of ochratoxin A occurrence as a function of season, year and handling procedures of the cereals. The level of ochratoxin A in the blood gives a direct measure of the exposure of the organism to the toxin. This possibility is used in an attempt to correlate the occurrence of Balkan endemic nephropathy with the exposure of humans to ochratoxin A. The distribution of ochratoxin A between different organs in a number of animals is well known. The toxin is accumulated in the blood of some animals. This accumulation is attributed to binding of the toxin to serum albumin. The rate of elimination from the blood stream is very different in different species. The half-life of ochratoxin A in the blood of pigs is about 25 times longer than in chicken. This difference in pharmacokinetics between different species is a problem/ when the exposure to the toxin is monitored by blood analysis. The persistence of ochratoxin A in the blood and the kinetics of the transport into the target organ, the kidney, are very important to know for full understanding of the analytical data.

S12 SYNTHETIC STUDIES ON MARINE TOXIC POLYETHERS TOTAL SYNTHESIS OF OKADAIC ACID

Minoru Isobe Laboratory of Organic Chemistry, Faculty of Agriculture Nagoya University, Chikusa, Nagoya 464, JAPAN

Okadaic acid (1) was found in two sponges Halichondria okadai and H. melanodocia, marine dinoflagellate Protocentrum lima and others. It is a carbo- xylic acid with a continuous 38 carbon chain having 17 asymmetric centers. This paper discusses on the organic synthesis of this molecule, the plan to which involves disconnection between C14-15 and C27-28 positions to lead into three segments. All of them have recently been synthesized in optically active form from glucose, respectively. Stereochemical proglems have been solved to control all the asymmetric centers involving those on its acyclic parts. The paper will deal with the syntheses of those segments and the connection of each segment in stereocontroiied manner, and hopefully deai with the total synthesis.

CMH<8013

OKADAIC ACID

S13 F1YC0T0XIC0SES OF LIVESTOCK IN SOUTH AFRICft

T.S. KELLERMAN

BSc(AGRIC), BVSc ONDERSTEPOORT VETERINARY RESEARCH INSTITUTE, P.O. ONDERSTEPOORT 0110, RSA

The aetiology, toxicology and pathology of mycotoxicoses known to cause, or suspected of causing, losses of livestock in South Africa are discussed. These include leukoencephalomalacia of horses (Fusariurorooniliforroe); stachy - botryotoxicosis of sheep (Stachybotrys chartarum); lupinosis (Phomopsis leptostromiformis), facial eczema (Pithoroyces chartarum) and diplodiosis (Diplodia maydis) of sheep and cattle; Paspalum staggers (Claviceps paspali) and a highly fatal tremorgenic nervous disorder (Asperqillus clavatus) of cattle; hyperoestrogenism (Fusarium qramineerum) and aflatoxicosis (Asper- qillus flavus) of swine. The possible role of P_. chartarum as a trigger for •geeldikkop', a hepatogenous photosensitization of sheep and goats grazing on wilted Tribulus terrestris in the Karoo, is described.

S14 ANTI-CYANOGINOSIN-LA MONOCLONAL ANTIBODY : PREPARATION AND CHARACTERIZATION

R KFIR, E JOHANNSEN and D P BOTES

The toxin cyanoginosin-LA (MW 909) was isrlated from a blue green algae Microcystis aeruginosa. The purified toxin was conjugated with polylysine and muramyl dipeptide to form a complex of an hapten, a carrier and a vaccine. This complex was used for the immunization of mice. Spleen cells of the immunized animals were fused with cells of a myeloma cell line resulting in 100% rate of fusion. Hybrid cells producing antibodies against cyanoginosin-LA were selected by enzjme-1inked immunosorbent assay. Positive hybrids were cloned and the confirmed clones were grown in vivo and in vitro to produce large amounts of monoclonal antitodies. The monoclonal antibodies of the most efficient producers were further purified and characterized.

S15 CHRONIC PATHOLOGICAL EFFECTS OF SOME FUSARIAL TOXINS

N P J KKIEK, W F O MARASAS, S J VAN RENSBURG, B YAGIN, A Z JOFFE M MED VET (PATH) DEPT. OF PATHOLOGY, FACULTY OF VETERINARY SCIENCE, MEDUNSA, P.O. MEDUNSA 0204, REPUBLIC OF SOUTH AFRICA.

Both T -toxin and the toxin(s) produced by certain isolates of Fusarium moni- 1iforme occur under natural conditions and are considered to be of the utmost importance in respect of human and veterinary public health.

The effect of the chronic administration to BD IX rats of fungal culture material of F_. moniliforme in the food is predictable; lesions are dose and time dependant. Effects progress from an initial nephrotoxicity to a cardiac- and hepatotoxicity and death after 3 weeks at dietary levels of the fungal material of up to 32%. The most important cause of death at this stage is a combination of cardiac and hepatic failure. At dietary levels of as low as 2%, death was associated with the development of hepatocellular and ductular carcinomas in 80 and 63%, respectively, of rats surviving for longer than 450 days. The progression of premalignant lesion to neoplasia is emphasized.

Chemically pure T -toxin was administered per os in DMSO 3 times weekly to vervet monkeys (Cercopithecus aethiops). A clear dose-response relationship was established. The morphological changes in the main target tissues (bone marrow, lymphoid tissue and gastrointestinal mucosa) varied over time: acute cases characteristically showed extensive necrosis and only slight abnorma- lities of various haematological parameters. Subacute and chronic cases were associated with lymphoid atrophy and an aplastic anaemia. A dosage rate of 350 >ug/kg 3 times weekly resulted in an extreme aplastic anaemic terminating in death after 1 year.

Both these intoxications are considered to be easily reproducable and may be used as experimental models.

S16 THE ANALYSIS AND OCCURRENCE OF PATULIN IN APPLE JUICES

S J KUBACK1

Ph.D. in chemistry, M.Sc. in biology Department of Food Analysis, Institute of the Fermentation Industry, Rakowiecka 36, 02-532 Warszawa, Poland

Patulin /4-hydroxy-4Hfuro /3,2-c/-pyran-2/6H/-one/ is a mycotoxin produced by numerous Aspergillus and Penicillium species frequently found in a variety of food especially in apples and apple products.

Occurrence of patulin in various crops and processed food is widely discussed with particular attention paid to apple juices. Apart from data published in literature the results which have been obtained in Poland for the last few years are included.

There are also described the methods used for analysis of patulin the majority of which are based on HPLC and TLC. The relevant IUPAC and ISO projects are presented too.

Additionally the lecture contains the experimental results on transformation of patulin into less toxic or non-toxic derivatives in reaction with sulfhydryl containing compounds and other food components as well as with some food additives.

S17 THE NATURAL CONTAMINATION OF CEREALS BY TOXIC TRICHOTHECENES RISK TO HUMAN HEALTH HIROSHI KURATA and MASAKATSU ICHINOE

Department of Microbiology National Institute of Hygienic Sciences 1-18-1 Kamiyouga, Setagaya-ku, Tokyo 158, Japan

Presently important and toxic trichothecenes which are mainly produced by Fusarium graminearum are deoxynivalenol(DON), and nivalenol(NIV). These two trichothecene mycotoxins are naturally and widely contaminated in cereal crops of wheat, barley, corn, and so on in Japan, but the natural contamination of NIV has not yet been found in the USA, Canada, and other world countries so far. T-2 toxin is also one of the representative toxic-trichothecenes which was firstly descovered in the USA and its natural occurrence have been found in some kinds of cereals and animal feeds in the USA, Canada, and India, although it not yet found in Japan and other Ascian countries. From this reason, the geographical differentiation of the occurence of the two toxins in different countries is likely to be a interesting problem on study of mycotoxin, especially on viewpoint of food hygiene. Since 1979, extensive survey for the natural occurrence and contamination of DON and NIV have been carried out by our research group in domestic cereal crops and in their products sampling from marketing channels. The data obtained from these surveys was reported by us in IUPAC Mycotoxin Meeting in Wiene in 1982. Then we want to report further information of present status of the DON and NIV. contaminating domestic cereals and their producs, including flours, noodls, pancake, popcorn, beer, and roasted barley tea, and to review the containation of world cereals and their poducts, finally to discuss about risk of DON and NIV to human health on the basis of these informations. The US, FDA has issued an advisary(8/15/82) recommended a level of concern of 1ppm for finished wheat product as use for human consumption. Canada governme- nt also seems to decide to limit human exposure of DON(except NIV) and more recently established a control level of 2 ppm for uncleaned soft wheat.

S1B FACTORS AFFECTING TOXIN PRODUCTION BY FUNGI

J. LACEY

B.Sc, Ph.D. Rothaicsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K.

Factors affecting toxin production by fungi may include strain variation in the fungus, interference by other fungi, the gaseous environment, temperature, water availability, any preservative treatment and their interactions. Water activity/temperature relationships have been defined for several fungus species/mycotoxin combinations and it has been shown that conditions for their production may vary for different toxins produced by one species and for the same toxins produced bv different species. Additionally, in growing crop plants, heat and water stress may affect the susceptibility of the host and its suitability as a substrate for toxin production. For instance, in both maize and peanuts, stress has been shown to increase the incidence of A. flavus and aflatoxin. However, our knowledge of the effects of these factors on many fungi and toxins is limited. This paper will review current progress and suggest areas for future research.

S19 MECHANISM OF ACTION OF THE TERRITREM,TREMORGENIC MYCOTOXIN ISOLATED FROM ASPERGILLUS TERREUS

LING,K.H.j LIOU,H.H.)FU,T.C.?KUO,L.?TSAI,M.C?and LIN, M.Y? Dr and Prof of Biochem. 12 3 Institutes of Biochem. Physiol. and Pharmacol,College of Med.Natio- nal Taiwan University,Taipei.Taiwan,Republic of China Territrem B (TRB) was given to rats and mice through different routs, such as intraperitoneal(ip), common carotid artery,lateral ventricle of the brain, lumbar subarachnoid space(T,13-L,1) femoral artery and by direct contact with the naked gastrocnemius muscle under various surgical treatments. TRB was also given by ip or from the left femoral artery to rats and mice after denervating the left sciatic nerve. Tremorgenic activity induced by TRB was qualitated by electromyo- graphy (EMG) from both gastrocnemius muscle of the animal. The results indicated that the action site of TRB was not the central but peripheral and that the functional integrity of the motor nerve ending was necessary for the tremorgenic activity of TRB. The effect of TRB on the neuromuscular transmission was further studied by electrophysiological methods with mouse phrenic nerve- diaphragm preparation and diaphragm from mice denervating the phrenic nerve at the cervical portion for 9-14 days. The results indicated that the action site of TRB was at the pre- synaptic area of the alpha-motor fibers in the endplate and potentiate the release of acetylcholine.

S20 HEPATIC METABOLISM AND ELIMINATICN OF TREMDRGENIC MYCOTOXTHS

P.G. MANTLE Ph.D. D.Sc. Biochemistry Department, imperial College, London SW7, U.K.

Tremorgenic compounds have stimulated research in the field of mycotoxicology in recent years since some are causal agents of naturally-occurring neurological disorders of animals (eg the paspalinines produced by Clayiceps paspali are the cause of Paspalum staggers). Structurally-analogous substances", the lolitrems, are regarded as causing ryegrass staggers and may be of fungal origin since they occur only in ryegrass infected by the endophytic fungus Acremonium loliae. The extreme potency of many tremorgenic mycotoxins generally precludes study of elimination in live animals unless the compounds can be radiolabelled, and in several instances biosynthetic radiolabelling is the method of choice. Verruculogen and penitrem A, together with the associated neurotoxin roquefortine, were, therefore, prepared to modest specific activity from C-labelled amino acid and/or isoprenoid precursors. In the perfused rat liver verruculogen is totally metabolised, with the loss of one isoprene, to the N-dealkylated compound TR-2 which is also the principal verruculogen metabolite eliminated by the sheep Into the bile! Although TR-2 is more polar (and less tremorgenic) than verruculogen, any TR-2 which is reabsorbed from the gut is not further dealkylated by the liver during its second cycle of elimination into the bile. TR-2 is also spontaneously epimerized to an isomer in solution and the precise structural elucidation of this transformation has been established by NMR spectroscopy. Both TR-2 and its isomer, however, disappear during passage through the intestines and this subsequent metabolic phase has been demonstrated in the laboratory using sheep ileum contents, presumably mediated by the ruminant microflora. By contrast, penitrem A is not metabolised by liver homogenates or slices but is excreted in the bile in the form of more polar degradation products, whether in the perfused rat liver or in a sheep with a cannulated bile duct. The molecule seems to be degraded in vivo directly in bile and the feasability of this proposition has been demonstrated _fjn_ vitro using fresh sheep bile, revealing virtually complete transformation of penitrem A within a few hours. The same occurs when the bile has been boiled to inactivate enzymes. A search of the literature suggests that this spontaneous non-enzymic degradation of a xenobiotic in bile 1s at most a rather unusual phenomenon. Verruculogen and penitrem A, having rather similar polarity and molecular weights (511 and 633, respectively), are eliminated principally in bile; their molecular size precludes significant renal elimination. Roquefortine (MW-389), however, is significantly eliminated 1n urine. Excretion in bile is without hepatic metabolism but roquefortine is subsequently degraded by the intestinal microflora. These results emphasise the Importance of liver function in elimination and provide the basis for discussing the putative elimination of lolitrems and its significance in the tolerance of ruminants to the challenge of forage-generated tremorgens.

S21 FUSARIUM MONILIFORME : A MyCOTOXICOLOGICAL MIASMA

W.F.O. MARASAS Ph.D. National Research Institute for Nutritional Diseases, S.A. Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa The word miasma is derived from the Greek word for pollution and has medieval connotations of "noxious exhalations from putrescent organic matter". Fusarium moniliforme Sheldon (F. m.) is a plant pathogenic fungus which can also grow saprophytically on dead organic matter. £. m. has many similarities to medieval miasmata with respect to its habitat, prevalence, toxicity, carcinogenicity and the confusion and ignorance surrounding its taxonomy and raycotoxicology. The reasons for calling F_. m. a "mycotoxicological miasma" can be summarised as follows:

1. _F. m. is one of the most prevalent fungi associated with maize (Zea mays L.), a major human and animal dietary staple, throughout the world.

2. £.•£>• causes field outbreak of equine leukoencephalomalacia (LEM), a neurotoxic disease characterised by liquefactive necrotic lesions in the white matter of the brain, in many countries.

3. F_. m. is correlated with human oesophageal cancer risk in Transkei and China.

4. F_. ro. is extremely toxic to many experimental animals and hepatocarcinogenic in rats.

5. F_. m. is surrounded by confusion and ignorance with respect to its taxonomy and mycotoxicology. Some authors recognise F_. m. as the only species in Fusarimn Section Liseola while others accept 10 taxa. The mycotoxin moniliformin named for F. m, is a misnomer because £. m. is in fact a weak producer of moniliformin. The production or not of trichothecenes and zearalenone by F. n. is controversial. A potent mutagen, fusarin C, is produced by F. m. and occurs naturally in Transkeian maize. It is, however, not known whether fusarin C is carcinogenic or not.

6. F_. m. has been grossly neglected mycotoxicologically and the chemical nature of the mycotoxin that causes equine LEM and the carcinogen that causes liver cancer in rats is unknown.

7. _F. m. causes diseases in search of mycotoxins and produces mycotoxins in search of diseases.

8. £. ni- represents a real threat to human and animal health and deserves to be elevated from the miasmatic to the scientific plane.

S22 METABOLISM AND RESIDUES OF TRICHQTHECENES IN DIFFERENT ANIMALS AND TISSUES

C. J. MIROCHA PROFESSOR Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA

The metabolic fate of T-2 toxin and diacetoxyscirpenol was studied in rats, monkeys, cats and bovine. The tissues monitored were the liver, lung, heart and kidney as well as the blood and urine. Monkeys dosed with T-2 (2.4-6.0 mg/kg) contained T-2, HT-2 and TC-3 and TC-6 in the heart. Monkey urine contained T-2 tetraol. Bovine urine collected from T-2 dosed cows contained T-2-tetraol, T-2-tetraol-Ml, TC-3, TC-1, IsoTC-1, T-2 and HT-2. Bovine blood contained traces of T-2, HT-2, TC-3, T-2-tetraol and T-2-tetraol-Ml. Cats dosed with T-2 toxin contained T-2, HT-2, TC-1, TC-3 and T-2-tetraol in the heart, lung and kidney. Their urine contained HT-2, TC-3, TC-1 and T-2-tetraol. T-2-tetraol was found in the bone marrow. Rats dosed with diacetoxyscirpenol contained tnonoacetoxyscirpenol and triacetoxyscirpenol in cardiac tissue.

S23 CHEMISTRY OF SOME CYTOREACTIVE MOLD METABOLITES A EPITETRATHIODIOXOPIPERAZINE AND TWO NEW CYTOCHALASANS

S. NATORI

Professor Meiji College of Pharmacy, Yato-cho, Tanashi-shi, Tokyo 188, Japan

Starting from the surveys on mycotoxin-production of food-borne fungi collected in Japan by cytotoxicity testing using HeLa cells, chaetomium spp. were found to produce indolyl-[13]cytochalasans named chaetoglobosins. Further surveys on 120 strains of the genus showed production of a variety of mycotoxins such as sterigmatocystin, o-methylsterigmatocystin, chaetochromin, chetomin, and 2) chaetocin. ' The three species, C. abuense, C. retardatum, and C. tenuissimum showed strong cytotoxicity and the causative agents were isolated and identified respectively as chetomin, cochliodinol and a new dioxopiperazine named chetracin A (I), and lla,11'a-dihydroxychaetocin and I. The structure of chetracin A was supposed to be a tetrathio derivative of the dihydroxychaetocin by physical and chemical methods. Finally single crystal X-ray analysis revealed the structure(I). In the course of reinvestigation of the molds showing cytotoxicity accompa- nying polynuclear cell formation, septoria sp,(68-G0-164 starin) CH-jOH was found to produce a series of phenyl-[ll]cytochalasans. Four %c of them were identified with epoxycytochalasin H and J and cytochalasin H and J. The other two named cytochalasin N and 0 were proved to be expressed by the formulae II and III by spectral A\ data and correlation reactions. ; Structure-activity relationship of cytochalasans will also be discussed. References: l) S. Sekita et al., Chem. Pharm. Bull., 1609 (1982). 2) S. Ddagawa et al., Canad. J. Microbiol., 25, 170 (1979); S. Sekita et al., ibid., TT_, 766 (1981). 3) T. Saito, K. Koyama, S. Natori, I. Ii- taka. Tetrahedron Lett., to be published. 4) T. Tomioka, K. Koyama, S. Natori, Phytochemistry, to be published. 5) S. Sekita, K. Yoshihira, S. Natori, F. Harada, K. Iida, I. Yahara, J. Pharm. Dyn., in the press.

S24 HEPATCARCINOGENICITY OF CORN SCREENINGS NATURALLY CONTAMINATED WITH FUSARIUM MONILIFORME

P. E. NELSON AND T. M. WILSON Professor of Plant Pathology and Associate Professor of Veterinary Science Fusarium Research Center, Department of Plant Pathology and Department of Veterinary Science, The Pennsylvania State Univ., University Park, Pa. 16802 USA In 1983-84 an outbreak of equine leukoencephalomalacia, in which 9 of 15 horses died, occurred in southeastern Pennsylvania. Clinical signs and neuropathological lesions were consistent with a diagnosis of leukoencephalomalacia. The corn was produced on the farm where the outbreak occurred and had no history of fungicide treatment. Thirty-five kg of the corn screenings being fed during this outbreak were obtained and samples were placed on a medium selective for Fusarium species. The predominant species recovered was F_. moniliforme. We recovered 64 cultures of this species along with 1 culture of F_. subglutinans, 1 culture of F. sporotrichioides, and 3cultures of F. equlseti. Analysis of a sample of this feed for aflatoxins at a level of 1.0 ppb was negative. The remaining corn screenings were ground finely and fed unsupplemented to 12 Fisher 344 rats for 176 days. A control group of 12 rats was fed commercial rodent chow. Control animals euthanized and evaluated after 176 days were free of significant gross lesions. Gross lesions in all test animals, sacrificed from day 123-176, were confined to the liver which contained multiple raised nodular lesions and pale depressed areas. These discrete brown nodules varied from 1-5 mm in diameter and were elevated above the liver surface. Multiple pale depressed lesions were also observed in all livers of test rats. These white lesions varied from 1-5 mm in size and were irregular in shape. The 3 distinct histopathological liver lesions observed in the test rats were nodular, adenofibrosis, and cholanglocarcinoma. The normal hepatic architecture was disrupted by multiple neoplastic nodules and foci of adenofibrosis. The hepatic nodules were composed of solid sheets of hepatocytes with variable shaped nuclei and enlarged nucleoli. Irregularly distributed throughout the liver were islands of two types of adenofibrosis composed of tubular structures surrounded in some cases by connective tissue. In one type regular tubular structures were lined by low cuboidal epithelial cells including goblet cells, which contained large bland nucleoli and small indistinct nucleoli, and the cytoplasm was clear. In the other type the tubular structures were irregularly branching or elongated and lined by plump anaplastic epithelial cells containing a large nucleus with an irregular nuclear membrane. Multiple nucleoli and abundant chromatin clumping were evident and mitosis was common. These transitional lesions were identified as cholangiocarcinomas. Eosinophilic material, inflammatory cells and detritus were evident in some lumens in areas of adenofibrosis and cholangiocarcinoma. Intertubular connective tissue and an admixture of inflammatory cells were present in areas of adenofilbrosis. This is the first report of the hepatocarcinogenicity of a natural sample of equine feed infested with F_. moniliforme.

S25 A RATIONALE FOR THE CONTROL OF AFLATOXIN IN FEEDS

DOUGLAS L. PARK, ALBERT E. POHLAND, LEONARD STOLOFF

Ph.D., University of Maryland, 1976, Food Toxicology

Food and Drug Administration Center for Food Safety and Applied Nutrition Washington, DC 20204

Since the discovery that aflatoxins can occur as food contaminants, they have presented a complex regulatory challenge in the area of food safety. Efforts to control marketing of foods highly susceptible to direct contamination and consumed by humans have been generally effective. Aflatoxins in animal feedstuffs, however, present a twofold regulatory problem. The health of animals ingesting contaminated feed as well as the health of humans consuming aflatoxin residues in foods derived from such animals must be considered. The Food and Drug Administration has developed a course of action for regulating aflatoxins in feedstuffs that assures a safe and plentiful food supply. Factors considered in developing a mycotoxin regulatory control program for animal feeds include analytical capability to detect, measure and confirm the identity of the toxin, assessment of the risk from consuming aflatoxins present in animal tissues, and the health of the food-producing animals.

