Administered 3H-Labeled T-2 Toxint

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1980, p /80/ /06$02.00/0 Vol. 39, No. 6 T-2 Metabolites in the Excreta of Broiler Chickens Administered 3-Labeled T-2 Toxint TAKUMI YOSIZAWA,: S. P. SWANSON, AND C. J. MIROCA* Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota A method for the detection of T-2 metabolites was developed and applied to analysis of metabolites in excreta of broiler chickens administered 3-labeled T- 2 toxin. The method used acetonitrile extraction and partitioning with petroleum ether followed by chromatography on Amberlite XAD-2, Florisil, and Sep-Pak C18. The recovery of T-2 toxin added to the chicken excreta was 73% at a concentration of 0.2,ug/g. About 80% of orally administered 3-labeled T-2 toxin was rapidly metabolized to more polar derivatives and eliminated in the excreta within 48 h. T-2 toxin, T-2 toxin, neosolaniol, and T-2 tetraol were detected at 0.06 to 1.13% of the total dose, 48 h after administration. Eight unknown derivatives, named TB-1 to TB-8, were quantitatively more significant than the metabolites above. TB-3 and TB-8 represented about 12 and 25% of the total dose, respectively. One of the metabolites (TB-6), 1.5% of the total dose, was identified as 4-deacetylneosolaniol (15-acetyl-3a, 4,8, 8a-trihydroxy-12, 13-epoxytrichothec- 9-ene). T-2 toxin (I, Fig. 1), 4fl,15-diacetoxy-8a-(3- methylbutyryloxy) - 3 a-hydroxy- 12, 13-epoxytrichothec-9-ene, is a toxic metabolite produced by Fusarium spp. and possesses various kinds of toxic effects in laboratory and farm animals. T- 2 toxin, as well as diacetoxyscirpenol, deoxynivalenol, and nivalenol, are some of the most important trichothecene mycotoxins occurring naturally in agricultural products (4, 5, 10, 12, 16, 18, 20). Serious mycotoxicoses, including moldy corn toxicosis in the north central United States (6) and Fusario-toxicosis in Canada (12) have been attributed to T-2-contaminated feed. The toxin is possibly involved in the bean-hull toxicosis of farm animals in the northern part of Japan (16). In addition, alimentary toxic aleukia, which had been a human health problem in Russia, was found to be associated primarily with ingestion of moldy cereals infected with T- 2-producing strains of Fusarium poae and Fusarium sporotrichioides (7, 16). Consequently, residues of T-2 toxin and its metabolites in animal products appeared to be an important human health problem. Recently, Chi et al. (2) estimated (based on radioactivity) that the muscle of broiler chickens contained 0.06 and 0.04,ug of T-2 toxin and its metabolites per g at 24 and 48 h, respectively, after dosing the birds transmission of radioactivity into eggs from laying hens administered 3-labeled T-2 toxin. Robison et al. (13) reported the transmission of T- 2 toxin into milk of a lactating cow intubated with T-2 toxin. Because of the high possibility of transmission of T-2 toxin and its metabolites into edible tissue of farm animals, the development of an analytical method for the detection of these mycotoxins in animal tissue and excreta is a prerequisite to not only metabolism studies, but also safety evaluation of animal foods for human health. In this paper, we describe the development of an analytical method for the detection of T-2 toxin and its metabolites and application of this method to analysis and identification of metabolites in the excreta of broiler chickens administered 3-labeled T-2 toxin. MATERIALS AND METODS Animal treatment. 3-labeled T-2 toxin (radiopurity, <99%; specific activity 100.6,uCi/mg) was synthesized by the method described by Wallace et al. (19) and was administered orally in a single dose of 1.6 mg/kg of body weight (3.53 x 10' dpm/kg) to 47-dayold broiler chickens, which had been fed a ration containing 10lg of nonradioactive T-2 toxin per g for 5 days before intubation of labeled T-2 toxin. Excreta were collected as described previously (2), flash frozen with 0.5 mg of 3-labeled T-2 toxin per kg of in liquid nitrogen, and stored at -20 C until required. body weight. Chi et al. (1) also investigated the Extraction and fractionation. The sample (25 g) t Paper No. 11,198, Scientific Joumal Series, Minnesota was extracted twice with 100 ml of acetonitrile in a Agricultural Experiment Station, St. Paul, MN blender and filtered by suction. The combined filtrate t Present address: Department of Food Science, Kagawa was partitioned with an equal volume of petroleum University, Kagawa, Japan. ether (bp 60 to 70 C) to defat, and the acetonitrile 1172