S26 AFLATOXIC SUPPRESSION OF CELL MEDIATED IMMUNE RESPONSES AND INTERACTION WITH T-2 TOXIN

A.C PIER M.J. VARMAN E.L. BELDEN D.V.M., Ph.D M.S. Ph.D. University of Wyoming, Department of Microbiology and Veterinary Medicine,

Laramie, WY 82071

Aflatoxin has been shown by several investigators to exert substantial suppressive effects on selected cell mediated immune (CMI) mechanisms. In animals experimentally dosed with purified and partially purified toxins significant reductions in thymic size and in delayed cutaneous hypersensitivity (DCH) have been demonstrated. Lesser but consistent suppression of lymphoblastogenesis and leukocyte migration inhibition has been observed in cells from aflatoxin treated donors and reduced phagocytic response has been observed in macrophages from such donors. Aflatoxic suppression of DCH has been shown to cross the highly complex porcine placenta and affect the unborne fetus. Adoptive transfer of the DCH response is reduced when cells from aflatoxin treated donors are employed. The T cell population of the peripheral blood lymphocyte pool has been shown to be undiminished in aflatoxin treated individuals. Other experiments on cultured lymphocytes have demonstrated ix^ vitro suppressive effects by some aflatoxin moieties, but not by all. Aflatoxins B. and Q. appear to be most suppressive of lymphoblastogenesis in in vitro cultures of peripheral blood lymphocytes. Aflatoxicol, a markedly mutagenic and carcinogenic moiety, has little effect on lytnphoblastogenesis suppression. No evidence of effect on cytotoxic or suppressor cell activity has been found.

This combination of events implies that aflatoxic immunosuppression of CMI responses may reside in suppressed lymphokine production as well as in reduced antigen recognition borne of suppressed macrophage function.

Current evidence implies that T-2 toxin also suppresses CMI responses, but, probably by different mechanisms than aflatoxin. Thymic aplasia, reduced graft vs_ host response and reduced lymphoblastogenic response have been noted in T-2 treated animals and cell cultures. The T-2 suppressive effects have been shown to cross the relatively simple rodent placental barrier and effect the unborne fetuses*CMI system. T-2 toxin effects on DCH have been reported in some systems to enhance that response thru reduction in suppressor cell activity. Other trichotbecenes have a suppressive effect on DCH. Our studies on DCH in guinea pigs show a reduced response in T-2 treated individuals. Inter- action between aflatoxin and T-2 toxin has been synergistic in terms of « lethal effect in mice. Our studies in guinea pigs confirm that finding but results to date imply only an additive effect in CMI suppression after _in vitro and in vivo exposures of cell cultures and intact animals to combinations of aflatoxin and T-2 toxin.

S27 MASS SPECTROMETRY/MASS SPECTROMETRY AS A TOOL FOR THE IDENTIFICATION AND QUANTITATION OF MYCOTOXINS

R. D. PLATTNER Research Chemist Northern Regional Research Center. ARS-USDA, 1815 North University street Peoria, Illinois 61604, USA Mass spectrometry/mass spectrometry (MS/MS) is a useful technique for the identification and quantitation of mycotoxins at low concentrations directly from crude extracts of biological samples with minimal chemical or chromatographic clean-up. Generally, a soft ionization technique such as chemical ionization

S28 CONFORMATION OF CYCLIC PEPTIOES : RHIZONIN AND PHOMOPSIN A

M. POTGIETER

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 393, Pretoria 0001, RSA

Rhizonin is a cyclic heptapeptide of which four amide groups are ^-methy- lated. X-ray crystallography confirmed its structure and the absolute configuration was established by derivatization of the acid hydrolysate and chromatographic matching with standards. Complete assignment and interpre- tation of the proton and carbon n.m.r. data of a chloroform solution of rhizonin indicated close similarity between its solid state and solution conformations. In solvents of increased polarity a second conformer is observed, i.e. 2b% in methanol and 50% in dimethyl sulfoxide. Phomopsin A, the principle toxic metabolite of Phomopsis leptostromiformis, is a cyclic hexapeptide of which the structural elucidation was facilitated by n.m.r. spectroscopy and amino acid analysis.

The solid state conformation of rhizonin will be discussed in comparison with cryatallographic results obtained for other cyclic peptides. The discussion of the three-dimensional solution conformations of rhizonin and phomopsin A includes reference to their conformational homogeneity, amide hydrogen bonding and the results obtained from n.O.e. experiments.

Ptiomoptin A Rhfzonin

S29 COMMERCIAL DETOXIFICATION OF AFLATOXIN-CONTAMINATED PEANUT MEAL

A. PREVOT Ingenieur E. N. S. C. P Directeur Regional de I1 INSTITUT DBS CORPS GRAS Rue Monge - Pare Industriel - 33600 PESSAC.France In spite of efforts of prevention -the best approach- (genetic improvement, agricultural practices, diversion, antifungal agents), aflatoxin contamination is often unavoidable. Removal by physical separation and by many chemicals have been tried. In 1979 an ammoniation semi-continuous plant was installed by the French LESIEUR Co in his oil mill in DAKAR (Senegal) with the financial support of European Fund of Deveioppement (F.E.D.). The capacity of the reactors was 16 m3 or 6| metric tons. Improvements of the process have been made by SONACOS, combining formol as antibinding agent and ammonia. The production of detoxified peanut meals was 600 metric tons a day but did not exceed 50 000 t a year, the whole production of the oil mill was small due to the low quota allowed. The price of the processing was 10 F CFA. In 1983, 20 000 t of PROFOR, the commercial name of this meal, were imported in France by the Federation of French Meal Producers and were succesfully tested (mean value 19 ppb). An official favourable opinion has been given.

In France a detoxification mill built in Saint-Gerand in Brittany using a similar processing has a capacity of 500 t a day for the begining and gives a peanut meal named AROFLOR with good performances.

S30 IMPORTANT LESSER KNCWN ICXECENIC FIM3

C.J. RABIE

D.Sc. Agric. Pta Univ, 1956; Ph.D Univ of Wise, 1965

S.A. Med. Res. Council, PO Box 70, Tygerberg 7505 The number of publications that have appeared since 1966 on 61 different mycotoxins and 28 toxic fungal taxa was enumerated by doing a retrospective search on both Biological and Chemical Abstracts. The object was to determine the state of the art, determine trends and project future developments. Results showed that the total number of publications are increasing, especially as far as some trichothocenes are concerned. Important but less well known toxic fungi include amongst others, Diplodia maydis, Rhizopus arrhizus, R. microsporus and R. microsporus var. chinensis. D. maydis is an important pathogen of maize in South Africa. The different Rhizopus species are amongst the most important fungal species contaminating sorghum malt and some of the lower grades of oats. Culture material of D. maydis isolates originating in the RSA, USA and Argenti- na induced the neuromuscular disease diplodiosis in cattle and sheep at low dose levels. The toxin involved is still unknown. No correlation was found between the acute toxicity of culture material of specific strains to ducklings and rats and the ability of these strains to induce diplodiosis in cattle and sheep. No neuromuscular symptoms were seen in rats and ducklings. A total of 47 out of 104 Rhizopus isolates representing 14 species were toxigenic to ducklings. Isolates of three species were also acutely toxic to rats, viz R. microsporus, R. microsporus var. chinensis and R. arrhizus. Rhizonin A, the sole mycotoxin chemically characterized in the genus Rhizopus, could not be detected in this culture material. In rats, R. arrhizus caused haemorrhagic gastroenteritis and enteritis with a predominance of mucosal erosive changes and subacute enteritis with secondary fibroblastic and hystiocytic infiltrations of affected villi. R. microsporus caused acute, partly erosive gastroenteritis. Myocardial disseminated cellular acidophilic necrotic and degenerative changes also occurred. R. microsporus var chinensis caused hepatocellular necrotic and degenerative changes. The results showed that these acutely toxic Rhizopus species induced totally different effects in rats, indicating that probably different unknown mycotoxins must be involved.

S31 ANALYSIS OF TOXINS OF FUSARIUM MONILIFORME

P.M. SCOTT M.A., Ph.D. Food Research Division, Health Protection Branch, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2 Fusarium moniliforme is a fungus of considerable international interest since its presence in corn is widespread and has been associated with human esophagal cancer and several animal mycotoxicoses, its cultures on corn or corn bread have caused cancer in rodents, and it produces the mutagenic metabolite fusarin C, among other mycotoxins. Therefore a method for the determination of fusarin C in corn and wheat flour by reverse phase liquid chromatography has been developed that is sensitive to 50 ng/g; samples are extracted with methylene chloride-acetonitrile (1+1), cleanup is by conventional silica gel column chromatography or, more rapidly, by small (3 mL capacity) bonded phase amino (NH2) columns. Precautions necessary to ensure good recoveries of fusarin C include restrictions on laboratory lighting ("Gold" fluorescent lamps). Stability studies on fusarin C in standard solutions under various lighting conditions, in ground corn at room temperature, and during cooking were carried out. No fusarin C was found in a small survey of 12 samples of Ontario corn that contained deoxynivalenol, but it was detected in a sample of unsterilized field corn incubated in the laboratory. Thin layer chromatography of moniliformin, another important mycotoxin from Fusarium moniliforme, and the stability of the free acid and sodium salt in various solvents have also been investigated.

S32 PHYSIOLOGICAL AND MORPHOLOGICAL INTERACTIONS IN THE TOXIC BLUE-GREEN ALGA (CYANOBACTERIUM) MICROCXSTIS AERUGINOSA

W.E. SCOTT M.Sc. (UOFS)

National Institute for Water Research, CSIR, P.O. Box 395, Pretoria 0001 Republic of South Africa

Natural populations of planktonic Microoystis often show a wide range of morphological forms. This has resulted in the description of about 40 species and varieties. Several taxonomic revisions have tried to reduce the number of species but there are still problems in deciding from a microscopical examination whether a specimen is likely to be toxic or non-toxic. It is possible to make a decision on the likelihood of toxicity from a microscopical investigation of field material by examining the form and shape of colonies and by measuring the diameter of single cells. Colony shape and cell diameter, however, are not constant characteristics and these can readily change, especially if Microcystis cells are cultured in the laboratory. It is well established that cultured Microeystis usually assume a single cell form. It is not possible to equate these forms with original field material. Doubts have therefore been expressed about the identity of single-celled cultures available from culture collections under the name of Microeystis. Various factors contribute to the production of sheath material (mucopolysaccharides) which in turn influences aggregation of cells and hence colony form. Estimates based on colony morphology were made of the amount of toxic Microcystis in Hartbeespoort Dam from weekly samples collected over a twenty-month period. These estimates show that sizeable populations of toxic M. aeruginosa are present in the dam for about 10 months of the year.

S33 BIOSYNTHETIC STUDIES ON MICKOBIAL METABOLITES CONTAINING A CARBON-PHOSPHORUS BOND

H. SETO Associate Professor Institute of Applied Microbiology, University of Tokyo Yayoi, Bunkyo-ku, Tokyo, Japan 113

Bialaphos is a metabolite of Streptomyces hygroscopicus SP- 1293 consisting of two moles of alanine and one mole of a new amlno acid, phosphinothricin. It shows very strong phytotoxic activities and is now being used as a herbicide in Japan. Bialaphos is the only natural product to have the unique C-P-C bond in the phosphinothricyl moiety. In view of this structural characteristics, our efforts were directed toward understanding the mechanism of formation of the C-P bonds. The results obtained so far are summarized as follows. (1) Labeling experiments using C-13 labeled precursors revealed that Cj and C2, and Co and C4 of phosphinothricin originated from acetic acid and phosphoenolpyruvate, respectively. (2) When the producing organism was cultivated in the absence of Co++, accumulation of new phosphinic acids were detected by ^^P-NMR spectroscopy and these compounds named demethylphosphinothricin and demethylbialaphos were isolated by ion exchange chromatography. (3) Further studies resulted in the isolation of several additional phosphinic acids and phosphonic acids from the fermentation broth of blocked mutants, and their structures have been determined. (4) Transformation experiments of these metabolites to bialaphos revealed the reaction sequence involved in the formation of the C-P-C. bond in bialaphos. Based on these results, the blosynthetic pathway of bialaphos will be discussed in detail.

0 NH, CH3 CH3

CHj-P-CH2CH2CHCO-NHCHCO-NHCHCOOH OH bialaphos

S34 STRUCTURAL AND BIOSYNTHETIC STUDIES ON POLYKETIDE MYCOTOXINS AND FUNGAL METABOLITES

T.J. SIMPSON B.Sc.,Ph•D. Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland

Some applications of H and C n.m.r. and H, C and O labelling to structural, mechanistic and biosynthetic problems associated with the polyketide-derived metabolites aspyrone (1) , grahamimycin (2), LL-D253a (3) and averufin (4) will be described.

(4)

S35 MOLECULAR ASPECTS OF AFLATOXIN B] MUTAGENESIS AND CARCIHOGENESIS

AVISHAY-ABRAHAM STARK

Dept. of Biochemistry Tel Aviv University, Ramat-Aviv, Tel Aviv 69978, ISRAEL

The adverse effects of the potent hepatocarcinoaen Aflatoxin B, (AFB,) occur in vivo after its activation by the microsomal cytochrome P450 system. Arrest of RNA and DIJA synthesis, mutagenicity and carcinogenicity can be attributed to the DNA-binding capability of activated AFB,. Thus, the type and rate of AFB, metabolism are major factors determining species and organ specificity of AFB, carcinogenesis. In vitro AFB, metabolism and DNA binding are like- wise responsible for mutagenicity and lethality exerted in bacterial systems. AFB^ can be activated in vitro also by near-UV light, resulting in covalent binding to DUA, mutagenicity and lethality in bacteria. AFB,-DNA binding levels are well correlated with mutagenicity and lethality in bacteria (irrespective of the activation system used) and with carcinogenicity in laboratory animals. Metabolically-activated AFB, binds exclusively to guanine in DNA, forming a variety of guanine adducts, the principal adduct being AFB-.-N Guanine.Short nucleotide sequence near guanine residues in DMA determine the relative susceptibility of those guanines to AFB, binding. Comparison of mutagenesis levels to AFB,-DNA binding levels in the mutagenized cells yield ratios much higher than unity. This fact, taken together with the strict depedence of AFB, mutagenesis on SOS repair mechanism and with the fact that AFB, efficiently and specifically mutagenizes DNA sites containing AT base pairs suggest that a considerable fraction of the mutations induced by AFB-j are non-targeted.

S36 TOTAL SYNTHESIS OF CORYNETOXINS AND TUNICAMYCINS

TETSUO SUAMI

Professor, Ph.D. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama 223, Japan 'orynetoxins have been discovered in toxic ryegrass seedheads infected by Corynebactepium vathayi, and tunicamycins were found in a fermentation broth of Streptonyees lysosuperifiaus nov. sp. as antibiotics. Corynetoxins are structurally closely related to the tunicamycin group of antibiotics which consist of uracil, a C-11 dialdose derivative named "tunicamine" N-acetyl-D-glucosamine and fatty acid. Corynetoxins differ from tunicamycins only in a chain length, a terminal branching and an additional hydroxyl group in the fatty acid side chains. Tunicamycins exhibit wide biological activities, such as antiviral and antimicrobial activities, a G| arrest of a cell culture, an alteration in a translocation of intracellular materials, and a modulation of cell differentia- tions. Biological activities of corynetoxins, the causative agents of annual ryegrass toxicity, are essentially identical with those of tunicamycins. There- fore, the terms, "corynetoxins and tunicamycins" are used often in a collective sense. Tunicamycins are a mixture of more than 16 homologs with different fatty acid residues, and so corynetoxins are. But both compounds have the same funda- mental structure: N-acetyl-a-D-glucosaminyl tunicaminyluracil. In the present study, I wish to report a total synthesis of corynetoxins and tunicamycins, as well as their biological activities. A tunicamine derivative has been synthesized by the Henry reaction of 3-0-acetyl-5-deoxy-l,2-0-isopropylidene-a-D-ribo- furanose and methyl 2-(benzyloxycarbonyl)amino-2-deoxy-3,4-0-isopropylidene-a- D-galactodialdopyranoside-(l,5) in acetonitril in the presence of KF, followed by a three steps reaction. Condensation of the tunicamine derivative with bis- (trimethylsilyi)uracil under the presence of SnCl4 afforded a tunicaminyluracil derivative. Further condensation of an anomeric chloride of the tunicaminyluracil derivative with N-acetyl-4,6-0-isopropylidene-3-0-propyl-a-D-glucosamine gave an a-D-glucosaminyl tunicaminyluracil derivative. O-Deacylation of the product in methanolic sodium methoxide, followed by catalytic hydrogenolysis of the N-protective group and successive N-acylation with an appropriate fatty acid and DCC yielded an O-isopropylidene derivative. Hydrolysis of the derivative in aqueous acetic acid gave corynetoxin or tunicamycin, depending on which fatty acid was used for the N-acylation. 0

H MO"H 9T" H H

0-\—-O-^i C - AcHN J

HO—trr^-0 OH OH CH2OH Corynetoxin U17i

S37 ANALYTICAL TECHNIQUES FOR DINOFLAGELLATE TOXINS

J. J. SULLIVAN Ph.D. - Food Chemistry U.S. Food and Drug Admin., 5009 Federal Off. Bldg. . . Seattle, WA 98174, U.S.A. Several marine dinoflagellate species are responsible for the production of a number of potent and diverse toxins. Traditional analytical techniques for these toxins involve bioassays in mammalian species. Due to inherent limitations of most bioassays, recent research has centered on the development of alternate chemical or biochemical methods for the determination of dinoflagellate toxins. The newer techniques have had mixed success due to the often unique nature and extremely low levels of marine toxins present in most natural samples. Of particular importance are the toxins associated with Paralytic Shellfish Poisoning (PSP), a problem endemic to much of the temperate coastal areas of the world. A number of newer techniques have been developed for the PSP toxins with one of the more sensitive based on conversion of the toxins to fluorescent derivatives by oxidation under alkaline conditions. Operational parameters and performance of two analytical techniques using fluorescence based detection systems will be discussed. These are a High Performance Liquid Chromatographic (HPLC) procedure which provides a total toxin profile for samples and an auto-analyzer (AA) method which can be used to determine relative toxin levels. Both methods exhibit good correlation with the standard mouse bioassay (r = .90) and have several advantages. Application of the new methods to research and shellfish toxicity monitoring programs will be discussed.

S38 HPLC DETERMINATION OF AFLATOXINS AND MAMMALIAN AFLATOXIN METABOLITES

P.G. THIEL

National Research Institute for Nutritional Diseases, S.A. Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa.

The development of sensitive and accurate analytical procedures for the determination of aflatoxins in foods and feeds is essential to implement existing regulations. Methods are also required for the analysis of human and animal samples for aflatoxins and their conversion products in studies on the exposure of populations to these highly toxic and carcinogenic fungal metabolites.

Various excellent TLC procedures for aflatoxin analysis are in use worldwide. In recent years HPLC techniques offer improved reproducibility, the possibility for automation and generally improved sensitivity. One of the major limitations of analysis by HPLC is that positive identification is mainly dependent on retention time which in some cases can lead to false positive observations. However, the development of techniques involving sample derivitization has contributed towards verification of results.

This contribution describes the use of both pre- and post-column derivitization in the analysis of aflatoxins in various agricultural commodities and mammalian tissue samples by reverse phase HPLC using fluorescence detection.

S39 SEQUENCE DETERMINATION OF CYCLIC PEPTIDES BY MASS SPECTROMETRY

A A TUINMAN

NATIONAL CHEMICAL RESEARCH LABORATORY, CSIR, P 0 BOX 395 PRETORIA OOO1, REPUBLIC OF SOUTH AFRICA

The number of possible amino acid sequences for a cyclic peptide of known amino acid content is usually given by:

(n-1)!

ra! x rfc! x ... rm! where n = number of amino acids present, m = number of different amino acid types, and r-j = number of repetitions of amino acid type "i".

In cases where a peptide bond can be specifically cleaved, either chemically or enzyjiatically, sequence determination may proceed by classical Edman degradation or low resolution mass spectrometry.

Where specific cleavage of a single bond of the cyclic peptide is not attainable, sequence determination is much more difficult. High resolution mass spectrometry of the cyclic peptide and/or some simple derivatives will often contain sufficient information to determine the sequence unambiguously. However, the sheer volume of data and possible solutions make manual evaluation tedious and risky.

A computer program will be described which assists in the evaluation of both low and high resolution ms data of such cyclic peptides. A few examples of the successful application of this program will be discussed, e.g. Rhizonin, Cyanoginosin, and DolastaJn.

S40 ROLE OF MYCOTOXINS IN ENDEMIC LIVER AND OESOPHAGEAL CANCER

SCHALK J VAN RENSBURG

B.V.Sc, D.V.Sc. National Research Institute for Nutritional Diseases Present address: Roodeplaat Research Labs, P O Box 13873, S5NOVILLE 0129 Early associations between aflatoxin exposure and the risk for primary liver cancer (PLC) were overshadowed by the demonstration that most cases were associated with Hepatitis B virus infection and that the virus has properties suggesting oncogenecity. However, examination of the alleged relationship between the virus carrier rate and PLC incidence revealed many discrepancies. The risk of PLC in virus carriers living where it was unusually cold or dry was very low. Re-evaluation of the role of aflatoxin has confirmed an excellent relationship between current exposure and PLC risk, both in Africa and Asia. Aflatoxin is unexpectedly frequently present in the livers and tissues of PLC cases, to an approximately equal extent in Africa, Asia and USA, according to existing data. Marked seasonal variation of PLC prevalence, in proportion to the seasonal variation of aflatoxin exposure, has been recorded north and south of the equator. In Mozambique a rapid decline of incidence over five years commenced within a few years of public awareness of the association between moulds and liver cancer. The expected rate declined markedly after 1 year in migrants experiencing reduced exposure. Some evidence exists suggesting that only a subset of the population is susceptable to the effects of aflatoxin. It is concluded that aflatoxin probably precipitates the majority of PLC cases occurring worldwide in individuals preinfected with Hepatitis B virus, acting particularly at a late stage in the carcinogenesis process. Clinical studies suggest that, when aflatoxin exposure is high, the cancerr grow larger and are more rapidly fatal.

All regions having a very high rate of oesophageal cancer such as Transkei, N.E. Iran and the Central Provinces of China are characterized by a subsistence economy. Furthermore, the disease is prevalent in areas where the agricultural circumstances are more adverse than in adjacent lower-risk regions. The quality of the dietary staples are low due to soil mineral deficiencies, lack of moisture and poor farming practices. Xerophyllic fungal invasion, particularly Fusarium moniliforme, is common on maize. Cultures of this organism induce oesophageal hyperplasia and markedly potentiate experimental oesophageal carcinogenesis in rats. The latter effect has also been shown with a strain of Alternaria common on wheat samples from Iran. Existing data suggests that fungal metabolites enhance.the malignant progression of initiated cells, especially when protective nutrients are deficient.

S41 BIOSYNTHETIC STUDIES ON MYCOTOXINS USING MULTIPLE STABLE ISOTOPE LABELLING AND NMR SPECTROSCOPY

J.C.VEDERAS

Chemistry Department, University of Alberta Edmonton, Alberta, Canada T6G 2G2

The bioysntheses of a series of polyketide metabolites from mycotoxin-producing Aspergillus species have been examined by incorporation of precursors multiply-labelled with 2H,1-*C and 180. Among the compounds under study are meroterpenoids like the austin, andibenin B and terretonin (in collaboration with Dr.T.J. Simpson, University of Edinburgh). A variety of one and two dimensional NMR techniques permit facile spectral assignment and location of labelled sites. A new method to determine relative stereochemistry of deuterium labelling is described which employs heteronuclear *H,13C NMR shift correlation spectroscopy in conjunction with deuterium decoupling. The technique allows detection of deuterium-labelled hydrogens in cases where ^H NMR fails because of resonance overlap.

S42 CONFORMATIONS OF MYCOTOXINS UTILIZING X-RAY DIFFRACTION AND MOLECULAR MECHANICS TECHNIQUES

WILLIAM H. WATSON Professor of Chemistry Department of Chemistry, Texas Christian University Box 32908, Fort Worth, Texas 76129

The solid state structures of mycotoxins can be determined by X-ray diffraction techniques. In general, the solid state structure is also the minimum energy conformation of the isolated molecule; however, this may not be true for flexible molecules such as macrocyclic mycotoxins. The conformation of a flexible mycotoxin at an active site may also differ from that of theisolated molecule. Molecular mechanics is a method by which the low energy conformations of a molecule can be explored. At present there is no general procedure for finding systematically all possbile conformations and considerable reliance must be placed upon the experience and intuition of the investigator. Studies of the low energy conformations may be useful in the aiappinrjof receptor sites or in relating the activities of a series of substrates. The structures of several macrocyclic mycotoxins determined by X-ray diffraction and molecular mechanics techniques will be presented. Alternate low energy conformations will be explored and the factors affecting conformational energies and barriers between conformations will be discussed.

S43 KINETIC PULSE LABELING IN THE BIOSYNTHESIS OF MYCOTOXINS

LOLITA 0. ZAMIR Professor of Chemistry Universite' du Quebec, IAF, 531 des Prairies Blvd., Laval (QC) Canada H7N 4Z3

The major metabolites produced by Fusarium culmorum are 3-acetyl-deoxynivalenol (I), sambucinol (II), culmorin (III) and 7 a-8 e-dihyclroxyca)enectrin (IV).

The biosynthesis of these compounds have been investigated in our laboratory. A kinetic pulse labeling experiment led to interesting intermediates. The structures of these putative precursors and the mechanisms of their conversion to 3-acetyldeoxynivalenol will be discussed.

S44 BASIC ASPECTS OF SECONDARY METABOLISM IN RELATION TO TOXIN PRODUCTION

B1-B7 EFFECT OF WATER ACTIVITY (A ) ON THE PRODUCTION OF ASTELTOXIN w BY EMERICELLA VARIECOLOR (BERK. ET BR.)

AMELIA E DE JESUS1, R M HORAK, ELLEN M LAWSON2 AND MRUDULA PATEL2 D. PHIL 1NCRL CSIR Box 395 PRETORIA 2 Dept of Microbiology, University of the Witwatersrand Box 1176 JOHANNESBURG

Emericella variecolor produces asteltoxin on 3% malt extract agar but not on 3% malt extract broth. A study of asteltoxin production by Emericella variecolor at 25°C showed that asteltoxin is produced within a narrow range of water activities.

P1 EFFECT OF DIFFERENT ANTIOXIDANTS AND FREE RADICAL SCAVENGERS ON AFLATOXIN PRODUCTION

C.FANELLI0, A.A.FABBRI". S.PIERETTI0, E.FINOTTI°°and S.PASSI00 0 University Researchers; oo Hospital Researchers ° Dipartimento di Biologia vegetale,Universita di Roma'La Sapienza' Largo Cristina di Svezia 24 00165 Roma,Italy We have previously shown that epoxides, lipoperoxides and haloge- nated free radicals markedly enhance aflatoxin production when incubated with Aspergillus parasiticus 'in vitro1. Following these results, we have analysed the effect of different antioxidants and free radical scavengers on aflatoxin production. The different compounds at different concentrations were used: buthylated hydro- xyanisole (BHA)0.05%,0.01%,0.02% w/v; buthylated hydroxytoluene (BHT)0.01%,0.02% w/vr tocopherol(vitamin E)0.1%w/v; ascorbic acid 0.1% w/v; sodium thiosulphate 0.05% , 0.1%,0 . 2% w/v; reduced glutathione 0.2% w/v; cysteine 0.2% w/v; mercaptosuccinic acid 0.2%w/v; cysteamine 0.2, 0.1% w/v. The above compounds were tested in culture of Aspergillus parasiticus supplemented with carbon tetrachloride, a potent stimulating agent of aflatoxin output. The production of aflatoxins was analysed by HPLC on RP-18 column. Cysteamine(0.2% w/v), sodium thiosulphate(0.1% w/v), BHA(0.01%w/v) highly inhibited the aflatoxin production induced by carbon tetrachloride; the inhibition decreased by lowering the concentration. On the contrary, vitamin E(0.1% w/v).vitamin C(0.1% w/v),reduced glutathione(0.2% w/v),cysteine(0.2% w/v)and mercaptosuccinic acid (0.2% w/v) further enhanced the carbon tetrachloride stimulating effect. The addition of the above compounds did not significantly affect the growth of the fungus. BHA and thiosulphate are then promising drugs to solve the problem of aflatoxin contamination.

P2 MICROSOMAL AND MITOCHONDRIAL INVOLVEMENT IN THE PRODUCTION OF AFLATOXINS INDUCED BY CARBON TETRACHLORIDE IN CULTURES OF ASPERGILLUS PARASITICUS o S . PASS I *M. NAZ Z ARO-PORRO* E. FINOTTI *G. PANFILI * A. A. FABBRFand C .FANELLI *Hospit^l Researchers; ° University Researchers * Istituto San Gallicano (IFO) , Via San Gallicano 25a,00165 Roma; ° Largo Cristina di Svezia 24 00165 Roma, Italy Carbon tetrachloride(CCl ) highly stimulates aflatoxin production when incubated in cultures of Aspergillus parasiticus. We postulated that the toxic free radical intermediate CCl • , formed by the action of cytochrome P-450 of the fungus on CCl , and consequently, the lipid peroxidation of membrane structural lipids in which cytochrome P-450 is embedded, could be the key events of the stimulation. Such hypothesis requires two basic conditions: 1) potent free radical scavengers such as cysteamine must inhibit the aflatoxin output induced by CCl ; 2) the addition of CCl to cultures must alter the fatty acid(FA)composition and unsaturated/saturated FA (U/S x FA)ratio of membrane lipids, particularly phospholipids (PL) of microsomes and partly of mitochondria of A. parasiticus. In the present work we have studied either the production of aflatoxins or FA composition and U/S x FA ratio of microsomal and mitochondrial PL of Aspergillus parasiticus in cultures supplemented with 0.5% v/v CCl in the absence or in the presence of 0.25% w/v cysteamine. Analyses of both aflatoxins and FA were performed by HPLC on RP-18 column with different eluent systems. Mitochondria and microsomes were fractionated by centrifugation of homogenized cells of fungus. Cysteamine was capable of fully inhibiting the production of aflatoxins induced by CCl.. The values of U/S x FA ratio of PL of microsomes and mitochondria were respectively 83.6 and 51.0 for control cells of A.parasiticus; 3.6 and 7.9 respectively in the case of cultures supplemented with CCl .The addition of cysteamine to CCl reverted the values to those of control cells. The above results would confirm the role played by microsomal enzymes of the fungus in the aflatoxin production induced by CCl,.. 4

P3 THE EFFECT OF HEAT AND RADIATION ON GERMINATION AND OCHRATOXIN PRODUCTION OF SCLEROTIA OF ASPERGILLUS OCHRACEUS.

N. PASTER1, R. BARKAI-GOLWAND R. PADOVA2

department of Stored Products, ARO, The Volcani Center, P.O.B. 6, Bet- Dagan 50250, Israel. 2Soreq Nuclear Research Center, Yavne, Israel. Aspergillus ochraceus NRRL 3174 is a sclerotia forming fungus which is capable of producing ochratoxin, a potent nephrotoxin. Ochratoxin content of sclerotia collected from /L ochraceus inoculated wheat grains was significantly higher than that found in sclerotia produced on synthetic medium (SM) (25 |jg vs. 1,75 ug/lg sclerotia respectively). However, when sclerotia harvested from grains were grown for 14 days on SM the amount of ochratoxin produced by the colonies formed was 15 ug/lOml of medium as compared with 55 yq/lOmi medium produced under the same growth conditions by sclerotia originating from the SM. Percentage of germination of sclerotia collected from SM and exposed to efther irradiation (25 or 50 Krad) or heat treatment (60° for 15 or 30 min) was almost identical to that of the untreated sclerotia (80%). Exposure of sclerotia from the SM to combined treatment of irradiation plus heat caused a significant reduction in germination (20-25% of the control). All of the above treatments (irradiation, heat or irradiation + heat) failed to cause any reduction in germination of sclerotia originating from wheat grains. A 25 or 50 Krad dose applied to sclerotia collected from grains as well as the heat treatment alone (60° for 30 min) did not affect ochratoxin yields from colonies developed from the treated sclerotia on SM. The combined treatments of heat plus radiation with 25 or 50 krad applied to sclerotia increased ochratoxin production in colonies developed from these sclerotia to up to 60-80% over the control. However, mycelial growth of the colonies (based on dry weight) was very similar to that of the control.

P4 PRODUCTION OF FUSARIN C IN LIQUID CULTURE

J.M. FARBER, G.W. SANDERS AND P.M. SCOTT B.Sc.,M.Sc..Ph.D. B.Sc. M.A., Ph.D. Food Directorate, Health Protection Branch Tunney's Pasture, Ottawa, Ontario K1A 0L2 Fusarium moniliforme is a major parasite of several Gramineae such as corn, rice, sugarcane and sorghum and is widespread in humid and sub-humid temperate zones and sub-tropical and tropical zones throughout the world. The organism, as well as being highly toxic to experimental animals, has been implicated in the etiology of esophageal cancer.

Recently a highly mutagenic compound, fusarin C, was isolated from a culture of the organism growing on corn. Since there is no information at present on the production of fusarin C in liquid culture or on the potential of North American isolates to produce the compound, a liquid culture medium suitable for screening potential fusarin C producers was developed.

Major factors which stimulated the production of fusarin C included pH, reduced 0~ levels, and possibly a low concentration of organic nitrogen. Of 7 sugars tested, sucrose and glucose appeared to be the best carbohydrate sources for mycotoxin production and levels of fusarin C greater than 10 ppm in liquid culture were obtained (28 C, 7 days). A time course study of fusarin C production was done over a 21-day period measured against pH, sugar concentration, nitrogen levels and fungal biomass.

Of the F. moniliforme isolates tested, 9/10 produced fusarin C in liquid culture, while none of 7 isolates of _F. moniliforme var. subglutinans studied was found to produce the compound. Additionally, fusarin C was not found in the growth medium of 10 F. graminearum isolates tested but was detected in corn cultures of this species.

P5 ZEARALENONE PRODUCTION BY FUSARIUM SPECIES ISOLATED FROM SOYBEANS

G VAAMONDE, N B BONERA and G T SCARMATO

Dr en Ciencias Qulmicas, Universidad de Buenos Aires Facultad Ciencias Exactas y Naturales, Dpto. Qulmica OrgSnica, Microbiologia de Alimentos, C. Uhiversitaria, Pabell6n II, 3°P, 1428 Buenos Aires, Argentina

To evaluate the incidence of species of Fusarium in soybeans and their ability to produce zearalenone, 936 beans belonging to samples of several varieties were analyzed. Fusarium species were present in the 5% of the beans. 43 isolates were tested for zearalenone production on autoclaved moist rice (moisture content 60%) incubated for two weeks at 25"C followed by eight weeks at 12°C. 41,8% of the strains were toxicogenic. They belonged to the following species (number of producers/number tested): F. equiseti (5/11), F. semitectum (13/24), F. moniliforme (0/6) and Fusarium spp (0/2). Two high- producing isolates, F. equiseti 1-80 and F. semitectum 4-63, produced on rice 2050 and 1370 ppm respectively. The same strains were inoculated on soybeans at different levels of water activity (aw):0,934; 0,967; 0,976; 0,981; 0,986 and 0,997 (moisture contents between 202 and 602). Although the fungus grew vigorously on soybeans with a 0,967 and higher, none of the two isolates produced a detectable amount of zearelenone on them. These data confirm the results obtained by other workers with F. roseum and suggest that soybeans are not a good substrate for the biosynthesis of zearalenone by these species.

P6 EFFECT OF CULTURE CONDITIONS ON THE TOXICITY AND CHEMICAL COMPOSITION OF THE TOXIN OF MICROCYSTIS AERUGINOSA (UV 006)

A J VAN PER WESTHUIZEN, J N ELOFF, G H J KRiiGER PhD Dept. of Botany, UOFS, Bloemfontein, 9300, South Africa

The effects of culture age, pH of medium, temperature and fluence rate were studied under controlled laboratory conditions on the growth, toxicity and toxin composition of axenic M. aeruginosa (UV 006), a South African blue- green algal strain from the Hartbeespoort Dam in the Transvaal. Different growth conditions greatly influenced the toxicity of M. UV-006 cells. Optimal conditions for growth did not coincide with optimal toxin production in all cases. Temperature had the most pronounced effect on the toxicity of the cells. The Microcystis toxin consists of a mixture of closely related toxic peptides of approximately similar molecular masses (ca. 1000) as determined by gel chromatography. More than 90% of the toxin consisted of two major peptides except for cells grown at 16CC. In this case a third major peptide was detected. . . . .

Changes in growth conditions often resulted in a change in the relative peptide composition of the toxin. Different growth conditions did not affect the amino acid composition of the toxic peptides. Changes in toxicity was probably mainly due to changes in the concentration of the constituent peptides and evidently not to changes in potency of the toxic peptides. The effect of a change in peptide composition per se on the toxicity of the toxin is still uncertain.

P7 BIOSYNTHESIS OF MYCOTOXINS AND PHYCOTOXINS

C1-C6 O-METHYLTRANSFERASES AND AFLATOXIN BIOSYNTHESIS

R K BERRY AND M F DUTTON M 5c (Natal) Department of Biochemistry, University of Natal P 0 Box 375, Pietermaritzburg, 3200

Sterigmatocystin, when incubated with cell-free extracts of Aspergillus parasiticus 1-11-105 Whl, is converted to aflatoxin B and O-Methylsterigmato- cystin. This observation has prompted an investigation into the role of methyltransferase enzymes in the biosynthesis of aflatoxin B .

The methyltransferase enzyme responsible for the 0-methylation of sterigmatocystin is being isolated and characterized, and this data will be presented. T'lic use of the purified nietli,y itr-ii'ioTt* ase as a probe in the elucidation of the mechanism of conversion of the anthraquinone moeity of versicolorin A to the xanthone derivative of sterigmatocystin will be discussed.

•V

P8 METABOLISM OF AFLATOXIN Bo BY ASPERGILLUS FLAVUS

M F DUTTON Ph D Department of Biochemistry, University of Natal P 0 Box 375, Pietermaritzburg, 3200

Aflatoxin B was originally shown to arise by chemical reaction from aflatoxin B ;a water being added across the terminal double bond in the dihydrobisfuran system of aflatoxin B.. Consequently aflatoxin B_ has been regarded as nothing more than an artifact having no role in aflatoxin metabolism.

On addition of labelled aflatoxin B? accumulating strain of Aspergillus flavus the added labelled compound rapidly disappeared and the activity was relocated in the aflatoxin B fraction by radioautography. Repeated attempts to separate the activity from aflatoxin B_ by thin layer chromatography failed and it was concluded that aflatoxin B? is metabolised by this strain of A flavus to aflatoxin B .

The implications of this finding may be quite significant as the reaction provides a link between aflatoxin B and B via B . Consequently aflatoxin B may well be a true intermediate in this sequence and similar hemiacetals such as those derived from versicolorin A and sterigmatocystin, may also have a role in aflatoxin biosynthesis.

P9 THE EFFECT OF ETHANOL ON THE METABOLITE PRODUCTION OF ASPERGILLUS USTUS

A.E. DE JESUS, R.M. HORAK, P.S. STEYN and R. VLEGGAAR

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa

Aspergillus ustus (MRC 1163) is a prolific producer of secondary metabolites, viz, the austalides, austocystins, and the pigments averufin and versicolorin C. The addition of sublethal doses of ethanol to growing cultures of the fungus severely inhibits the production of these metabolites and induces the formation of a new metabolite, ethyl 2,4-dihydroxy-6-ethyl-3-methylbenzoate (1). The corresponding methyl ester is formed if methanol is substituted for ethanol, whereas the addition of propanol results in a mixture of ethyl and propyl esters. The substitution pattern of the benzoate (1) was determined by selective population inversion (5PI) C n.m.r. experiments.

The biosynthetic origin of the metabolite (1) was studied using [l- Cj- and [1- 3C,"Hjjacetate, and (2S)-[methyl- C]methionine as precursors.

P10 ISOLATION, PURIFICATION, AND CHARACTERIZATION OF EC OXIDASE THAT TRANSFORMS EREMOFORTIN C TO PR TOXIN

Y. H. WEI, S. C. CHANG, AND R. D. WEI Ph.D. M.S. Ph.D. Department of Biochemistry, National Yang-Ming Medical College 155 Li-Long St., Sec. 2, Shih-Pai, Taipei, Taiwan 112, R.O.C. Eremofortin C and PR toxin are secondary metabolites of PznitUZLLum ^ohZi. These two compounds are secreted into the culture medium by the fungi, and the peak amount of EC occurs earlier than that of PR toxin. The decrease in the amount of EC is always associated with a rapid increase in PR toxin production. Recently, we discovered the enzyme that transforms EC to PR toxin in the culture medium that had been grown with the fungi. The enzyme started to appear from the 7th day after inoculation and reached to the maximum on the 13th day, which corresponded to the maximal production of PR toxin in the medium. The enzyme was isolated and purified from the culture medium by a procedure that involved ammonium sulfate fractionation and DEAE-cellulose column chromatographies. The molecular weight of the enzyme was estimated to be about 40,000 daltons. The optimal pH for the enzyme reaction was about pH 5-6. The enzyme reaction was temperature-dependent, and the optimal reaction temperature was between 30 to 40 C. The K^ and Vmax of the enzyme as determined at 30°C were 0.02 mM and 4.0 ymol/min/mg, respectively. The enzyme did not require either NAD* or NADP+ as coenzyme to carry out the transformation of EC to PR toxin. However, the optical spectra of the purified enzyme preparations exhibited an absorption peak at about 450 nm, which indicates that the enzyme is a flavoenzyme. Moreover, we found that during transformation of EC to PR toxin as catalyzed by the enzyme hydrogen peroxide was produced as that found in glucose oxidase reaction. We have thus concluded that the enzyme participates in the oxidation of eremofortin C to PR toxin, and that the enzyme is a flavin-containing oxidase. We tentatively name the enzyme EC oxidase.

P11 13C NMR STUDY OF THE BIOSYNTHESIS OF SECONDARY METABOLITES OF FUSARIUM CULMORUM

B.A. BLACKWELL, J.D. MILLER, R. GREENHALGH AND ]L. ZAMIR

Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Ont. K1A 0C6. Institute Armand-Frappier, Universite du Quebec, Ville de Laval, Quebec H7N 4Z3 C NMR spectroscopic investigations of the biosynthesis of secondary metabolites produced by3Fusariunuculmorum (CMI 14764) were carried out through the incorporation of [ C-l J, L C-3J and [ C-1,2] sodium acetate. A variety of secondary metabolites were produced by this fungus, of which 3-acetyldeoxynivalenol, dihydroxycalonectrin, sambucinol and culmorin are the most abundant. Butanolide was also produced, but only in small quantity. The addition of several aliquots of labelled precursor at the onset of stationary phase of the culture, resulted in high enrichment {8-10 fold) in the four major metabolites. The C NMR spectra of the metabolites were unambiguously assigned with 2D NMR techniques. The specific incorporation pattern in the major metabolites was determined by C NMR analysis of crude extracts of the culture medium. The enrichment patterns of the trichothecenes and sambucinol,are,consistent with the condensation of three mevalonate units. The C- C couplings observed between C-5 and C-l2 and C-6 and C-l5 of these species confirm the current hypothesis that the trichothecenes are derived via a common biosynthetic pathway. Culmorin is also enriched in a manner consistent with a mevalonate origin. Fungal extracts enriched with [ C-1,2] acetate and analysed by C homonuclear 2D NMR gave complimentary information to that from specific enriched precursors. The incorporation sites of intact acetate units within each molecular species indicate the manner in which the trichothecene structure is formed from farnesyl pyrophosphate. The results are discussed in terms of competition for the labelled precursors in an organism whose secondary metabolic pathways are predominantly mevalonate in origin and are compared with F. graminearum, in which mevalonate, polyketide and amino acid biosynthetic pathways are active.