2 VOL. 39, 1980 T-2 METABOLITES IN CICKEN EXCRETA 1173 (I) (II) (III) (IV) (V) (VI) T-2 toxin T-2 toxin Deacetyl-T-2 toxin (Triol) Neosolaniol (NS) 4-Deacetyl-neosolaniol (TB-6) T-2 tetraol (Tetraol) 020R2 R' R2 FIG. 1. Chemical structures of T-2 metabolites. R3 COC2C(C3)2 layer was evaporated to dryness. The residue was dissolved in 10 ml of methanol, and 50 ml of water was added later. The mixture was concentrated to about 20 ml and chromatographed on XAD and Florisil columns by a modified procedure of Kamimura's (8) method (Fig. 2). The aqueous solution was charged onto an Amberlite XAD-2 resin column (1 by 14 cm, mesh, Mallinckrodt Inc., Paris, Ky.). The column was rinsed with 100 ml of water, followed by 100 ml of 90% methanol in water. The methanol eluate was concentrated to dryness in vacuo, dissolved in 10 ml of chloroform-methanol (3:1, vol/vol), and introduced onto a column containing Florisil (10 g, 1.5 by 12 cm, mesh, Fisher Scientific Co., Pittsburgh, Pa.), packed in chloroform-methanol (3:1, vol/vol), and topped with a layer of anhydrous sodium sulfate (5 g). The column was eluted with an additional 90 ml of chloroform-methanol (3:1, vol/vol) and then with 100 ml of methanol. The chloroform-methanol eluate was concentrated, dissolved in 2 ml of water, and introduced onto a Sep-Pak C18 cartridge (Waters Associates Inc., Milford, Mass.). The cartridge was developed with a step gradient which involved in sequence (2 ml each): water, 20, 50, and 70% methanol in water, and methanol. TLC analysis. Aliquots of the individual C18 eluates were spotted onto half of precoated silica gel 60 chromatoplates (5 by 20 cm, 0.25 mm gel thickness), and standard compounds were spotted onto the opposite half of the plates. The C18-50% and 70% MeO fractions were developed in chloroform-methanol (9:1, vol/vol), whereas the C18-20% MeO fraction was developed in chloroform-methanol (5:1, vol/vol). After development, the radioactive region of the plate containing the sample was covered with a glass plate, and the standards were sprayed with 20% sulfuric acid in methanol, and heated and made visible by means of an ultraviolet lamp. The bands corresponding to the following Rf values were scraped from the thin-layer chromatography (TLC) plates (Rf values in parentheses): T-2 toxin (0.68), TB-1 (ca. 0.62), T-2 toxin (0.45), and deacyl T-2 toxin (0.30) from the C18-70% MeO eluate; TB-2 (ca. 0.62), neosolaniol (0.51), TB- 3 (ca. 0.38), TB-4 (ca. 0.30), and TB-5 (ca. 0.22) from the C18-50% MeO eluate; TB-6 (ca. 0.45), T-2 tetraol (0.30), TB-7 (ca. 0.15), and TB-8 (0.00) from the C08-20% MeO eluate. The bands named TB-1 to TB-8 did not correspond to any known T-2 metabolites (Fig. 3). To obtain radiochromatographic profiles of the individual fractions extracted, aliquots of sample solutions were applied on high-performance silica gel TLC plates (200 Im gel thickness, Whatman Inc., Clifton, N.J.) and developed in the same solvent systems as described above. For each sample, 25 bands (3 mm width) were scraped from the TLC plates directly into scintillation vials, each containing a few drops of water. A 1-ml volume of ethanol was then added to the vials followed by 10 ml of Aquasol 2 (New England Nuclear Corp., Boston, Mass.). The counting of radioactivity was carried out in a Beckman LS-8000 liquid scintillation counter. All data were corrected for background, dilution, quenching, and counting efficiency. GLC and GC-MS analysis. Constituents of the extracts were derivatized to trimethylsilyl (TMS) ethers or trifluoroacetic acid (TFA) esters with Tri-Sil BT or MBTFA (Pierce Chemical Co., Rockford, Ill.), respectively, before analyses by gas-liquid chromatography (GLC) and gas chromatography-mass spectroscopy (GC-MS). GLC analysis was performed on a ewlett-packard model 5710A gas chromatograph equipped with a hydrogen flame detector under the following conditions: 1 m by 3 mm stainless-steel column packed with 3% OV-17 on mesh Gas Chrom Q; temperatures: injection 300 C, detection 3000C, oven programmed at 150 to 2900C at 8 C/min. Mass spectroscopy was carried out at 20 ev in an LKB-9000 GC-MS system equipped with a PDP-8e minicomputer based data system, selected ion monitoring mode. RESULTS AND DISCUSSION Extraction and fractionation of 3 residues from chicken excreta. Table 1 shows the recovery of 3 of the various fractions extracted from the chicken excreta. About 70% of