P12 BIOSYNTHESIS OF 3-ACETYLDE0XYNIVALEN0L AND OTHER METABOLITES BY FUSARIUM CULMORUM IN A STIRRED JAR FERMENTOR

J.D. MILLER AND B.A. BLACKWELL

Chemistry and Biology Research Institute, Agriculture Canada Ottawa, Ont. Canada.

The formation of 3-acetyldeoxynivalenol (ADON) and other secondary metabolites of Fusarium culroorum (CMI 14764) including culmorin, sambucinol, sambucoin and 3,15 diacetoxy -7,8,dihydroxy 12,13-epoxytrichothec-9-ene in a stirred jar fermentor is described in relation to nutritional (nitrogen, sucrose, glucose, fructose, glycerol), physical (pH, 0-) and cellular parameters (hyphaKdry weight, protein, isocitrate dehydogenase activity). The addition.,of [ C-l] sodium acetate to the fermentation was used to demonstrate with C NMR techniques that the conditions used result in the biosynthesis of one compound, ADON, at the expense of other primary and secondary metabolites. Biosynthesis of ADON was apparently induced by nitrogen limitation. At the end of the fermentation ca. 710 mgL* ADON was obtained.

P13 STRUCTURE AND CHEMICAL PROPERTIES OF MYCOTOXINS AND PHYCOTOXINS

E1-E11 13-CARBON AND PROTON RESONANCES ASSIGNMENT IN LEUCINOSTATIN A

C.G. CASINOVI, L. RADICS, C. ROSSI, L. TUTTOBELLO

DIRETTORE DEL LABORATORIO DI CHIMICA DEL FARMACO

ISTITUTO SUPERIORE DI SANITA' - VIALE REGINA ELENA, 299 - 00166 ROMA

LEUCINOSTATINE A is a peculiar peptide, containing (from the amino end) one

residue of 4-Me-Pro, one of 1-NH , 4-Me, 6 OH, 8 oxo-decanoate, one of 3-OH-

Leu, one of •£.-Aba, two of Leu, two of o^-Aba and one of JS-Ala; the amino end

is "naturally protected" by a 4-Me, hexen-2-oate residue and the carboxyl end

by 2-araino, 3-dimethylamino, 2-propyl group. It is a compound particularly

active against P388/S cells cultures. A complete assignment of carbon and

proton chemical shifts is reported, basing on COSY, RELAYH (400 MHz) HETCOR

(300/75 MHz).

P14 STRUCTURE/ACTIVITY RELATIONSHIPS OF THE FUSARINS

W.C.A. GELDERBLOM, P.G. THIEL, K.J. VAN DER MERWE

National Research Institute for Nutritional Diseases, S.A. Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa.

A mutagenic compound, fusarin C, was isolated and purified from culture material of Fusarium moniliforme strain MRC 826 using the Salmonella/micro- some mutagenicity assay as a monitoring system (1). Fusarin C occurs naturally on maize and is also produced by £. graminearum in culture (2).

Structural and biological studies on fusarin C and related mutagenic and non-mutagenic forms revealed that the C _-C epoxide is likely to be involved in the biological activity of fusarin C (3, 4). The chemical and enzymatic reactions of this epoxide with glutathione lead to inactivation of fusarin C i_n vitro.

The mode of activation and deactivation will be discussed in relation to the chemical structures of the fusarins.

1. Gelderblom, W.C.A., Thiel, P.G., Van der Merwe, K.J., Marasas, W.F.O. and Spies, H.S.C. Toxicon 2X_, 467 (1963).

2. Gelderblom, W.C.A., Thiel, P.G., Marasas, W.F.O. and Van der Merwe, K.J. J. Agric. Food Chem. J2^, 1064 (1984).

3. Gelderblom, W.C.A., Marasas, W.F.O., Steyn, P.S., Thiel, P.G., Van der Merwa, K.J., Van Rooyen, P.H., Vleggaar, RV and Wessels, P.L. J. Chem. Soc. Chem. Commun. 122 (1984).

4. Gelderblom, W.C.A., Thiel, P.G. and Van der Merwe, K.J. Biochem. Pharmacol. 33, 1601 (1984).

PIS STRUCTURE ELUCIDATION OF THE FIRST NATURALLY OCCURRING TRICHOTHECENE GLYCOSIDE, A METABOLITE OF FUSARIUM SULPHUREUM

C.P. GORST-ALLMAN, P.S. STEYN AND R. VLEGGAAR (National Chemical Research Laboratory, CSIR, P 0 Box 395 Pretoria OOO1 South Africa)

AND C.J. RABIE (National Research Institute for Nutritional Diseases, S.A.M.R.C, P 0 Box 70, Tygerberg 7505, South Africa)

The structure elucidation of the first naturally occurring trichothecene glycoside, a metabolite of Fusarium sulphureum, is described. The compound is a glucoside derivative of monoacetoxyscirpenoi, and possesses interesting biological properties. It is considerably less toxic that its parent trichothecene. The structure elucidation is based on *H and 13C n.m.r. spectroscopy, and some chemical reactions of the metabolite.

P16 UNIQUE INSECTICIDÄL rïYCOTOXIN FROM ASPERGILLUS MET-T Fire YUKAWA ISOLATED FROM CADAVER OF SILKW3RM, BGMBYX MORI I,

TOSHICHIKA OHTOMO, HIROSHI SATO AND KOSAKU YOSHIDA (Ph. D, M. D) (M. S) (M. D)

Départirent of Microbiology, St. Marianna University School of Medicine, 2095 Sugao, Miyamaye-ku, Kawasaki 213, Japan

Nine strains of Aspergillus nelleus isolated from naturally infected cadavers and feoes of the silkworm (Bortoyx nori L) showed high pathogenicity and oral toxicity to the insects in their culture broth. Five strains of A. melleus produced unique insecticidal mycotoxin on a liquid medium. This substance was designated as mellenatoxin A. The substance was purified by chloroform- rrethanol fraction, sephadex-G 10, and thin-layer chromatography. Chemical properties and IR, UV and mass-spectra of the substance were discussed in terms of its chemical structure. From the cultures of 10 Fenbach flasks, 380 mg of pure substance was purified showing [oCJn23 74.0 C(G=1.0 MetCH) and a maximum absorption at 300 nm by UV. In the IR spectrum, absorption was observed at_3330, 2950, 1640, 1520, 1420, 13R0, 1260, 1150, 1090, 1000, 920, and 840 cm" suggesting the presence of peptide likages. Analysis hydrolysate of mellenatoxin A by amino acid analyzer demonstrated the following composition : N-methylvaline, N-methylleucine, and N-methylalanine. Particularly, the amide (C=0) absorption at 1640 cm" and the NH absorption at approximately 3330 cm"1, would probably be characteristic of cyclic peptides. This materials melting point is greater than 137 C and it reacts with chlorine-0 tolidinepotassium iodide, P-anisalaldehyde- H_S0. and ethanolic ferric chrolide exhibiting a pinkish color but is negative to ninhydrin. A negative anthrone reaction of this substance denotes a presence of carbohydrate in the molecule. Little structural information can be derived from the mass spectrum. These results indicate that the unique insecticidal mycotoxin weis different from the other insecticidal substances. Insecticidal activities of mellenatoxin A were examined in different insects. With the typical application each sample could be dissolved in a definite amount of ethylacetate. Even at a dose of 2 ppm, mellenatoxin A caused immediate poisoning manifested by sudden weakness, shock -like manifestation and occasionally followed by death. Also, mellenatoxin A caused the death of final inster larvae of silkworm in 30 hrs, by oral adminstration at a dosage of 1 ppm and other insects such as Arctia hyphantria, Aprina germari and Periplaneta suliginesa were similar effect. However, mellenatoxin A did not show any significant toxic activity in mice even, at a high dose of 160 mg of the substance either by oral administration or intraperitoneal injection. It is well known that numerous isolates of genus Aspergillus are pathogenic to the silkworm, Bombyx mori I, and other insects. This paper describes the isolation, purification and some biological and biochemical properties of mellenatoxin A from A. malleus.

P17 QUANTUM CHEMICAL STUDIES OF AFLATOXIN Bj, STERIGMATOCYSTIN AND VERSICOLORIN A, AND A COMPARISON WITH THEIR MUTAGENIC ACTIVITY

RUTH PACHTER AND PIETER S. STEYN

NCRL, CSIR, BOX 395, PRETORIA

Aflatoxin Bj exhibits significant mutagenic, carcinogenic and cellular transformation ability, coupled to the ability to bind covalently to nucleic cells. However, structural differences of the moieties attached to the bisdihydrofuran, e^. coumarin in the case of aflatoxin Bi(AFBi), xanthone for sterigmatocystin (ST), and anthraquinone for versicolorin A(VA) markedly affect the carcinogenity and mutagenicity of these molecules.

Semi-empirical INOO calculations of atomic charges and bond orders were performed for AFB \, ST and VA, and were analysed in order to establish a possible structure-activity relationship for aflatoxin Bi and its biosynthetic precursors. No significant variation is observed in the calculated Pc-2-C-3 bond orders for AFBi, ST and VA. The reduced biological activity of ST and VA compared to AFBi is, therefore, not related to a reduced susceptibility to epoxidation of the 2,3-bond. However, the proximity of a higher electron density to the C-2 carbon atom in AFBi, will result in a relatively higher stability of the AFBi carbocation formed from the corresponding epoxide, than the respective ones of ST and VA, accounting at least partially for the higher biological activity of AFBj. The electronic effects do not explain the observed differences in mutagenicity and toxicity. In the case of these substances the ease of epoxidation of the C-2-C-3 vinyl ether bond must be related to how these substances fit the active enzyme sites.

P18 UNEXPECTED CYCLIZATION UPON OXIDATION OF DIPLODIATOXIN

M. POTGIETER and P.S. STEYN

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, R5A

Diplodiatoxin (1), a toxic metabolite isolated from Diplodia maydis, was subjected to oxidation with osmium tetroxide and sodium periodate. In contrast to the starting material, the major product formed (2) lacked carbonyl functions (IR, NMR). The minor component of the product mixture contained a single carbonyl group and was reduced in molecular mass.

Compound (2) was identified by proton spin-spin correlated spectroscopy (COSY) to be a tetracyclic system, geminally substituted at C-2 with methyl and hydroxyl groups. The minor product (3) contained C resonances indicative of hemi-ketal and anomeric carbons, which suggested the presence of two aldehyde functions before cyclization. This indicated sugar-like degradation of the carbon chain in the presence of sodium periodate.

CH,

HO CH3 CH3COOH

P19 CHEMICAL REACTIONS OF THE PENITREMS

P.S. STEYN, F.R. VAN HEERDEN, AND R. VLEGGAAR

National Chemical Research Laboratory, CSIR, P.O. Box 395, Pretoria OOO1, Republic of South Africa

The penitrems constitute a group of closely related complex neurotoxins produced by PeniciIlium crustosum. Results obtained upon the treatment of tetrahydropenitreni A with RuOi,, and those obtained upon reaction of penitrem A with LiAlHi,will be presented. The penitrems are extremely labile upon exposure to mineral acids. The preliminary findings on the structures of the degradation products will be reported.

Penitrem A

P20 TRICHOTHECENES FROM FUSARIUM COMPACTUM

CM. MAES, R.M. HORAK, W.F.O. MARASAS*, C.J. RAB1E*, P.S. STEYN AND R. VLEGGAAR National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa

•National Research Institute for Nutritional Diseases, South African Medical Research Council, P.O. Box 70, Tygerberg 7505, Republic of South Africa

Fusarium conpactum is a frequent contaminant of agricultural commodities such as millet and maize. The toxinogenic isolate, MRC 1293, was cultivated on sterilized maize and the toxic principles removed from the mouldered material by solvent extraction. The subsequent isolation processes were guided by bio-assay in ducklings and brine shrimp larvae. The acute toxigenicity of this strain of F. compactum can be ascribed to the presence of a large number of trichothecenes of the neosolaniol type. The structural elucidation of these compounds is based on the complete assignment of their high-field H and 3C n.m.r. data, using two-dimensional ( H, H) and ( 3C, H) correlation spectroscopy.

P21 SOME SECONDARY METABOLITES OF FUSARIUM CULMORUM AND F. ROSEUM

R. GREENHALGH, ]R.-M. J1EIER, D. LEVANDIER, B.A. BLACKWELL, J.D. MILLER, ^A. TAYLOR AND 'j.W. APSIMON

Chemistry and Biology Research, Institute, Agriculture Canada, Ottawa,Ont. ARL/NRC, Halifax, N.S., and Carleton University, Ottawa, Ont. Canada.

Both Fusariuro culmorum (CMI 14764) and F. roseum (ATCC 28114) have been used to produce 3-acetyldeoxynivalenol (ADON) orPa large scale in liquid culture. The crude fungal extract remaining after removal of ADON, contains a variety of other minor secondary metabolites, some of which have been isolated using ,, various chromatographic techniques and characterized by their MS and H and C NMR spectra. Several known metabolites, including butenolide, culmorin, culmorone, sambucoin and sambucinol were found to be common to both species. Zearalenone was detected only in the crude fungal extract of F^ roseum. In addition to ADON which is a type B trichothecene, eleven minor type A trichothecene metabolites have been isolated. They include hydroxylated derivatives of isotrichodermin and calonectrin, together with deacetylated derivatives of calonectrin. Both the type A and B trichothecenes isolated from these two species have an oxygen at the C-3 position, either as a hydroxy or acetyl moiety. No C-3, C-4 oxygen substituted compound was found under the experimental conditions employed, which are optimized for ADON production. This fact sugests that both fungal species are 3-oxytrichothecene producers, a fact which may have chemotaxonomic significance.

P22 REDUCTION IN LEVELS OF DEOXYNIVALENOL AND ITS FATE IN CONTAMINATED SOFT WHEAT AFTER CHEMICAL TREATMENT

J.C. YOUNG, \. SUBRYAN AND 1D. POTTS

Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Ont. Diversified Research Laboratory, Toronto, Ont., Canada.

Various chemical reagents have been tested to reduce the levels of deoxynivalenol (DON) in contaminated soft white winter wheat. Of those tested, sodium bisulfite effected the greatest reduction of DON. The extent of the reduction was dependant upon the concentration and contact time. Flour from the milling of bisulfite treated wheat contained only low amounts (ca. 5%) of that in untreated wheat. When the treated contaminated flour was baited into a variety of products, DON reappeared at a level about half of that originally present in the wheat. Studies with pure mycotoxins revealed that type B trichothecenes rapidly form sulfonite products by reaction with the keto moiety. These sulfonite products are stable under acid conditions. However, under alkaline conditions, they revert to the parent compounds, especially at higher temperature and pH. In the presencen of 0.1M NaOH at 75 C, DON rapidly rearranges and degrades to at least five products, of which some are transient. The characterization of these skeletally modified products will be discussed.

P23 SOME MYCOTOXIN STRUCTURES DETERMINED BY X-RAY CRYSTALLOGRAPHY AT THE NCRL

J.L.M. DILLEN and P.H. VAN ROOYEN

NCRL, CSIR, P 0 Box 395, PRETORIA OOO1 Republic of South Africa

The structures of six mycotoxins that were determined by using single crystal X-ray crystallography, will be presented. These mycotoxins include rhizonin A, a novel cyclic heptapeptide from Rhizopus microsporus; fusarin C, a mutagen from Fusarium rooniliforme; diplosporin, the mycotoxin produced by Diplodia macrospora; ochratoxin A, the nephrotoxin from Aspergillus ochraceus; asticolorin A, a new type of mycotoxin from Aspergillus multicolor; and austalide A, produced by Asperqillus ustus. The structural elucidation of asticolorin A (2.C33 H3^07*3 MeCOMe ^OJby crystallography was a major achievement since the asymmetric unit contained 93 non hydrogen atoms and the two toxin molecules were linked by acetone and water molecules.

The solid state conformations as well as other structurally significant features of these toxins will be described.

Asymmetric unit of asticolorin A

P24 ANALYSIS OF MYCOTOXINS AND PHYCOTOXINS

G1-G18 !j\PID I.ET!;OE FOR TIE DSTSIiriKATIC" CF I-'ACEOCYCLIC TRICKOT!IECi:i:E TOXIITS Hi FIELD 5AITL2S

ARPAE BATA.AITBKAS VAIIVI

Technical I.'niversity Budapest,Department of Biochemistry and Pood Technology, 11-1521 BUDAPEST, T.O.BOX 91, FJHOARY It is generally accepted and widely known that the stachybotriotoxicosis is caused by macrocyclic trichothecene toxins. Due to the heterogeneity of toxins produced by Stachybotrys atra /minimum four different highly toxic inacrocyclic trichotheccnc toxins are produced/ determination of a very low concentration for each compound is needed to the no effect level. According to our experience the toxic level in case of naturally occuring toxicosis lies between 0.2-0,5 ppm /the sum of the amount of four different toxins aatratoxins H and G, verrucarin J, roridin E/. Accordingly the no effect level must be for each toxin 0.01 ppm or below. Determination of these compounds at such a low concentration range is very difficult,expensive and claims high experience with chemical methods.

To find an accurate but less complicated method of determination wo used after extraction of the toxins a transesterification procedure. As the re- cult of this reaction all the toxins mentioned above were transformed to verrucarol. Determination of verrucarol in this case gives the sum of the four nacrocyclic trichothecenes.

The new rapid method /extraction of samples with methanol treatment of extract with petroleum ether, transesterification using sodium methoxyde reagent, purification by silicagel column ciiromatography, TLC determination or sylilation with Ms /chloromethyl/-tetramethyl-disilazane reagent and ECTi-GLC determination/ was used for screening field samples. The detection limit of the verrucarol using TLC technique is about 0.05 ppm. In case of capillary GLC with specifice detector /ECD/ detection limit is 0.C1 ppm. About 20 samples v/cre detected causing stachybotriotoxicosis among horses, sheeps and calves.

P25 A Monoclonal Antibody Based Enzyme Immunoassay for Aflatoxin 8.

A.A.G. Candlish, W.H. Stimson and J.E. Smith

B.Sc; B.Sc, Ph.D.; B.Sc, Ph.D. Immunology Division, University of Strathclyde, 31 Taylor Street, GLASGOW. G4 ONR, Scotland.

A direct competitive enzyme-linked immunosorbent assay (ELISA) utilising a monoclonal antibody (McAb) specific for aflatoxin B, (AFB,) has been developed. The McAb was selected after fusion of the mouse x63.Ag8.653 myeloma cell line with spleen cells obtained from female NZB/Balb-C Fl hybrid mice. Selection of hybridomas secreting specific McAb was achieved by utilising an indirect non-competitive ELISA in which an AFB.-oxime derivative conjugated to bovine serum albumin was used as the solid phase antigen and the mouse was immunized via intraperitoneal injection with the AFB.-oxime derivative conjugated to keyhole limpet haemocyanin. Both AFB,-oxime protein conjugates were formed by the water soluble carbodiimide method.

Hybridoma cells secreting McAb specific for AFB, have been successfully grown in tissue culture, stored by freezing in liquid nitrogen and grown as ascites in histucunipaliult; rniee. The (NH.LSG, fraction of the ascites fluid was then conjugated to horse radish peroxidase by the two-step glutaraldehyde method. The McAb-peroxidase conjugate was then used at a dilution of 1:1,000 in a direct competitive ELISA for AFB,and the assay was shown to have a range of 0.2 to lOng/ml in a typical standard curve. Although the sensitivity of this assay is suitable, the range is rather narrow for quantitative analysis without serial dilutions of samples. Nevertheless it does allow distinct differences between positive and negative samples to be obtained.

The specificity of various aflatoxin metabolites for the assay system was measured by the 50% displacement method. Cross reactivity of G,,B2,M,,M2 and G2 for the assay was 15.25%, 14.18%, 7.41%, 5.46% and 2.05%, respectively. Thus, the assay system is very specific for AFB, and shows very little cross-reactivity for other aflatoxin metabolites likely to be found in natural substrates. The affinity of the McAb was determined using a procedure similar to the Farr assay and calculating the equilibrium affinity constant to be 1.3 x 10 litre mole" by the Langmuir adsorption isotherm. Thus, the affinity of the McAb is suitably high to allow it to be used in various immunoassay systems.

In conclusion, the assay system has suitable sensitivity and specificity for it to be applicable for the routine detection of AFBj in natural substances.

P26 THIN LAYER IMMUNOASSAYS FOR MYCOTOXINS

W.V. DASHEK, J.E. MAYFIELD, N.T. LAPPAS, C.E. O'REAR AND G.C. LLEWELLYN Assoc. Profs. Biol., Assoc. & Full Profs. Foren. Sei., Üir. Bur. Toxic Substances Atlanta Univ., Biol. Dept., Atlanta, GA 30314, GWU, Forensic Sciences, Washing- ton, DC, 20052 and Commonwealth of VA, Health Dept., Richmond, VA 23219

The trichothecenes are biologically-active fungal metabolites which can be synthesized both in vitro and in vivo by various Fusarium, Tricho- thecium, Trichoderma, Cephalosporium, Myrothecium, Stachybotrys, and possibly Dendrodochium species. Interest in the biological effects of T-2 toxin has been heightened as a result of its alleged use as a biological/chemical warfare agent. Therefore, it is necessary to develop rapid, inexpensive, reliable, accurate and sensitive assays for the toxin. Here, we propose an ionunological method for both the detection and quantification of T-2 toxin. This method, which is being revamped as a generic procedure for a variety of mycotoxins, involves the production of a monolayer of T-2 toxin within polyvinyl chloride microtiter wells. The subsequent steps include in sequence: a) washing and drying of the monolayer, b) incubation of the layer with bovine serum albumin (BSA), c) washing and drying of theBSA-toxin layer, d) incubation of the layer with anti-T-2 toxin and e) washing and drying of the anti-T-2 toxin- BSA-toxin layer. Finally, the toxin-antitoxin complex is both visual- ized and quantified via a water vapor technique. Both the historical development and value of the thin layer immunoassay as an analytical tool for quantifying picogram quantities of Xenobiotics as well as a mini- review of the chemistry, currently-available screening methods and biological effects of the trichothecenes will be detailed.

P27 STUDIES ON AFLATOXIN ANALYSIS USING LIQUID CHROMATOGRAPHY

W. R. DAY and Y. HAROON M.Sc. Ph.D. Waters Chromatography Division of Millipore Corp., 34 Maple St., Mil ford, MA 01757 U.S.A.

The separation of aflatoxins in sample extracts can be complicated by the elution of compounds other than aflatoxins, which interfere with the estimation.

Chromatograms of aflatoxins, using a number of different mobile phases and columns, both normal and reverse phase, are shown which illustrate how successful separations can be accomplished with different chemistries. The different separation conditions show how the chromatography can be modified to deal with difficult samples.

Limits of detection are affected by the column geometry and the type of detector used. Some examples of different column geometry are shown. Fluorescence and UV detection are compared.

Sensitive detection of aflatoxins in reverse-phase eluents requires the formation of derivatives, usually formed by treatment of the sample with water and trifluoroacetic acid (TFA). This procedure can be used to confirm the identity of aflatoxins B. and G,. Other derivatives can be formed using iodine and alcohols. The alcohol derivatives can produce inadvertent interference and loss of the aflatoxins if alcohols are present during the derivatization procedure. Chromatograms are presented to illustrate separation of the alcohol derivatives.

The formation of iodine derivatives, prior to injection, is shown to be unreliable and offers a detectabi1ity no better than the water adducts.

P28 IMMUNOASSAY OF THE MYCOTOXIN ZEARALENONE (F2)

S. N. DIXON and K. CLARKE CChem, MRIC, PhD. BSc. AFRC Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, RG16 ONN, U.K.

Synthetic derivatives of zearalenone and the anabolic agent zeranol were linked to bovine serum albumin and human serum albumin, these antigens were used to produce antibodies in rabbits.

Using ( H) zeranol the cross reactivity of antibodies raised against a zeranol-7ct-hemisuccinate-bovine serum albumin conjugate was 48% for zearalenone compared with 100% for the anabolic agent zeranol and its metabolite zearalanone. A radioimmunoassay for zearalenone has been developed using this antibody. In the assay of edible tissues from farm animals which may have been treated with zeranol, HPLC separation of zearalenone from zeranol and related compounds is necessary prior to radioimmunoassay. For analysis of animal feed and grain which would almost certainly not be contaminated with zeranol, the HPLC separation is not required. The standard curve has a useful working range between 0 and 600 pg of zearalenone with a lower limit of sensitivity of 25 pg. This assay is more sensitive than any existing method.

Antibodies raised in rabbits against synthetic derivatives of zearalenone (F2) have been evaluated and used to develop a non-isotopic (ELISA) immunoassay which has a sensitivity comparable to the radioimmunoassay.

These assays are being applied to the quantitative determination of zearalenone in animal feed and grain and to the edible tissues, faeces and body fluids of farm animals.

P29 THE INTERNATIONAL MYCOTOXIN CHECK SAMPLE PROGRAMME

M.D. FRIESEN

International Agency for Research on Cancer 150 cours Albert Thomas, 69372 Lyon Cedex 08 FRANCE

Each year, laboratories in more than 40 countries around the world, involved in the analysis of aflatoxins in food, control the quality of their analytical results by participating in the International Mycotoxin Check Sample Programme. Identical portions of homogenized samples of peanuts or maize, contaminated with aflatoxins B , B , G. and G_, and of lyophilized milk, contaminated with aflatoxaii H., are distributed to over 250 laboratories. By analysing these samples, the participating analyst can compare his own results, obtained using the method of his choice, to those of a large number of other laboratories and can take steps to modify and improve techniques if his results do not compare favourably.

An attempt is also made to evaluate and compare the performance of sub-groups of laboratories using particular methods and to evaluate trends in their use. For example, a group of laboratories utilizing the BF method for aflatoxin B. in maize or peanuts has generally reported significantly lower levels than have groups using the CB or EEC methods. The use of the BF method has decreased over the years while the use of HPLC for mycotoxin analysis has increased significantly. The reproducibility of results, expressed as percent coefficient of variation, has generally been better for sub-groups of laboratories using HPLC or 2-dimensional TLC methods than for sub-groups using 1-dimensional TLC.

Participation in the programme is without charge and samples are distributed about once each year. Interested laboratories are encouraged to participate in future surveys.

P30 HPLC AND GC-MS ANALYSIS OF MONILIFORMIN IN MAIZE

J. GILBERT, M.J. SHEPHERD, J.R. STARTIN AND I. PARKER

MSc. Ph.D. BSc. Ph.D. BSc. Ph.D. MSc. Ministry of Agriculture, Fisheries and Food Food Science Laboratory, Norwich NR2 *tSX,U.K. An HPLC analytical procedure has been developed for screening at low levels for the presence of moniliformin in samples of maize. The method involves solvent extraction of the ground material followed by a dual column clean- up involving high performance aqueous size exclusion chromatography directly switching the eluted component to reverse phase HPLC with U.V. detection. Good sensitivity and adequate specificity has been demonstrated and the method has been applied to a small number of commercially important maize samples.

For confirmation,a number of derivatization procedures have been critically evaluated prior to capillary column GC-MS. Selected ion monitoring of ^_-butyldimethylsilyl derivatives has been demonstrated as being a suitable approach to confirmation of moniliformin positive samples detected by the initial HPLC screening.

P31 BIOASSAY OF CYTOCHALASIN E AND OTHER ELEVEN MYCOTOXINS USING SKIN OF ONE-DAY-OLD MOUSE

T. GLINSUKON1, S. SINLAPANAPAPORN1 AND S. SRIURAIRATANA2

Department of Physiology , Faculty of Science and Department of Pathology', Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand.

Total 16 mycotoxins were used to develope skin bioassay in one-day- old mouse. Cytochalasins B, C, D and E, patulin, roridin A and verrucarin A induced erythematous lesion in dermis of one-day-old mice when given s.c. whereas aflatoxin Bj, citrinin, cytochalasin A, diacetoxyscirpenol, luteo- skyrin, rubratoxin B, sterigmatocystin, T-2 toxin and zearalenone had no capability of producing skin erythema at a dose as high as 8.48 mg/kg BW or a lethal dose. Cytochalasin E at a dose of only 0.106 mg/kg BW (0.16 yg/ mouse) was the most potent derivative among cytochalasins to induce erythema with an area of 6.6 mm2 within 30 min atter: injection. Patulin (1.06 mg/kg BW) also induced erythematous lesion to a maximum area of 9.2 mm^ that accom- panied by middle white zone within 30 min after which it disappeared within 3 days with desquamated skin and scar. In contrast, roridin A (0.27 mg/kg BW) and verrucarin A (1.06 mg/kg BW) could induce severe edema and expensive erythematous lesions with maximum area of 42.2 and 39.3 mm^ respectively at about 12 hr after injection. These lesions disappeared at the same time but without any scar. Cytochalasin E induced doae-dependent(0.106-1.06 mg/kg BW) response of skin erythema. Thus,skin erythema was employed to estimate amount of cytochalasin E produced by Aspergillus clavatus (AG5890) on various substrates including corn, peanut, rice, mung bean,soybean and sorghum. This isolate of A.clavatus produced cytochalasin E at a highest concentration of 125 mg/kg in sorghum. According to the lesion of skin erythema, cytochalasin E caused extravascular effusion of most red and some white blood cells and also plasma through the widening of intercellular gaps between endothelial cells of capillaries and venules as observed under electron microscope. Therefore, this implies that a possible cause of death in rats given i.p. is extravascular effusion of plasma through the enlargement of these intercellular gaps of capillaries and venules in the peritoneal cavity. In addition, two other methods of bioassay were also developed to estimate cytochalasin E content by using an inhibition of spermatozoa motility and glucose absorption in red blood cells. The sensitivity and specificity of these methods are discussed.

P32 PROBLEMS ENCOUNTERED IN THE ROUTINE ANALYbIS OF FOODSTUFFS FOR AFLATOXINS.

HANS KROHM, B.Sc. Honn. (Biochemistry) CITY HEALTH DEPARTMENT, P.O. BOX 1477, JOHANNESBURG, 2000

A HPLC method for the clean-up of foodstuff samples prior to quantitative analysis for aflatoxins is described. Analysis of a variety of peanut, sorghum and maize products for aflatoxins at the level of the maximum legal limit of lOug/kg (total) has shown that both clean-up by silica gel column chromatography and gel permeation chromatography are inadequate for analysis at these low levels. A second clean-up step is introduced which makes use of the fact that the trifluoroacetic acid derivatives of aflatoxins B-jand Gj have significantly different retention times from the parent compounds en a C-18 HPLC column. Clean-up of underivatised samples is achieved by dumping peaks, eluting before the aflatoxins which are then collected. Subsequent trifluoroacetic acid derivatisation of the collected fraction shifts the G2aand BggPeaks into the previously cleaned up region of the chromatogram, allowing them to be quantitatively determined. This clean-up procedure can be extended for methods incorporating the Iodine derivative of aflatoxin B-j. In this case, peaks eluting before aflatoxin B, are dumped prior to derivatisation with Iodine. This latter method is used routinely to confirm the presence of aflatoxin in samples containing residues in excess of the limit. These combined techniques allow trace amounts of aflatoxin residues to be determined, with confidence.

P33 AFLATOXIN METABOLITES IN HUMAN URINE AND LIVER IN ZAMBIA

J S N KAGGWA, L A OIL, A C BAYLEY AND C E A LOVELACE

B.Sc, M.Sc, Ph.D. CHEMISTRY DEPARTMENT, UNIVERSITY OF ZAMBIA, P 0 BOX 32379, LUSAKA, ZAMBIA.

Human urine and autopsy liver samples were investigated for their aflatoxin content by thin layer chronatography. Urine samples were taken from 136 newly admitted patients at the University Teaching Hospital, Lusaka, between September 1983 and August 1984; 67 were patients showing liver pathology (hepatitis, cirrhosis, hepatoma) and 69 were controls with no liver pathology, selected according to age and sex. During the same period 3k autopsy liver samples were collected; one was from a subject who died of hepatoma and 33 were from subjects who showed no liver pathology at the time of death. All the samples were analysed for aflatoxin B. and free metabolites

(Mj, Gj, B2 , G2 and aflatoxicol). Sulphate and glucuronide conjugates were looked for after enzymic hydrolysis of 50 urine samples, which included all the samples which were positive for B. and free metabolites. Aflatoxins B. , M. and aflatoxicoi were detected in urine but no conjugates were observed. The incidence of urinary aflatoxins was 3.7%; 2.2% were liver patients and 1.5% were controls. The level of urinary aflatoxins was 't.O to 20.0 ng/100 cm'. Aflatoxin B. was the only species detected in liver, at an incidence of 1*».7%. The level of B. in liver was 0.3 to 9.5 ng/g. The maximum, 9.5 ng/g, was observed in a liver sample from a subject who had died of hepatoma, and was over 10 times higher than the highest level (0.86 ng/g) detected in samples from subjects who had no liver pathology at the time of death. All the samples which contained aflatoxins were collected during the rainy season, November to Apr i1.

P34 DETOXICATION OF AFLATOXINS BY ETOX®-GASING

J LEIBETSEDER. J.BOHM and CH NOONPUGDEE

Professor D.V.M.; engineer; D.V.M. Institute of Nutrition, University of Vet.Medicine Vienna Linke Bahngasse 11, A-1030 Vienna, AUSTRIA

INTRODUCTION Detoxication of mycotoxins has a considerable ecological and economical significance. Because of the toxic and carcinogenic effects aflatoxin (AF) content of feed and food has to be reduced to the maximum allowance of 10 to 50 ppb in feed without impairment of feed quality and at a reasonable expense. Gasing with ETOX (Ethylenoxid/C02 =9/1) is not only effective against microorganisms but also seems to reduce AF in contaminated substrates (4). This effect should be tested under definite experimental conditions. METHOD 10 samples of paenut expeller each containing different amounts of AFB1 were evacuated and gased with ETOX (concentration: 1500g/ms substrate moisture: 10-16 %, temperature: 20, 50, 80°C resp.) for 16 hours (2,3). Afterwards AFB1 was determined by HPLC (1,5). In order to test the stability of the AFBI/ETOX-reaction products under physiological conditions gased samples were digested in vitro (pepsin- HC1, subsequenty increasing pH to 8) and AFB1 was then analyzed. Additio- nally the HPLC fraction containing the main reaction products was collected for MS in order to clarify the chemical nature of the reaction products. RESULTS Detoxication of AFB1 by ETOX-gasing is possible. The effect depends upon temperature and moisture. At 80°C the reduction of AFB1 amounts to 90 % and even more. Stability testing and clarifying the reaction products is still under work. These data will be presented at the syirposium. DISCUSSION The presented method of detoxication is highly effective, creates no risks for human or animal health by residues of ETOX, is applicable to large amounts of food and feed and reasonable in regard to the expense. REFERENCES 1) B0H4,J.,NOONPUGDEE,Ch.,and LEIBETSEDER,J.< 1984bErnahrung/toutrition 8:675. 2) DEGESCH Ges.m.b.H., Frankfurt: (1979): patent specification Nr. 34 9875, Austria 3) DEGESCH Ges.nub.H.,Frankfurt: (1979):patent specification Mr. 353589, Austria 4) MAYR,G. (1973): Thesis, Univ. Bonn, Faculty of Agriculture 5) SCHWEIGHARDT, H. and LEIBETSEDER, J. (1981): Wien. tierarztl. Mschr. 68_: 302.

P35 THE INVESTIGATION OF SOME ASPECTS OF A HPLC AFLATOXIN ANALYSIS METHOD FOR GROUNDNUTS

S.A.F. MEYER, L.H.W. VERHOEF AND P.J. GROBLER

OILSEEDS BOARD, BOX 211, PRETORIA, OOO1, SOUTH AFRICA

The effect of particle size, as produced by the milling of raw nuts, on the repeatability of an analysis was investigated. Repeated analysis on the meal produced by a popular mill proved to have a very poor repeatability (coefficient of variation 36%). The repeated analysis of fines produced by sieving the meal through a ,89 mm aperture sieve proved to have the same average value and a much improved repeatability (coefficient of variation 15%).

The AOAC BF method for the extraction of aflatoxins from groundnuts was complemented by the addition of a scaled down mini column cleanup utilising silica gell packed in a pasteur pipette. This resulted in all the advantages associated with a mini column cleanup plus a reduction in the cost associated with the commercially available equivalent.

As a final stage in the sample preparation afalatoxins Bl and Gl is derivatised with Trifluoroacetic acid, the resulting derivatives B2A and G2A was evaluated as to their stability in some commonly used solvents as well as to their sensitivity to ordinary fluorescent lighting and direct UV Light. The derivatives were found to be unstable in 100% methanol and 100% ethanol as well as sensitive to fluorescent light and very sensitive toward UV light thus making it necessary to avoid these solvents when making up samples and to protect samples from light.

P36 DECONTAMINATION OF GROUNDNUT MEAL CONTAINING AFLATOXIN B1 BY TREATMENT WITH CAL- CIUM HYDROXIDE AND PARAFORMALDEHYDE AND CONSEQUENCES ON AFLATOXIN Ml LEVEL IN

THE MILK

G. PIVA , A. PIETRI and E. CARINI

1 Full Frofessor in Animal Nutrition Institute of Animal Nutrition, University of Agricultural Sciences, U.C.S.C.,

29100 Piacenza - Italy

A groundnut meal containing aflatoxin B1 (401 -ug/kg, ppb) was adjusted to 1,

moisture, mixed with calcium hydroxide (4%) and paraformaldehyde (0.5%) auto-

claved at 2 atm. (I21°C) for 20 min.. The treatment reduced the detectable con

centration of aflatoxin to 29.5 ppb (-92.62%). The contaminated and treated

meals were incorporated into two mixed feeds, that showed a final aflatoxin

Bi level of 160 and 11.8 ppb respectively. Two trials for a period of 10 and

22 days, with a withdrawal period of 12 days between them, were carried out:

in the first trial two milking cows were fed the concentrate containing conta-

minated groundnut meal; in the second, the same cows were fed the concentrate

containing detoxified groundnut meal.

In both trials, aflatoxin Ml appeared in milk in the milking following the ad-

ministration of the contaminated feeds. The daily mean concentration of afla-

toxin M1 in the milk of the two cows was 651+323 and 1442+755 ng/1 (ppt) in

the first and 66+25 and 94+32 ppt in the second trial (P<0.001). The aflatoxin

M1 percentages excreted in the milk with respect to the aflatoxin B1 ingested

were similar in the two experimental periods (1.20-1.15 in cow 1 and 3.42-2.18

in cow 2) and in the second trial they were in fact slightly lower. On the

other hand some authors reported that ammoniated groundnut meal fed to dairy

cows caused 10-20% elimination of aflatoxin Ml in the milk, while these per-

centages were 10 times lower with untreated meal. In conclusion, our feeding

trials confirmed the effectiveness of the process in reducing the biological

activity of the aflatoxin B1, as shown by the reduction in the chemical assay.

P37 Effects of four Trichothecenes to Larvae of Brine Shrimps (Artemia salina L.) and of Mosquitos (Aedes vexans Meigen)

H. RAINER SCHMIDT

Dipl.-Chemist, Dr. Fa. Hans W. Schmidt, Saarstr. 52, P.O.B. 3628, D- 6500 Mainz F.R.G.

Different trichothecenes (T-2 toxin, HT-2 toxin, acetyl-T-2 toxin and neosolaniol) were isolated by HPLC from cultures of Fusarium sporotrichioides Sp 941 grown on rice. Bio-assays of the purified mycotoxins were performed with larvae of brine shrimps (Artemia salina L.) and mosquitos of the Rhine area (Aedes vexans Meigen), which are phylogenetically separate species. In both systems trichothecenes showed similar toxicities. The LC_g-values were obtained graphically by dose-response relationships. The LC5~-values after 24 hrs exposure were between 0.65 and 6.0ug/ml for brine shrimps and between 2.1 and 12.5 jig/ml for mosquito larvae T-2 toxin was the most toxic of the trichothecenes. An increase of hydro- phobicity by acetylation of hydroxyl groups (acetyl-T-2 toxin) does not yield in an increase in toxicity. Incubation of brine shrimps with trichothecenes for 48 hrs showed only small differences (0.24 ug/ml T-2 toxin , acetyl-T-2 toxin and HT-2 toxin both 0.32 ug/ml and neosolaniol 0.40pg/ml). This effect is likely due to a metabolic conversion of these trichothecenes into the same product, perhaps by esterases. This is supported by the LC^p-value for T-2 tetraol, which is 0.75ug/ml. New researches measuring the motility by an optical counter instead of the microscopical determination of the lethality offer the observation over the whole period of investigation. In addition more objective results can be obtained. The applicability of this method was tested with brine shrimps and T-2 toxin.

P38 RAPID THIN LAYER CHROMATOGRAPHIC DETERMINATION OF AFLATOXIN Mi IN MILK

M.L.SERRALHEIRO and M.L.QUINTA Licenciate in chemistry.Licenciate in biology LNETI - DCEAI, Biologia. Azinhaga dos Lameiros a Estrada do Pago do Lumiar, 1699 Lisboa Codex. PORTUGAL

A method is described for the detection of aflatoxin M. in milk. The toxin is extracted with chloroform. The extract is e- vaporated and the residue partitioned between carbon tetrachloride and an aqueous saline methanol solution. The toxin is once again extracted from the methanol solution with chloroform and the ana - lysis is performed by 1 - dimensional thin layer chromatography. The detection limit of powdered milk is OjS^ug/Kg and the recove - ries of M^ added are around 83%. The detection limit can be lowred to 0,3^ig/Kg if the plate is sprayed with a solution of H_SO. after the development.

P39 CHARACTERIZATION AND ANALYSIS OF TRICHOTHECENE MYCOTOXINS BY MASS SPECTROMETRY

J.R.J. PARE, R. GREENHALGH, P. LAFONTAINE AND ^.W. APSIMON

Cheraistry and Biology Research Institute, Agriculture Canada, Ottawa, Ont. Carleton University, Ottawa, Ont. Canada

Crude fungal extracts of Fusarium culmorum (CMI 14764) and F. roseum (ATCC 28114) have been analyzed by fast atom bombardment mass spectroscopy (FAB/MS). This technique has proven to be a fast, efficient and reliable screening test for the presence of known secondary metabolites. The EI/MS fragmentation patterns of the trichothecenes, nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, T-2, HT-2, diacetoxy scirpenol, 7,8-dihydroxy calonectrin, sambucinol, sambucoin, roridin A and verrucarin A have been established using linked scan techniques. Although no single fragmentation pattern emerged characteristic of the trichothecene ring system, it is possible to fully characterize all the trichothecene mycotoxin under study, despite the complexity of each fragmentation pattern. These data are evaluated in terms of a definitive GC/MS/MID analytical method for quantitative analysis and characterization. The use of both FAB/MS and EI/MS in that sequence permits the complete analysis of mixtures of trichothecene tnycotoxins without the need for extensive preliminary separation and isolation procedures.

P40 THE ROLE OF TECHNOLOGICAL PROCESSES IN DECREASING PATULIN CONTAMINATION OF APPLE JUICES AND APPLE WINES

T LIPOWSKA and H GOSZCZ

M.Sc in food technology * B.SC in food technology Department of Food Analysis, Institute of the Fermentation Industry, Rakowiecka 35, 02-532 Warszawa, Poland

The influence of the following technological processes on patulin content of concentrated apple juices has been investigated: pasteurization, depectiniza- tion, filtration and concentration. Disappearance of patulin contamination during alcoholic fermentation has been also studied.

25% decreasing patulin content in the course of concentrated apple juice processing has been found. It is ascertained that the reduction mostly takes place at the concentration stage. Contrary to the above patulin was not detected in apple wines after A9 hours of fermentation. At the same time the effect of addition of sulphur dioxide to stum has been observed. No patulin was detected after two weeks when 0,125% of sulphur dioxide was added and ca. 50% reduction of patulin was noticed at the dose of 0,05% of sulphur dioxide

P41 BIOCHEMICAL MECHANISM OF ACTION OF MYCOTOXINS AND PHYCOTOXINS

H1-H17 THE PATHOLOGICAL EFFECTS OF IMMUNOSUPPRESSION OF PLASMODIUM BERGHEI - INFECTED RATS, WITH PARTICULAR REFERENCE TO SURVIVAL, HEPATOCARCINOGENESIS AFTER AFLATOXIN B TREATMENT.

SUBHKIJ ANGSUBHAKORN, DVM.MS, PERMSIN SATHIROPAS, MS, NATTH BHAMARAPRAVATI, MD, DSc, SOMPHONG SAHAPHONG, MD, Ph.D. AND PHICHAI THUVASETHAKUL*, Ph.D. Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400 and Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10A00.

ABSTRACT The prevalence of aflatoxins are common in man in malaria endemic region such as in Africa and Southeast Asia. The interactions between the mycotoxin and the parasite were studied in animals as followed: A.Weanling male and female Buffalo rats were divided into 3 groups: Groupl, AFB.. (2 mg/kg body weight) followed by 10* I\berghei-treated (AFB /Malaria); Group 2, DMSO followed by 10* P.berghei-treated and Group 3, 10* P.berghei-treated. B. Adult male Buffalo rats were divided into 4 groups: Group 1, AFB. (2 mg/kg body weight) followed 10& P.berghei-treated (AFB.,/Malaria) ; Group 2, AFB1 (2 mg/kg body weight)-treated; Group 3, 10^ P.berghei-treated; and Group 4, untreated.

When plasmodia were injected into 24 hr-AFB^ treated animals (AFB / Malaria). The different pattern of malarial disease was observed in both weanling and adult animals. In weanling rats, instead of recovering as normally expected, they died in about three weeks after infection. The percents parasitemia were 60.4*9.2 and 56.5*4.9% while their mortality in male and female rats was 62.5 and 75% respectively. When AFB^ was administered into male adult animals 24 hr before the plasmodium, similar results were noted but in lesser degree, i.e. the animals died on days 14 and 18 with 28.6% mortality and 24.0 6.3 x 103 instead of 12.6 1.3 x 103 parasites. Of interest, it was that 6 adult animals in AFB./Malaria group developed hepatocellular carcinomas on days 17, 23 and 29, while only 1 animal with AFB alone did on day 29.

These findings suggested that the parasite might facilitate AFB toxicity and proliferation of AFB..-induced neoplastic cells by possible lmmuno- suppression. To strengthen our suggestion, furhter experiments need to be done in order to establish more firmly the interaction between the mycotoxin and malarial parasites in rats.

P42 COMPARATIVE STUDY ON THE METABOLISM OF DIACETOXYSCIRPENOL AND ZEARALENONE IN PIGS

BAUER, J. , M. GAREIS, C. ENDERS and B. GEDEK Dr. Institute for Medical Microbiology, Infectious- and Epidemic Diseases, University Munich, Veterinary Faculty VeterinarstraBe 13, 8 Munich 22, F.R.G. Two groups of 4 female pigs each (approx. 20 kg b.w.) were orally dosed with diacetoxyscirpenol (2.0 mg/kg b.w.) or zearalenone (0.5 mg/kg b.w.). Samples of blood serum, urine and feces were collected over a period of 7 days. The analysis for zearalenone and its metabolites •<- and ^-zearalenol was carried out following the method of MIROCHA et al 1981. For the detection of diacetoxyscirpenol and its metabolites, the samples were extracted with ethylacetate (serum,urine) or acetonitrile (feces). A Sep-Pak C18 cartridge and a florisil column were used for clean up the crude extracts. Extracts of feces were further separated by HPLC on a LiChrosorb RP 18 (10|im) column into 20 fractions using a gradient program (20% methanol to 100% methanol in 20 minutes). All extracts were derivatized with n-methylbis(trifluoroacetamide) and analyzed by GC-PID or GS-MS (chemical ionization in methane). Only low concentrations of zearalenone were found in serum - up to 7.0 ng/ml 6 hours after toxin administration - over a period of 48 hours (3 pigs) and 72 hours (1 pig) respectively. Free and glucuronide forms of zearalenone and oC-zearalenol were detected in urine and feces, while no 4-zearalenol was found. Highest amounts of zearalenone and«£-zearalenol were excreted during the first 24 hours in the urine (395.5 \ig/ 62.1 ug) and from 24 - 48 hours in the feces (1203.7 ug/ 164.8 ng) • An excretion of zearalenone in urine and feces was still detectable *' 7 days after toxin treatment. At the same time o6-zearalenol was j found in the feces of only one animal, whereas it was still t present in the urine of all pigs. ' After oral administration of diacetoxyscirpenol, this mycotoxin j as well as monoacetoxyscirpenol and scirpenetriol were detectable • in blood sera with highest levels during the first hour. Mono- acetoxyscirpenol and scirpenetriol were excreted in feces (26.9 iig/ 442.6 ug) and urine (12.7 ug/ 9.0 ug) for 24 hours (3 pigs) and 48 hours (1 pig) respectively. Diacetoxyscirpenol was found during the same period in urine (8.7 ug) whereas small amounts (0.9 ug) were analyzed in feces only during the first 6 hours. On the contrary to zearalenone, these results indicate a rapid metabolism of diacetoxyscirpenol.

MIROCHA et al 1981: Fd Cosmet. Toxicol., 19, 25.

P43 ANATOXIN-A(S): A NEUROTOXIN PRODUCED BY THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS-AQUAE NRC 525-17

Nik A. Mahmood and Wayne W. Carmichael Doctoral Candidate; Associate Professor Biomedical Sciences Program and Department of Biological Sciences Wright State University Dayton, Ohio 45435 U.S.A.

Anatoxin-a(S) (Antx-a(S)) is produced by Anabaena flos-aquae clone NRC-525-17, which was isolated from Buffalo Pound Lake, Saskatchewan, Canada in 1965. Mice injected intraperitoneally (i.p.) with toxic lyophilized cells (LD__ - 14 mg/kg) showed typical muscarinic and nicotinic effects. These included salivation, urinary incontinence, muscular weakness, fasciculation, convulsion (including opisthotonus in chicks) and respiratory arrest. The toxin was extracted from lyophillzed cells with 1.0 M acetic acid: absolute ethanol (80:20) and purified through column chromatography; Sephadex G-15, CM-Sephadex C-25 and high performance liquid chromatography. Purified toxin has an LDj-n i.p. mouse of approximately 50 (ig/kg and gives a linear relationship on a dose vs. duration curve from 0.1 to 1.0 mg/kg. On isolated chick biventor-cervicis and frog rectus-abdominis muscles, Antx-a(S) did not show any direct agonistic effect but potentiated the acetylcholine response and antagonized the d-tubo- curarine actions. Twitch potential and tetanic fade were observed on isolated phrenic nerve diaphragm muscles when stimulated indirectly at different frequencies. Thus Antx-a(S)might be affecting the acetylcholine concentration at the cholinoceptors. Mice pretreated with atropine sulfate showed prolonged survival time. Atropine sulfate eliminated the parasympathomimetic signs of toxicity but did not prevent the eventual death with convulsions and respiratory arrest. This indicated that the toxicity of Antx-a(S) cannot be solely attributed to a peripheral muscarinic action.

P44 INHIBITION OF CHOLIKESTEHASE BY ANATOXIN-A(S)

Nik A. Mahmood and Wayne W. Carmichael Doctoral Candidate; Associate Professor Bioraedical Sciences Program and Department of Biological Sciences Wright State University Dayton, Ohio 45435 U.S.A.

Anatoxin-a(S) (Antx-a(S)) produced by Anabaena flos-aquae NRC-525-17 was found to be a potent neurotoxin acting on the cholinergic system. Gross pharmacological tests on isolated nerve-muscle tissue preparations showed no agonistic effect by Antx-a(S). Results did indicate that it induced a prolonged acetylcholine release, _In vitro enzyme assays showed Antx-a(S) to be a strong, partially reversible inhibitor of true and pseudocholinesterase. The type of inhibition showed by Lineweaver-Burk and Dlxon plots is noncom- petitive with an inhibition constant for acetylcholinesterase (E.C. 3.1.1.7; electric eel) three times that of butyrylcholinesterase (E.C. 3.1.1.8; horse serum). The inhibition was instantaneous and did not diminish with prolonged incubation of the toxin and cholinesterase.

P45 THE INTERACTION OF AFLATOXIN B, WITH RAT PLASMA ALBUMIN IN VIVO AND IN VITRO

HEINI w. DIRR AND JOHAN C. SCHABORT M. SC. (R.A.U.) Department of Biochemistry, Rand Afrikaans University, P.O. Box 524, Johannesburg, 2000, SOUTH AFRICA The in vivo and in vitro binding of [3H]-aflatoxin B, to rat plasma was investigated. Column chromatographic analyses and discontinuous polyacrylamide gel electrophoresis clearly demonstrated [3H]-aflatoxin B, to bind primarily to plasma albumin. Other plasma proteins indicated very little binding activity. The aflatoxin-albumin complex was purified from rat plasma to apparent homogeneity. Spectrof1uorometric studies were undertaken to gain a better insight into the nature of the aflatoxin-albumin interaction. The lone tryptophan fluorescence intensity was quenched by binding of aflatoxin B,. This quenching was due, at least partially, to a ligand-induced conformationai change in the protein molecule. Aflatoxin B, binds to an apolar site with an association constant of 3 x 104 M~' at pH7.4 and 20°C. Neither charcoal treatment of rat albumin nor the presence of 0.15M NaCI had any significant effect on the binding of aflatoxin B,. The association constant was pH-dependent, increasing about 1.7-fold as the pH was increased from 6.1 to 8.4. This pH-dependence can be ascribed to a pH-induced conformational change in the albumin molecule. The association constant varied from 3.8 to 2 x 10* M"' at temperatures from 8° to 37°C. The aflatoxin-albumin interaction is exothermic (AH =- 3.8 kcal /mole) with an entropy change, AS, value of + 7 cal. mole ~'K~'. Both hydrogen and hydrophobic bonding are suggested to be involved in the aflatoxin-albumin binding mechanism.

P46 THE MECHANISM OF INHIBITION OF PYRUVATE DEHYDROGENASE COMPLEX BY THE MYCOTOXIN MONILIFORMIN

P.S. GATHERCOLE, P.G. THIEL, J.H.S. HOFMEYR

National Research Institute for Nutritional Diseases, S.A. Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa. Moniliformin is a highly toxic fungal metabolite produced by several species of Fusaria, some of which are commonly found on basic foodstuffs (1). It can be isolated as the potassium or sodium salt of semi-squaric acid (1-hydroxy- cyclobut-l-ene-3,4-dione) (2) and has been shown to occur naturally on maize (3). In an attempt to establish the nature of the toxic action of this compound, Thiel (4) measured the effect of monilifonnin on oxygen consumption after the addition of various substrates to isolated rat liver mitochondria. Very low concentrations of moniliformin were found to selectively inhibit pyruvate and a-ketoglutarate oxidation. This study was undertaken to investigate the precise mechanism of action of moniliformin. The a-keto acid dehydrogenase complexes were isolated from bovine heart using a modified method of Stanley and Perham (5). Thiamine pyrophosphate proved to be necessary for the inhibitory action of moniliformin. However, at higher concentrations thiamine pyrophosphate reduced this inhibitory effect. The inhibition reaction was shown to be time dependent and to follow saturation kinetics indicating a mechanism of suicide inactivation. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin inactivated complex only partially reversed inactivation. Moniliformin inhibited the pyruvate dehydrogenase component of the enzyme complex but had no detectable inhibitory effect on the dihydrolipoyl trans- acetylase and dehydrogenase components. These results together with the structural analogy between pyruvate and moniliformin would indicate that moniliformin could be classified as a suicide enzyme inactivator. This would imply that moniliformin is activated in a similar manner to the reaction of pyruvate with thiamine pyrophosphate, forming an active intermediate which binds irreversibly to the pyruvate dehydrogenase component of the enzyme complex.

1. Rabie, C.J., Marasas, W.F.O., Thiel, P.G., Lilbben, A. and Vleggaar, R. Appl. Environ. Mircobiol. 43 (3), 517 (1982).

2. Springer, J.P., Clardy, J., Cole, R.J., Kirksey, J.W., Hill, R.K., Carlson, R.M. and Isidor, J.L. J. Am. Chem. Soc. 96 (7), 2267 (1974).

3. Thiel, P.G., Meyer, C.J. and Marasas, W.F.O. J. Agric. Food Chen. 30, 308

(1982).

4. Thiel, P.G. Biochem. Pharmacol. 27, 483 (1982).

5. Stanley, C.J. and Perham, R.N. Biochem. J. 191, 14? (1980).

P47 MUTAGENIC ACTIVITY OF SECONDARY METABOLITES OF ASPERGILLUS USTUS

E JOHANNSEN, R KFIR and R VLE6GAAR

Several secondary metabolites isolated from a fungus Aspergillus ustus were tested for mutagenic activity using the Salmonella mutagenecity assay (Ames test). The tests were carried out using two tester strains of Salmonel 1 a typhimurium, namely TA 98 and TA 100 with and without 1 iver activation (microsomal fraction Sg). The presence or absence of mutagenic activity as well as the type of mutagenic action (frame shift, base pair substitution) were correlated with the chemical structure of these metabolites.

P48 ACTION OF FATÜXIN ON THE GLUTATHIONE POOL IN A YEAST

P. THONART*, J. BECHET**, S. SENE* and Z. LEZI SUMBÜ*

*Département de Technologie Agro-alimentaire et Forestière Faculté des Sciences Agronomiques, B-5800 Gembloux, Belgique **Institut de Recherches et Institut des Industries de Fermentation C.E.R.I.A., B-1070 Bruxelles, Belgique

Patulin is a mycotoxin produced by numerous molds, especially Pénicillium patu- lum and Byssochlamys nivea. Our purpose in this study was to specify the action of patulin on Saccharomyces cerevisiae. We chose this yeast because of the well documented fact that patulin disappears during fermentation of apple juice; so far, no model to explain this degradation has been proposed. At weak doses, the drug inhibited growth, but inhibition was transient. After recovery of growth, patulin disappeared from the medium and induced cells resisted at very high patulin concentrations (1).

Glutathione concentration in Saccharomyces cerevisiae has been studied in function of patulin addition in the growth medium. At weak doses (100 ug/ml), the drug inhibited the growth and the glutathione concentration decreased from 5 to 2,5 yg/mg of dry weight. After recovery of growth, the glutathione concentration increased until 7,5 yg/mg and patulin disappeared from the medium. At 200 pg/ml of patulin, glutathione concentration decreased and no induction appeared.

However, if the cells were incubated for 3 hours at 50 yg/ml of patulin, they become resistant to doses of > 200 yg/ml patulin and the glutathione concentration in the cells reached 7 yg/mg. These experiments suggest the action of glutathione as a detoxifying agent against patulin in Saccharomyces cerevisiae.

(1) Z. Lezi SUMBU, Ph. THONART and J. BECHET (1983) Action of Patulin on a yeast. Applied and Environmental Microbiology, 45, 110-115.

P49 PHOMOPSIN A, THE CAUSATIVE AGENT OF LUPINOSIS, INTERACTS WITH MICROTUBULES _IN VIVO AND I_N VITRO

E.M. TONSING, D.J.J. POTGIETER, I.W. SIMSON, P.S. STEYN, M. OSBORN & K WEBER

BSc(Hons), P.U. for C.H.E. Department of Biochemistry, University of Pretoria, Hillcrest, PRETORIA 0002

Phomopsin A, a cyclic hexapeptide , is a metabolite of a fungal parasite of lupins. It is known to be the causative agent of lupinosis, a severe liver disease of sheep which ingest infected lupins. Histological studies showed that phomopsin A blocks hepatocyte cell division in late metaphase, followed by fragmentation of chromatin and the formation of micronuclei.

Phomopsin A acts as a mitotic drug both ^jn vivo and jj^ vitro. Immuno= fluorescence microscopy with tubulin-specific antibodies shows that phomopsin A interferes with the expression of cytoplasmic and spindle microtubules in a time- and concentration-dependent manner. Phomopsin A induces C-mitotic arrest as well as a total loss of microtubules from interphase cells. Depoly= merization seems to start at the membrane. Parallel biochemical and electron microscopical studies show that phomopsin A inhibits the jjn vitro polymeriza= tion of microtubules from both microtubular protein and from pure tubulin. Phomopsin A also depolymerizes preformed microtubules, probably from the ends. Our results stongly suggest that several known symptoms of lupinosis arise from microtubular dysfunction.

1. Culvenor, C.C. J., et_ al_. (1983). J. Chem. Soc. Chem. Commun. 2J_, 1259-1262.

2. Tonsing, E.M., Steyn, P.S., Osborn, M. & Weber, K. (1984). Eur. J. Cell Biol. 35, 156-164.

P50 INFLUENCE OF PHOMOPSIN AND IVALIN ON STEROID HORMONE BINDING AND MCF-7 CELL PROLIFERATION

C.H. VAN ASWEGEN, W.M. LEWKO? J.J. VAN DER WATT, H.C. POTGIETER, N.M.J. VERMEULEN, D.J.J. POTGIETER, J.L. WITTLIFF*

Dept. of Biochemistry & Inst. of Life Sciences, Univ. of Pretoria, PRETORIA. *Dept. of Biochemistry, Cancer Center, Univ. of Louisville, LOUISVILLE, KY, USA

The influence of the toxins, phomopsin and ivalin, which are reported to exhibit carcinogenic and antitumour activities respectively, were studied on steroid hormone receptor binding. Competitive binding analyses were conducted with three different levels of radiolabelled steroids and concentrations of unlabelled toxins ranging from 0,85 nM to 14 uM. No effect was observed on either the binding capacity or on the rate of association of [3HJ estradiol- 173, [3H]R5020, a synthetic progestin and [}H] dexamethasone to their respective receptors in cytosol of human breast cancer and rat liver.

When the action of these toxins was studied on steroid receptors of intact MCF-7 cells, the antitumour toxin ivalin, had an inhibitory effect on [3H] = estradiol-17ß binding, while an increase in binding was observed with [3H]= dexamethasone. In contrast to ivalin, phomopsin had an inhibitory effect on [3H]R5020 binding, while no significant effects were exercised on the £3H]= estradiol-17ß binding and [3H] dexamethasone binding.

Labelled thymidine and glycine incorporation in a human breast cancer cell line (MCF-7) were both inhibited by phomopsin, but not with ivalih. In fact, stimulation of glycine incorporation was observed in the presence of ivalin.

Plating efficiencies of MCF-7 breast cancer cells grown in culture were decreased to 50% by 1,5 x 10~5 M ivalin and 6,5 x 10~5 M phomopsin. Also the number of dead or released cells increased in the presence of 10~7 M ivalin and 10~5 M phomopsin.

When MCF-7 cells were incubated with increasing concentrations of either ivalin or phomopsin, a dose dependent decrease in total cell proliferation was seen.

These data suggested that ivalin exhibits antitumour properties similar to tamoxifen at the same concentrations. Although phomopsin may exhibit carcino= genie properties in certain cases, these data clearly indicate its antitumour properties when given in vitro.

P51 THE INFLUENCE OF AFLATOXIN B, ON PROTEIN PHOSPHORYLATION IN RAT LIVERS

J. VIVIERS AND J.C. SCHABORT B. SC. HONS. (R.A.U.) Department of Biochemistry, Rand Afrikaans University P.O. Box 524, Johannesburg, 2000, SOUTH AFRICA

A study was conducted on the effect of aflatoxin B, on protein phosphorylation in rat livers by incubation of soluble and insoluble cell fractions with [Y32P] ATP. SDS-polyacrylamide gel electrophoresis indicated a total of eight rat liver phosphoproteins were affected during a feeding period of 36 weeks on a carcinogenic aflatoxin B, containing diet compared to the phosphoprotein patterns obtained from the livers of rats on a normal noncarcinogenic diet. The appearance of only two of these phosphoproteins were c-AMP dependent. DEAE-cellulose chromatogranhy pmnlnyed to separate the different histone kinase activities in soluble rat liver cell fractions showed that the specific activity of histone kinase I activity was increased but its total activity decreased while the histone kinase II activity stayed unchanged for rats submitted to the aflatoxin B, containing diet compared to those on the normal noncarcinogenic diet. In view of the fact that this is the first report on the effect of the very potent hepatocarcinogenic compound, aflatoxin B,, on protein phosphorylation, the phosphoprotein patterns obtained in the abovementioned study were compared with these reported for other chemical carcinogens and oncogenic viruses. The aflatoxin B, induced alteration of the phosphoprotein patterns in the soluble and insoluble rat liver fractions may be due to changes in the regulation of protein phosphorylation in one or more of many possible ways including altering the amount and properties of specific protein kinases that phosphorylate specific substrate proteins; the substrate proteins themselves and specific phosphoprotein phosphatases. The possible involvement of alterations with respect to protein kinases as well as de novo protein synthesis are suggested by the results obtained in this study.

P52 TRICHOTHECENE DEGRADATION BY PURE CULTURES OF RUMEN BACTERIA

K WESTLAKE, M F DUTTON AND R I MACKIE M Phil (Trent Polytechnic) Department of Biochemistry, University of Natal P 0 Box 375, Pietermaritzburg, 3200

Four strains of Butyrivibrio fibriosolvens have been tested for their ability to degrade T-2 toxin and a further strain, isolated en a tributyrin mediun, tested for its ability to degrade a number of other trichothecenes.

The latter strain was shown to be resistant to T-2 toxin at concentrations as high as 1 ppm and was able to degrade acetyl T-2 toxin and T-2 toxin to HT-2 toxin over a period of approximately 8 hours.

The former strains were all shown to be able to degrade T-2 toxin to HT-2 toxin but to varying degrees.

The above results are discussed in terms of the low susceptibility of ruminants to trichothecene toxicoses.

P53 PROTECTIVE EFFECT OF VITAMINS AGAINST TRICHOTHECENES TOXICITY TOWARD SACHAROMYCES CEREVISIAE

B. YAGEN and S. HALEVY Ph.D. Department of Natural Products and Laboratory of Chemotherapy, Hebrew University, Pharmacy School, P. 0. Box 12065, Jerusalem 91120, Israel.

Abstract Several trichothecene mycotoxins inhibited the orowth of Sacharomyces cerevisiae. This effect was most pronounced with the macrocyclic trichothecenes, specially verrucarin. Sliqht effects were observed by usinq T-2 toxin and very little with metabolites of T-2 toxin and diacetoxyscirpenol. Incubation of S. cerevisiae treated with verrucarin, in the presence of several vitamins resulted in a decreased toxicity. Pyridoxine-HCl, Ca- panthothenate, thiamine-HCl and tocopherol acetate were amona the most potent orowth initiators, overcominq the toxicity induced in S. cerevisiae. It seems that addition of certain vitamins enables the cells to reconstruct their enzymatic activity and overcome the mycotoxin poisoninq.

P54 DIFFERENTIAL INHIBITION BY T-2 TOXIN OF TOTAL PROTEIN, DNA AND ISOPRENOID SYNTHESIS IN THE CULTURED MACROPHAGE CELL LINE J774

RAPHAEL N. MELMED AND BORIS YAGEN

PROFESSOR, M.D. AND PROFESSOR CHEMISTRY Lipid Research Laboratory, Hadassah-University Hospital and Department of Natural Products in the School of Pharmacy, H.U. Med. School, Jerusalem, Israel T-2 toxin (4£,15-diacetoxy-8K-(3-methylbutyryloxy)-12,13-epoxytrichothec-9- ene-3o{-ol) is part of a major family of mycotoxins known as trichothecenes, which have worldwide distribution and which are implicated in a diverse variety of disease processes in animals and man. The clinical picture in both man and animals is determined principally by arrest of normally rapidly proliferating tissues followed by their necrosis, as in bone-marrow, skin and gastrointestinal tract. Of major interest is the effect of T-2 toxin on immune function, as leukopenia and agranulocytosis are life threatening complications of toxicity states. In vitro studies have shown a number of the trichothecene toxins to be potent inhibitors of protein synthesis. These compounds, including T-2 toxin, would bind to ribosome thereby inhibiting peptidyl transferase and blocking initiation of synthesized polypeptides. Although the mechanism of protein synthesis inhibition has been studied in vitro, there have been very few detailed studies on whole cells aimed at accurately elucidating the effects of T-2 toxin on integrated cell function. As protein, DNA and isoprenoid synthesis are all essential elements of the normal proliferative apparatus of the cell, we studied the kinetics of T-2 toxin inhibition of these pathways. We found that in these cells, protein and DNA synthesis were more than 50% inhibited within one hour by extremely low doses of the toxin («el ng/ml). Isoprenoid synthesis, unlike protein and DNA synthesis, showed delayed inhibition (i.e. after 3-4 hours). This effect of T-2 toxin on protein, DNA and. isoprenoid biosynthesis presumably reflects the differential stability of the enzymes involved in these biosynthetic pathways.

P5S STUDY OP THE METABOLIZATIOH OF TRICHOTHECENE TOXINS

AKPAD BATA, AHDRAS VAIIYI

Technical University Budapest, Department of Biochemistry and Pood Technology, H-1521 BUDAPEST, P;0.3ox 91-, HUNGARY

Some years ago very surprising infertility symptoms were observed. Prom the aggs of parents seeing to be healthy the snail birds did not hatch. It was detected that the feed used for feeding of gooses contained T-2 toxin. Nevertheless we could not found reliably T-2 toxin or its metabolites in the goose eggs.

To clarify the statements mentioned above in vitro experiments were carried out. T-2 toxin and diacetoxyscirpenol /DAS/ were injected into the yolk of embryonated hen's eggs and the eggs were incubated. The toxins and metabolites formed during incubation v/ere analysed by method presented in an earlier paper. T-2 toxin, neosolaniol, T-2 triol toxin, T-2 Ictraol toxin, DAS, monoacetoxyscirpenol and scirpentriol woro found in eggs. All the examined metabolites are toxic but less toxic than T-2 toxin or DAS. After 10 days incubation trichothecenes v/ere found in eggs. This result shows that trichothecer.es are very stable in eggs and they can develop their toxic action in the later period of incubation.

To determine the lowest concentration range having a toxic effect very low amounts of these trichothecenes v/ere injected into hen's eggs. If a quantity corresponding to LD,._ value was injected the segmentation did not begin

or stop almost at the beginning. If an amount less than LDtQ was injected the segmentation stopped on the 5 th or 10 th day or the embryo could not hatch out of the egg.

Because trichothecenec are very stable in egg they could show their toxic properties for a long time and they are toxic much lower concentration than their 1D<-Q value moreover they could kill the embryo even through the goose.

P56 PATHOLOGY OF MYCOTOXINS AND PHYCOTOXINS

11-16 TOXICITY OF ALTERNARIA TO CHICKENS

W.L. BRYDEN, K.D. BARROW1 and D.A.I. SUTER2

Department of Animal Husbandry, University of Sydney, Camden, N.S.W., 2570, Australia. department of Biochemistry, University of New South Wales, Kensington, N.S.W., 2033, Australia. 2N.B. Love Industries, Enfield, N.S.W., 2136, Australia.

Alternaria are major fungal contaminants of many cereal grain crops. Secondary metabolites of the genus are toxic to mice, rats, ducklings and chickens, and yet very little research has been conducted on the effects of these mycotoxins in domestic animals. In the present studies the response of chickens to sorghum contaminated with Alternaria prior to harvest and the effect of feeding purified toxins was evaluated.

In the first experiment day old male and female broiler chickens were fed diets containing either contaminated or unccutaminated sorghom. The mouldy sorghum was contaminated with alternariol (AOH; 10 mg/kg) and alternariol monomethyl ether (AME; 7 mg/kg). During the 5 weeks of the growth study the chickens appeared clinically normal but growth rate and food conversion efficiency were depressed (P<0.05). At necropsy, liver, spleen and pancreas weights were found to be increased, while the bursa was regressed (P< 0.05).

In the second series of experiments a number of isolates of Alternaria alternata were grown in bulk either on rice or modified Czapek Dox agar at 25°C for 28 days. Toxins were extracted by organic solvents and purified from both neutral and acidic fractions on a milligram scale by chromatography on silica gel. Final purification of AOH and AME was by HPLC. Chick bioassays of these toxins confirmed their low toxicity when compared with aflatoxin B}. Results of both studies will be presented along with chick bioassay results of subsequent toxin isolations.

P60 COMPARATIVE PERFORMANCE OP BOARS AND GILTS FED DEOXYNIVALENOL-CONTAMINATED DIETS

FRIEND, D.W., TRENHOLM, H.L., THOMPSON, B.K., FISER, P .S ., ANDHARTIN, K .E .

B.Sc, M.S., Ph.D- AGRICULTURE CANADA, ANIMAL RESEARCH CENTRE OTTAWA, ONTARIO, CANADA K1A 0C6 In recent studies at A.R.C., diets containing 70% wheat were fed to 30 kg growing pigs. When deoxynivaleno1 (DON)-contaminated wheat (5 mg/kg diet) was used, feed consumption was reduced 15% compared with controls; when the wheat diet was supplemented with Fusariurn inoculated corn (14 mg DON/ kg), consumption fell 50%. In the present study, DON-contaminated diets were fed to entire boars and gilts;the diets contained clean wheat (control), and contaminated wheat or inoculated corn as the DON source. The concentration of DON was as nearly as possible the same (4 mg/kg) in each contaminated diet. The experimental design was a split-plot with sex at the whole plot and diet at the split-plot level, totalling 18 pigs fed the DON- wheat diet (VW), 18 pigs fed the inoculated corn diet (IC) and 12 pigs fed the clean wheat control diet (CW). Pigs weighed approximately 2 3 kg at the start, feed intakes and bodyweights were recorded weekly; the diets were fed for 7 weeks at the end of which time each pig was necropsied. The weights of the major body organs were recorded and the observed condition of the cardiac, fundic and esophageal areas of the stomach wall was scored numerically. Tissue sections from the ovary and testis were fixed in Carnoy's solution and stained by a modification of the Feulgen reaction.

The effect of feeding DON-contaminated wheat was to reduce total feed intake by 23%, compared with the control diet; the reduction was more pronounced (30%) when feeding the IC diet. Weight gain data showed, significantly,the VW pigs gained 30% less and the IC pigs 72% less than the control pigs. Feed:gain ratios were 2.5, 2.8 and 8.2:1 for the CW, VW and IC diets, respectively. Boars gained more than gilts when fed the CW and VW diets, but less than gilts when fed the IC diet; none of these sex differences was significant (P>0 .05) . The feed and weight gain means did not reflect the considerable variation occurring among pigs fed the contaminated diets. This phenomenon of extreme variation in response to mycotoxin-contaminated diets has been reported by us before. There was a consistent trend for the organ weights at necropsy to follow that of the carcass weight, i.e. CW > VW > IC. Even when adjusted for carcass weight, liver weights were heavier (P<0.01) for the CW than for the VW and IC diets. The data showed pigs fed the IC diet to have lighter weight testes and ovaries than the controls (P<0.05). Dietary differences were shown by the necropsy scores for the fundic region (P<0.01) in which the IC diet caused less mucosal redness than either of the other two diets, and in the esophageal area where the IC and VW diets seemed to have maintained better, the integrity of the mucosa than the control diet. Histological examination of testis and ovary preparations is in progress.

P61 TOXICITY STUDIES ON TOXIN BE-4 FROM THE BLUE-GREEN ALGA, MICROCYSTIS AERUGINOSA

P.G. THIEL, K. JASKIEWICZ, J.E. FINCHAM, D.P. BOTES

National Research Institute for Nutritional Diseases, S.A. Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa.

Toxic blooms of Microcystis aeruginosa have caused several cases of animal poisoning in various parts of the world. Evidence from Australia (1) suggests that liver damage occurred amongst residents in a city during the period of a bloom of M. aeruginosa in the water supply reservoir. Only one study has thus far been done on the toxicity of M. aeruginosa to primates (2) where repeated doses of 200 mg/kg of lyophilized culture material proved to be lethal to vervet monkeys when administered by intragastric intubation. Toxicity studies with the purified toxin BE-4 (3) were done on rats, mice and primates. The lesions in rats and mice were basically similar to those described by Falconer et^ al. (4) causing haemorrhage in the liver as well as necrosis of hepatocytes. Focal degenerative changes were also seen in the myocardium. From the primate experiments it appears that primates are more resistant to toxin BE-4 than rodents as judged by histological investigations on tissues as well as levels of serum enzymes indicative of liver damage.

1. Falconer, I.R., Beresford, A.M. and Runnegar, M.T.C. Med. J. Australia 1, 511 (1983).

2. Tustin, R.C., van Rensburg, S.J. and Eloff, J.N. 2H: Liver. Saun- ders, J. and Terblanche, J. (eds), S.A. Medical Research Council (1973).

3. Botes, D.P., Viljoen, C.C., Kruger, H., Wessels, P.L. and Williams, D.H. Toxicon 2£, 1037 (1982).

4. Falconer, I.R., Jackson, A.R.B., Langley, J. and Runnegar, M.T. Aust. J. Biol. Sci. 34, 179 (1981).

P62 CHRONIC EXPERIMENTAL AFLATOXICOSIS (B1) IN DOGS

*T.W. NAUDE, P. BLAND VAN DEN BERG, F. REYERS & R.C. TUSTIN "B.V.Sc, M.Sc.(Agric) Faculty of Veterinary Science, University of Pretoria, P.O. Box 12580, 0110, ONDERSTEPOORT, R.S.A.

An extensive outbreak of suspected aflatoxicosis in dogs was traced to a commercial brand of dog pellets containing from £.100 to 300 ppb (pg/kg) aflatoxin in different batches. The allowable upper limit in dogfood in South Africa is 50 ppb.

Only data on the acute LD of aflatoxin in dogs could be obtained and this was in the order of 0,5-1 mg/kg in single doses.

A pilot trial was conducted with 3 groups of beagles ( a dog and bitch in each group). Zero, 250 ppb and 500 ppb aflatoxin B1 was artificially mixed into a known aflatoxin-free commercial dogmeal and fed to them for a maximum of 6 months.

The control group was unaffected. At 500 ppb both dogs died rather suddenly £. 2 months after total cumulative dosages of 0,6 and 1,2 mg aflatoxin Bi/kg in the dog and bitch respectively. At 250 ppb the dogs succumbed after 5i and 7 months at cumulative dosages of 0,9 and 1,8 mg/kg in the dog and bitch respectively.

Aflatoxicosis was confirmed macro- and histopathologically. Clinical chemistry on the 500 ppb group revealed one severe, lethal bout of hepatocellular disease. The 250 ppb group showed several bouts of hepatocellular damage with progressively decreasing maximum enzyme levels.

If the published acute LD in dogs is compared with the chronic LD obtained in this pilot experiment, aflatoxin B1 appears to be significantly cumulative in this species.

P63 AN EXPERIMENTAL MVCOTOXICOSIS IN SHEEP AND GOATS CAUSED BY DRECHSLERA CAMPANULATA, A FUNGAL PATHOGEN OF GREEN OATS

D.J. SCHNEIDER1, W.F.O. MARASAS2, M. COLLETT^, G.C.A. VAN DER WESTHUIZEN4, P.S. STEYN5, R.M. HORAK^ Regional Veterinary Laboratory, Private Bag X5020, Stellenbosch 7600. Nation- al Research Institute for Nutritional Diseases, P.O. Box 70, Tygerberg 7505. 3Department of Pathology, Faculty of Veterinary Science, University of Pretoria, Pretoria 0002. 4Plant Protection Research Institute, Private Bag XI34, Pretoria 0001. ^National Chemical Research Laboratory, CSIR, P.O. Box 395, Pretoria 0001.

Field outbreaks of a syndrome of unknown etiology in the south-western Cape Province associated with the ingestion of green oat (Avena sativa) leaves were characterised by diarrhoea, photosensitivity and mortality in goats and diarrhoea and milkdrop in cows. In both cases, conspicuous reddish-brown leaf spots were seen on the oat plants suspected of being toxic and the plant pathogenic fungus Drechslera campanulata (teleomorph Pyrenophora semeniperda) was isolated from these spots. Pure cultures on autoclaved maize of 5 strains of jD. campanulata, isolated from oat leaves implicated in both field outbreaks and a Canadian isolate, proved to be highly toxic to ducklings, goats and sheep. Characteristic clinical signs of the fatal mycotoxicosis caused by El. campanulata culture material in goats and sheep were anorexia, apathy, diarrhoea and ruminal stasis. Necrosis of the forestomach was the most characteristic gross pathological change. Histopathological findings included mild focal erosions to severe, diffuse coagulative necrosis of the mucosa in the rumen, reticulum and omasum and congestion and haemorrhages in the abo- masum. Cytochalasin B was isolated from the toxic culture material of all 5 strains of £. campanulata and the highest yield (2.8 g/kg) was obtained from strain MRC 2855. Dosing of a sheep (29 kg) over a period of 3 days with 5 g of crystalline cytochalasin B isolated from this strain caused a transient diarrhoea on the second day but no other clinical signs. Thus cytochalasin B was apparently not responsible for the toxicity of I), campanulata. Further investigations are in progress to isolate and characterise the responsible mycotoxin(s). These results provide circumstantial evidence that green oat leaves infected by D. campanulata may cause outbreaks of a mycotoxicosis in grazing animals.

P64 NATURALLY OCCURRING TOXICANTS IN HUMAN AND ANIMAL HEALTH

J1-J16 OCCURRENCE OF fJ/aiÄJüW-MYCOTOXYNS IN CEREALS, IN ITALY

BOTTAL ICO A., A.LOGRIECO, A.VISCONTI, and M.SOLFRIZZO

Dipartimento di Patologia végétale dell'Université degli Studi, and Istituto Tossine e Hiootossine da parassiti vegetali del Consiglio Nazionale delle Ricerahe, Via G.Amendola 197/F, 70126 Bari, Italy.

Surveys of field and commercial cereal samples collected all over Italy for many years to investigate the presence of zearalenones, trichothecenes and moniliformin, indicated the occurrence of zearalenone and deoxynivalenol in Fuaariwn-infected corn ears and, to a much lesser extent, in commercial corn grains. The zearalenols (diastereomeric mixture) along with zearalenone and deoxynivalenol, were found to be associated with Fusarivon stalk rot in Corn. Among the twelve Fusariim species isolated from cereals (vegetative parts and kernels), the toxigenic ones were F.eulmorum (zearalenone, zearalenols, deoxynivalenol, and 3-acetoxydeoxynivalenol), F.graminearvm (zearalenone, deoxynivalenol, and 3-acetoxydeoxynivalenol), F.equiseti (zearalenone, and zearalenols), F.moniliforme var. sübglutinans (moniliformin), F.avenaeeim (moniliformin), and F.sporotriahioides (T-2 toxin, HT-2 toxin, acetyl T-2 toxin, T-2 triol, and neosolaniol).

P65 NATURAL OCCURRENCE OF TRICHOTHECENES AND ZEARALENONE IN FEEDSTUFFS IN THE FEDERAL REPUBLIC OF GERMANY

GAREIS, M., J. BAUER AND B. GEDEK

Dr. Institute for Medical Microbiology, Infectious- and Epidemic Diseases, University Munich, Veterinary Faculty, Veterinarstr. 13, 8 Munich 22, F.R.G.

Samples of feedstuffs (commercial source or submitted from veterinarians from suspected cases of intoxications) have been analyzed since 1982 for the natural occurrence of trichothecenes (type A) and zearalenone. Extraction and clean-up of the samples were made by modified methods of Romer et al. (1978) and/or Eppley et al. (1974). The guinea pig skin bioassay was used for screening trichothecenes. Type A trichothecenes (T-2 toxin,HT-2 toxin,T-2 triol,verrucarol,neosolaniol,diacetoxyscirpenol,monoacetoxyscirpenol) were detected as their TFA-esters after reaction with n-methylbis(trifluoroacetamide) by gas liquid chromatography - flame ionization detection using DB-5 capillary columns. Determination was carried out by GLC - quadrupole mass spectronetry (full scan and selected ion monitorinq of two or three key ions). In comparison to the electron impact ionization the chemical ionization technique in methane showed to be more suitable as fragmentation of the toxins is weak and molecule ions (protonated) were detected for all trichothecenes analyzed. Extracts from 42 samples out of 295 (14.2%) caused a dermal reaction by the skin bioassay. Substances interfering with T-2 toxin and HT-2 toxin caused 5 false positives by GLC-FID analysis. Nine samples contained at last trichothecenes as determined by mass spectrometry: T-2 toxin in mixed feed (65ug/kg), oats (80 and 86ug/kg) and wheat (100ng/kg); DAS in mixed feed (125ug/kg) and wheat (50ug/kg); neosolaniol in oats (310 and 350ug/kg); HT-2 toxin in oats (700ug/kg). Oats proved to be more frequently contaminated as compared to the other cereals and the analyzed mixed feed. Analysis of zearalenone was performed by high-pressure liquid chromatography with fluorescence detection (excitation 275 nm; emission 445 nm). A 5-u.m LiChrosorb RP-18 column was used and eluted at a rate of 0.4 ml of solvent per min. The mobile phase was initially 50% methanol, which was linear programmed to 80% methanol in 25 min. Forty-four out of 421 samples (10.5%) contained zearalenone at levels ranging from 1.0 to 179.9 ug/kg (mean cone. 16.6 ug/kg). The toxin was found in 11.7% of 290 samples of mixed feed (1.0-65.4 ug/kg) and 6.7% of 89 grain samples (1.0- 179.9 ug/kg). Zearalenone was also detected in one sample each of crushed soybeans (3.0 ugAg), concentrated protein (21.5 ugAg) » maize silage (5.1 (ig/kg) and grass silage (9.0 ugAg).

Eppley et al., 1974: J. Ass. Off. Anal. Chem., 57, 633-635 Romer et al., 1978: J. Ass. Off. Anal. Chem., 61, 801-808

P66 TOXICITY OF FUSARIUM SPECIES ASSOCIATED WITH SEEDS OF ANNUAL MEDICAGO SPECIES IN SOUTH AFRICA

1 2 2 3 S.C. LAMPRECHT , W.F.O. MARASAS , P.G. THIEL , D.J. SCHNEIDER Plant Protection Research Institute, Private Bag X5017, Stellenbosch 7600. 2 National Research Institute for Nutritional Diseases, P.O. Box 70, Tygerberg 7505. Regional Veterinary Laboratory, Private Bag X5020, Stellenbosch 7600.

Annual Medicago species (medics} are important pasture legumes in the winter rainfall areas of the south-western Cape Province. During the dry summer months (October to March) sheep on some farms survive almost exclusively on the medic seedpods. Six Fusarimn species were isolated from surface- sterilised medic seeds collected in the Swellendam district during 1984: F^. acuminatum, B\ avenaceum, f\ equiseti, F_. graminearum Gr. 1, £. reticulatum and F_. sambucinum. The predominant Fusarium species isolated from seeds collected during the summer was £. acuminatum which was isolated from 22.3% of seeds examined. Cultures on autoclaved maize of 5 single-conidial isolates of each of the 6 Fusarium species were tested for toxicity in day-old Pekin ducklings. Maize cultures were incubated at 25°C for 21 days and fed £d lib, to groups of 4 ducklings for 14 days. With the exception of 4 isolates of F_. equiseti that proved to be nontoxic, all isolates were highly toxic and caused 100% mortality of ducklings. Culture material of one isolate of each species was also dosed to sheep by means of a stomach tube. The isolate of F_. acmni- natum proved to be extremely toxic to sheep and a single dose of 5 g of culture material Ag body weight caused death within 2 hours. Culture material of £. avenaceum administered twice at a dosage rate of 5 gAg caused death within 48 hours. Analysis of the £. acuminatum and F_. avenaceum culture material by high performance liquid chromatography revealed the presence of 4.2 and 0.5 gAg of monilifonnin, respectively. Dosing of a sheep with a single dose of 10 mgAg of crystalline moniliformin resulted in death within 18 hours. These results indicate that toxigenic Fusarium species associated with medic seeds present a potential hazard to grazing sheep.

P67 NATURAL OCCURRENCE OF xMYCOTOXINS IN AUSTRIA J.LEIBETSEDER, J.BOHM, A.M.ABDELHAMID AND CH.NOONPUGDEE Professor, D.V.M., engineer; D.agr.; D.V.M.

Institute of Nutrition, University of Vet.Medicine Vienna Linke Bahngasse 11, A-1030 Wien, AUSTRIA

Because of the moderate climate fusariotoxins (zearalenone (Z.) vomitoxin (V.)) and ochratoxin A (0.) are the most frequently occurring mycotoxins in Austria. Aflatoxins (A.) are detected almost only in feed imported from tropical or subtropical areas. Z. and V. cause quite often clinical symptoms in live-stock and tremendous economical losses in animal production. Since 1979 2752 samples were analyzed for mycotoxins by HPLC and HPLC-GC methods. The samples were transmitted partially by feed manufacturers for surveying partially by veterinarians and fanners under suspicion of mycotoxicosis in the live-stock. Table 1: Samples of the survey (A = number of samples, B=% positive, C = % of B containing 0,1-1 ppm, D = % of B containing > 1 ppm) Feeds Zearalenone Vomitoxin Ochratoxin A Aflatoxins A B C D A B C D A B C D A B C D maize 755 31 23 6 661 66 62 26 26 12 33 0 15 0 0 0 barley 134 16 19 0 86 24 76 10 32 16 0 0 4 0 0 0 oat 268 16 25 0 196 37 62 21 37 38 14 0 9 0 0 0 rye 16 50 0 0 12 33 75 0 10 10 0 0 9 0 0 0 wheat 204 31 33 5 112 75 68 18 35 6 0 0 15 0 0 0 corn silage 183 63 22 0 157 71 63 27 7 29 0 0 4 0 0 0 peanut meal 48 77 24 62 mixed feed 214 22 25 4 252 59 61 21 23 13 0 0 75 29 27 18 others 70 21 13 7 64 19 50 0 6 0 0 0 58 14 38 13 Table 2: Suspicious samples (A,-B, CD as in Table 1 ) Feeds Zearalenone Vomitoxin Ochratoxin A Aflatoxins maize 51 37 26 0 35 46 63 19 2 0 0 0 - - - - barley 72 14 30 0 16 13 100 0 8 25 100 0 3 0 0 0 oat 72 13 11 0 20 40 63 38 2 0 0 0 1 0 0 0 rye 3 0 0 0 wheat 18 22 0 25 4 25 100 0 ------corn silage 57 49 21 4 50 70 71 6 3 0 0 0 - - - - peanut meal ------5 60 0 0 mixed feed 166 25 15 5 125 38 49 9 9 100 33 0 32 16 60 0 others 28 18 40 0 14 14 100 0 3 33 0 0 14 29 25 0

P68 A STUDY ON MILK CONTAMINATION BY AFLATOXIN Ml IN A RESTRICTED AREA IN CENTRAL ITALY

Boccia A., Micco C, Miraglia H., Scioli M.

ISTITUTO SUPER!ORE DI SANITA1 - V.le Regina Elena, 299 00161 ROHA

Fifty-two samples of raw-whole milk were taken in the period February-June 1984 fro« different farms in a restricted zone, and were examined for their content in Aflatoxin Ml (AFM1) according to the method of Blanc B. The »ycotoxin was not detectable in 25 samples (49X), and was in the range 5-146 ppt in the remaining 27 samples (51X). The higher values correspond to the first months, which are colder, when there is a larger use of concentrates meal and also a greater chance of mould growth on stored forage. The overall level of contamination is however rather low, three samples only being contaminated at levels between 80 and 160 ppt. No relationship was found between the composition of feedstuff used in the farms and level of contamination, but peanut flour was present only in one case. A relatively higher percentage of contamination was found among 18 commercial samples of milk, 6 of which presented undetectable levels of AFM1. The other 12 contained between 5 and 30 ppt of AFM1. Furthermore, 5 samples of raw-whole milk were taken from bulky lots collected from different farms near Rone. They contained averagely 6 ppt of AFM1 (span 0-10 ppt). In conclusion, the results indicate that the contamination of milk by AFM1 is often found but mostly at low levels.

P69 LONG TERM ADMINISTRATION OF LOW DOSES OF MYCOTOXINS IN POULTRY. RESIDUES OF AFLATOXIN Bl AND ITS METABOLITES IN BROILERS AND LAYING HENS.

Micco C.1. Hiraglia M.1, Benelli L1., loppolo A.2, Mantovani Al.2. Stasolla D.2 (1) Laboratório Aliroenti - (2) Servizio Stabulario ISTITUTO SUPERIORE DI SANITA' - V.le Regina Elena, 299 - 00161 ROMA

The occurrence and importance of residues of Aflatoxin was investigated in a situation approaching contamination found in the field (i.e. small doses for long periods). Residues < 0.1 ppb were found in target organs (liver and kidneys) of broilers and hens fed, respectively, for 64 and 169 days with commercial diets contaminated with 50 ppb of Aflatoxin Bl. Broilers and hens withdrawn from contamination for, respectively, 28 and 82 days did not show residues. Treatment did not cause apparent adverse effects on the health of the animals. It is possible that edible tissues from apparently healthy poultry constitute a source of Aflatoxin intake for humans; however, it does not seem to be a very important public health risk.

P70 AFLATOXIN CONTAMINATION IN ZAMBIAN FOODS AND FEEDS: PRELIMINARY STUDIES

H NJAPAU, E WALUBITA, A C BAYLEY and FLC KAPUMPE B.Sc. B.Sc. B.A., M.B., B.Chir.F.R.C.S National Council for Scientific Research, P 0 Box 350049, Chilanga, Zambia

550 human food samples varying in type (maize based, beans and milk), and source (village and industry) were collected, from Katete, Chipata and Lusaka districts of Zambia, towards the end of rainy seasons of 1982 and 1983. Analysis for aflatoxins revealed a low incidence (7%) with a range of 1 to 50 ppb and a mean contamination lying between 16.38 and 32.92 ppb total aflatoxin. Some isolated cases falling outside this range were recorded.

During 1984, 34 poultry feed samples were collected from the Lusaka district and analysed for aflatoxin and diplosporin. This survey showed a higher incidence (80%) of aflatoxin contamination, despite a lower average contamination (12.43 <_ u _< 21.47 ppb total aflatoxin). 70% of these samples were positive for diplosporin and almost all contained spores of Fusarium, Rhizopus, Penici- llium and Aspergillus. In addition the majority of the samples had low levels of calcium, phosphorus and magnesium. In this district, most farmers store (before exhaustion) their feeds for a period of 7 to 10 days, at an average moisture of 4.6 j* 1.3%. Indications are that, contamination may have occurred at an earlier stage prior to milling. There is, therefore, a need to study the effects of low nutrient levels in combination with the probable synergestic effects of mycotoxins, on poultry.

P71 VARIATION IN THE LEVELS OF AFLATOXIN IN COW MILK CONSUMED IN THE CITY OF SAO PAULO, BRAZIL

SABINO, MYRNA; PURCHIO, A.; ZORZETTO, M.A.P. Chemist, M.S in Food Science Instituto Adolfo Lutz - Av. Dr. Arnaldo, 355 - Sao Paulo - Brazil

100 samples of commercially avaiable cow milk, collected in the state of Sao Paulo during the period from July/79 to September/81, were analysed for determination of the levels of aflatoxins Ml and M2 by the method of the AOAC. This investigation was also un dertaken in 50 samples of cow milk proceeding from two farms of the Medio Vale do Paraiba, the animals of which had ingested stored feed. Aflatoxin Ml was detected in only one sample of commercially avaiable cow milk, while those from the farms were found to contain a minimum of Q ,1 ug/1 and a maximum of 1,68 ug/1. Reversed phase high pressure liquid chromatography (HPLC) was u- sed for characterization and guantitation of the aflatoxin Ml.

P72 A RATIONALE FOR THE CONTROL OF AFLATOXIN IN HUMAN FOODS

LEONARD STOLOFF

Consultant, Sliver Spring, MD, USA

The regulation of aflatoxin in foods is part of the risk management process that, in it's initial stages In the period 1964-1969, required considerable speculation to supplement the meager knowledge that formed the basis of the risk assessment. The regulatory stance of the U. S. Food and Drug Adminis- tration is still based on on that original speculation, even though a risk assessment using current knowledge does not support the assumptions that were used. The original assumption that aflatoxin might be a potent hepato- carcinogen for humans cannot be supported by the considerable data on aflatoxin that has since evolved. But there is clear evidence that humans can suffer acute liver damage from aflatoxin ingestion. By considering the relation between the dose levels at which the acute effects are manifest, and the frequency distribution of the contamination levels in the ingested foods, a maximum tolerated level can be established that best protects the consumers health, pocket book and food choices.

P73 I CO-EXISTENCE OF NIVALENOL, DEOXYNIVALENOL AND ZEARALENONE IN CEREAL GRAINS

Y. UENO1), U.S. LEE1*, T. TANAKA2) and A. HASEGAWA2)

Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science , and Division of Food Chemistry, Public Health Research Institute of Kobe City2) 1) 2) Ichigaya, Tokyo 162 and Chuo-ku, Kobe 650, Japan A simple and sensitive method has been developed for the simultaneous analysis of trichothecenes (nivalenol NIV and deoxynivalenol DON) and zearalenone (ZEN) in cereals. These toxins were extracted with acetonitrile/water (3:1), defatted with n-hexane and purified by a two-step chromatographic procedure using Florisil and Sep-pak columns. The column eluates were dissolved in methanol, and the amounts of NIV and DON were quantitated by GC- ECD and confirmed by GC/MS. The detection limits were 2.0 jig/kg with recoveries over 87%. The amount of ZEN was determined by HPLC attached with a fluorescence detector (Nucleosil 50-10 column and 90% water-saturated chloroform-cyclohexane-acetonitrile-ethanol, 50:15:2:1). The detection limit was 1 jig/kg. Employing this improved method, the amounts of NIV, DON and ZEN in cereal grains produced in Japan, Korea and China (Taiwan) were analysed. The data indicated the co-existence of these mycotoxins in barley, wheat, oat and others, and their incidence and levels were significantly high. The lethal toxicity and cytotoxicity of NIV were about ten-fold higher than those of DON. The contamination of cereals with NIV is therefore a great problem in food hygiene.

P74 AFLATOXIN QUALITY CONTROL ON SOUTH AFRICAN GROUNDNUTS

L.H.W.VERHOEF, P.J. GROBLER AND D. DE JAGER

OILSEEDS BOARD, BOX 211, PRETORIA, OOO1, SOUTH AFRICA

The South African groundnut is known worldwide for its high quality standards and excellent taste. The high quality standards of groundnuts mainly revolves around the outstanding precautions the Oilseeds Board, as agent of the producer, takes to reduce the aflatoxin content. The pros and cons of various techniques used by the Oilseeds Board to eliminate aflatoxin contamination will be discussed.

The current physical grading regulations will be presented in short. The value of the colour and mechanical separation techniques as well as the hand picked method will be highlighted.

The successful use of these cleaning techniques produces highly contaminated groundnuts as a waste. The oil in the contaminated groundnuts is then recovered by expressing whilst the majority of the aflatoxin contamination will end up in the meal. The contaminated meal is then treated with ammonia to detoxify the aflatoxin. The emphasis of the detoxification process is placed on the techno- economical aspects of it, as it was adopted and applied for South African needs.

P75 IDENTIFICATION OF VARIOUS T-2 TOXIN METABOLITES IN CHICKEN EXCRETA AND TISSUES

ANGELO VISCONTI and CHESTER J. MIROCHA

Istituto Tossine e Micotossine, C.N.R., Via Aroendola 1971F, 70126-Bari, Italy

Gas chromatography - mass spectrometry has been used for identification of various T-2 toxin metabolites in chicken excreta and organs 18 hr after intraperitoneal injection of 3.5 mg of toxin per kg of body weight. The simultaneous detection of 11 trichothecenes present in a very complex matrix (such as chicken feces) could be performed by GC/MS using a convenient selection of monitored ions. No trichothecenes were detected in the heart and kidney, and only traces in lung. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also present in the liver. 3'-hydroxy HT-2 toxin (1.4 ppm), HT-2 toxin (0.2 ppm) and T-2 triol (0.2 ppm) were the major metabolites detected in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15-acetoxy-T-2 tetraol and T-2 tetraol, present at levels of 3.6, 2.1 and 0.7 ppm, respectively) we found 3'- hydroxy HT-2 toxin (8.2 ppm), 3'-hydroxy T-2 toxin (3.3 ppm), 4-acetoxy T-2 tetraol (1.0 ppm), and traces of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin and T-2 triol. Unmetabolized T-2 toxin (0.4 ppm) and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin.

P76 SURVEY OF MILK FOR AFLATOXIN

VISCONTI A., A.BOTTALICO, and M.SOLFRIZZO

Istituto Tossine e Mieotossine da parassiti vegetali del Consiglio Nazionale delle Riaerahe, Via G.Amendola 197/F, 70126 Bari, Italy.

A survey of seventy samples of farm and commercial milk and milk products, carried out in 1983-84 in Bari (Apulia, Italy), showed the following incidence of aflatoxin Mi

No. of No. of Range of aflatoxin level (ng/1) Milk sample sample positive analyzed sample 1-10 11-100 >100

Raw (farm) 19 6 3 3 - Whole pasteurized 2 2 2 - - U.H.T. min 3.5% 9 9 3 6 - U.H.T. max 1.8* 18 18 5 13 - U.H.T. max 0.3% 9 8 2 5 1 Milk cocoa drink (U.H.T.) 2 2 - 2 - Dietetic milk 2 2 - 2 - Infant milk powder formulae 9 9 4 5

P77 STUDIES ON THE TOXICITY OF SUBSTANCES ISOLATED FROM FUNGI GROWING ON PLANT MATERIAL

H. BRESCH H.K. FRANK R. MUNZNER Dr. rer. nat. Prof. Dr. rer. nat. Dr. rer. nat. Federal Research Centre for Nutrition, Engesser Strasse 20, D-75OO Karlsruhe 1, Fed. Rep. of Germany We are studying the toxic effects of secondary metabolites of different fungi growing as parasites on fruit, vegetables and cereals and which may cause storage decay. At present we are particularly interested in the products synthetized by Alternaria -, Venturia - and Botrytis species.

Alternaria spp. grow mainly on stored fruit and vegetables, but also on wheat ears in harvest periods with high relative humidity. Venturia inaequalis produces scab on apples and pears. Botrytis cinerea is predominantly found on stored fruit and vegetables and causes the so-called noble rot in grapes. Alternaria toxins have been known for long; whereas the chemical structure of the majority of metabolites has been described, little is known of the long-term toxicity of these products. We are studying alternariol and alternariol monomethyl ether, the main components of toxic metabolites produced by the majority of Alternaria species.

So far it has been unknown whether strains of Venturia produce toxic metabolites. We isolated five compounds each of 3 strains. Presently we are trying to get insight into the chemical structure of these and are subjecting the individual compounds to the toxicity tests mentioned below.

Of Botrytis, six fractions were isolated which are being studied as described before for Venturia. We are presenting first results obtained by the fish embryo- larval test and by the chicken embryo test. Although short-term tests, they still provide valuable information with regard to the toxicity of a certain compound since they include a great many differentiation reactions during the early development of the test organisms. In addition, the substances are also being tested for mutagenic effect.

P78 A STUDY OF STORAGE PROBLEMS UNDER LOCAL CONDITIONS WITH OR WITHOUT THE ADDITION OF SODIUM CARBONATE SPECIFICALLY OF RICE AND MAIZE IN RELATION TO MOULD GROWTH AND AFLATOXIN FORMATION

K TOPSY, G K CHUKOWRY, A REESAUL and M KASSEE Professor Doctor 17 Dr Moliere Street/Vuillemaln Beau-Bassin, Mauritius

Moulded and/or aflatoxin contaminated food and feedstuffs represent health hazards to humans and animals. Bad agricultural practice and storage conditions are the more important incriminating factors, even though driers, which raise production cost, are used at post harvest stage.

The antimould and antiaflatoxicogenetic effects of sodium carbonate in admixtures with rice and maize at post harvest and storage stages have been studied. The research programme has shown that these commodities with 202 + moisture contents stored at ambient temperatures under various hygroscopic conditions will be contaminated with moulds and aflatoxin B.. Such contaminations do not occur under similar conditions when these commodities are mixed with A to 6% sodium carbonate.

The data collected show that the sodium carbonate admixture process offers promising possibilities for post harvest and storage applications on the basis of technological and economic criteria as well as from the point of view of safety and efficiency.

P79 INDEX OF AUTHORS

Page Abdelhamid, AM P68 Edgar, JA S8 Angsubhakorn, S P42 Eloff, J N P7 ApSimon, J W S1,P22,P4O Enders, C P43

Barkai-Golan, R P4 Fabbri, A A P2.P3 Barrow, K D P60 Fanelli, C P2.P3 Bata, A P25.P56 Färber, J M P5 Bath, G F S6 Fincham, J E P62 Bauer, J P43.P66 Finotti, E P2,P3 Bayley, AC P34.P71 Fiser, PS P61 Bechet, J P49 Franck, B S9 Belden E L S27 Frank, H K P78 Benelli, I P70 Friend, D W P61 Bennett, J W S2 Friesen, MD P30 Berry, R K P8 Fu, T C S20 Bhamarapravati, N .... P42 Blackwell, B A S1,P12,P13,P22 Bland van den Berg P . P63 Gareis, M P43.P66 Boccia, A P69 Gathercole, PS P47 Böhm, J P35.P68 Gedek, B P43.P66 Bonera, N B P6 Gelderblom, W C A ... P15 Botes, D P S3.S15.P62 Gilbert, J P31 Bottalico, A P65.P77 Glinsukon, T P32 Bresch, H P78 Gorst-Allman, C P ... P16 Bryden, W L P60 Goszcz, H P41 Biichi, G PLI Greenhalgh, Sl,P12,P22,P40 Grobler, P J P36.P75 Candllsh, A A G P26 Carini, E P37 Halevy, S P54 Carmichael, W W S4.P44.P45 Haroon, Y P28 Casinovi, C G P14 Harris, C M PL3 Chang, S C Pll Harris, T M PL3 Chu, FS S5 Hartin, K E P61 Chukowry, G K P79 Hasegawa, A P74 Clarke, K P29 Hesseltine, C W PL4 Coetzer, JAW S6 Hofmeyr, J H S P47 Cole, R J S7 Holzapfel, C W S10 Collett, M P64 Horak, RM Pl,P10,P21,P64 Hsieh, D P H SU Huit, K S12 Dashek, W V P27 Day, W R P28 Ichinoe, M S18 de Jager, D P75 Ioppolo, A . P70 de Jesus, A E P1.P10 Isobe, M S13 Demain, A L PL2 DU, L A P34 Dillen, J L M P24 Jaskiewicz, K P62 Dirr, H W P46 Joffe, A Z S16 Dixon, S N P29 Johannsen, E P48,S15 Dutton, M F P8,P53,P9 Page

Kaggwa, J S N P34 O'Rear, C E P27 Kapumpe, F L C P71 Ohtomo, T P17 Kassee, M P79 Osborn, M P50 Keilerman, T S S6.S14 Kendrick, B PL5 Pachter, R P18 Kfir, R S15.P48 Padova, R P4 Kiessling, K-H PL6 Panfili, G P3 King, R R SI Paré, J R J S1.P40 Kriek, N P J S16 Park, D L S26 Krohrs, H P33 Parker, I P31 Krüger, G H J P7 Fassi, S P2.P3 Kubacki, S J S17 Paster, N P4 Kuo, L S20 Patel, M PI Kurata, H S18 Pauisch, W E PL13 Pier, A C S27 Pieretti, S P2 Lacey, J S19 Pietri, A P37 Lafontaine, P P40 Piva, G P37 Lamprecht, S C P67 Plattner, R D S28 Lappas, NT P27 Pohland, A E S 26 Lawson, EM...... PI Potgieter, D J J .... P5O.P51 Lee, Ü S P74 Potgieter, M S29.P19 Leibetseder, J P35,P68 Potgieter, H C P51 Levandier, D P22 Potts, D P23 Lewko, W M PSI Prevot, A S30 Lin, MY S20 Purchio, A P72 Ling, K H S20 Liou, H H S20 Lipowska, T P41 Quinta, ML P39 Llewellyn, G C P27 Logrieco, A P65 Lovelace, C E A ..... P34 Rabie, C J S31.P16.P21 Radies, L P14 Reesaul, A P79 Mackie, R I P53 Reyers, F P63 Mae s, C M P21 Rossi, C P14 Mahmood, N A P44.P45 Mantle, P G S21 Mantovani, Al P70 Sabino, M P72 Marasas, W F 0 P16.S22.P21 Sadler, W S6 P64.P67 Sahaphong, S P42 Mayfield, JE P27 Sanders, GW P5 Meier, R-M S1.P22 Sathiropas, P P42 Helmed, R N P55 Sato, H PI 7 Meyer, S A F P36 Scannato, GT P6 Micco, C P69.P70 Schabort, J C ...... P46.P52 Miller, J D S1,P12,P13,P22 Schiefer, H B PL8 Miraglia, M P69.P70 Schmidt, H R P38 Mirocha, C J S23.P76 Schneider, D J P64.P67 Moore, R E PL7 Scioli, M P69 Münzner, R P78 Scott, W E S33 Scott, P M P5.S32 Natori, S S24 Sene, S ...... P49 Naudé, T W P63 Serralhelro, M L .... P39 Nazzaro-porro, M .... P3 Seto, H S 34 Nelson, P E S2S Shepherd, M J P31 Njapau, H P71 Shimizu, Y PL9 Noonpugdee, C H P35,P68 Slason, I W P50 Simpson, T J ...... S3S Page Sinlapanapaporn, S .... P32 Walubita, E P71 Smith, J E P26 Watson, WH S43 Solfrizzo, M P65.P77 Weber, K P50 Sriurairatana, S P32 Wei, Y H PH Stark, A A S36 Wei, R D Pli Startin, J R P31 Westlake, K P53 Stasolla, D P70 Wilson, T M S25 Steyn, P S P10.P16.P18 Wittliff, J L P51 P19,P20,P21,P50,P6A Stimson, Vf H P26 Stoloff, L S26.P73 Yagen, B S16,P54,P55 Suami, T S37 Yoshida, K P17 Subryan, L P23 Young, J C P23 Sullivan, J J S38 Surabu, Z L P49 Suter, D A I P60 Zamir, L 0 S45.P12 Zorzetto, MAP P72

Tamm, Ch PL10 Tanaka, T ...„...., P74 Taylor, A S1.P22 Thiel, P G S39,P15,P47 P62.P67 Thompson, B K P61 Thonart, P P49 Thuvasethakul, P ...... P42 Tönsing, EM P50 Topsy, K P79 Townsend, C A ...... PL11 Trenholm, k L P61 Tsai, MC S20 Tuinman, A A S40 Tustin, R C P63 Tuttobello, L P14 J Ueno, Y PL12.P74 Vaamonde, G P6 van Aswegen, C H P51 van der Merwe, K J .... P15 van der Watt, J J P51 van der Westhuizen, G C A P7.P64 van Egmond, H PLI3 van Heerden, F R P20 van Rensburg, S J ..... S16,S41 van Rooyen, P H P24 Vanyi, A P25.P56 Var man, M J S27 Vederas, J C S42 Verhoef, L H W P36.P75 Vermeulen, N M J P51 •3 '• Visconti, A P65,P76,P77 Viviers, J P52 Vleggaar, R P10.PL14.P16 P20,P21,P48