3 1174 YOSIZAWA, SWANSON, AND MIROCA Residue Petroleum ether extract I C3CN distillate XAD-20 eluate Florisil-CCl3/MeO eluate C18-20 Dissolve in water Sep-Pak C18 column 08-20% MeO Frozen sample C3CN the 3 residue was recovered from the excreta at 48 h after administration. About 10% of the administered radioactivity (dpm) remained in the extracted residue. The extraction efficiency with acetonitrile was approximately 83 to 90%. Most of the radioactivity remaining in the residue was extractable with more polar solvents such as aqueous methanol and water (data not shown). An insignificant amount of 3 was found in the petroleum ether extract and the acetonitrile distillate, indicating that 3 located in the C-3 position of the trichothecene nucleus was stable in biological systems. About 86 to 90% of the radioactivity was concentrated in the XAD- 90% MeO eluate, and only a small amount of the activity was found in the XAD-20 eluate. When the XAD-90% MeO eluate was chromatographed on Florisil columns, the majority (56 to 60%) of the extracted radioactivity was eluted with chloroform-methanol (3:1), whereas 17 to 24% was eluted with the more polar solvent, Extract with CXCN Zxtract C3CN concentrate XAD-90% MeO eluate Partition with petroleum ether Evaporate to dryness Dissolve in water Amberlite XAD-2 column Rinse with water Elute with MeO-20 (9:1, v/v) Florisil column Elute with CC13-MeO (3:1, v/v) Elute with MeO Florisil-MeO eluate C'8-50% MeO C,8-70% MeO FIG. 2. Fractionation and chromatography scheme. APPL. ENVIRON. MICROBIOL. C18-MeO methanol. Radiochromatographic profiles of both eluates on TLC plates indicated that most of the radioactivity in the methanol eluate remained at the origin of plates and less polar metabolites such as T-2 toxin and partially deacylated metabolites were found in the chloroform-methanol eluate. When the chloroform-methanol eluates were separated on C18 columns, the majority of the radioactivity was concentrated in the Sep-Pak C18-50% MeO (31 to 40%) and C18-70% MeO (5 to 12%) eluates, whereas less than 10% of the radioactivity extracted was found in the C18-20% MeO fraction. When 3 T-2 toxin was added to control excreta at 0.2 and 3.1 jug/g, it was concentrated in the C18-70% MeO fraction, and 77.5 and 78%, respectively, was recovered by the method outlined in Fig. 2. Overall recovery, after TLC analysis, of T-2 toxin added to control excreta was 73.6%. Quantification of T-2 metabolites in the

4 VOL. 39, C) O 80C> d 2 6X 0 D a} "p IN 10' mc- III 1-o -1. ORIGiN FRACTION NUMBER FIG. 3. Representative radiochromatograms of C18 fractions from excreta of broiler chickens administered 3-labeled T-2 toxin. (A) C18-20% MeO eluate developed in CCl3-MeO (5:1); (B and C) C18-50% and C18-70% eluates, respectively, developed in CCl3-MeO (9:1). Excreta were collected at 12 h after administration of the toxin. Each fraction was chromatographed on a high-performance silica gel TLC plate and scraped in 3-mm-width bands. excreta of broiler chickens. Figure 3 shows the thin-layer profiles of the C18 eluates. Each metabolite was scraped from the TLC plates as described above. In a preliminary experiment, it was confirmed that T-2 toxin, T-2 toxin, and deacetyl T-2 toxin eluted with the C08-70% fractions, whereas neosolaniol and T-2 tetraol were collected in the C18-50% and C18-20% MeO fractions, respectively. Other unknown radioactive peaks were named as shown in Fig. 3 (see Materials and Methods for Rf value of each metabolite). Table 2 shows the concentrations of individual metabolites in the chicken excreta at different time periods. The major metabolites were unknown compounds such as TB-3, TB-4, T-2 METABOLITES IN CICKEN EXCRETA 1175 TB-5, and TB-6, the concentrations of which were estimated as 12.25, 5.31, 4.94, and 2.09%, respectively, of the total dose at 48 h after administration. About 25% of the total dose after 48 h eluted from the Florisil column with methanol. When the methanol eluate was chromatographed on TLC and developed in chloroformmethanol (5:1, vol/vol), the majority of the radioactivity remained at the origin and this Rf value corresponds to no known trichothecene. We have named these polar metabolites TB-8. T-2 toxin administered orally to animals is eliminated primarily into the intestine from the liver through the bilial excretion system (2, 9). The result (Table 2) indicates that T-2 toxin was rapidly metabolized to the more polar derivatives and about 80% of the radioactivity administered was eliminated in the chicken excreta within 48 h. Ellison and Kotsonis (3) reported that bovine and human liver homogenates were capable of metabolizing T-2 toxin into T-2 toxin. Matsumoto et al. (9) detected a considerable amount of T-2 toxin, T-2 toxin, and neosolaniol from excreta of rats orally administered with 3-labeled T-2 toxin. The data in the present study, however, indicate that known derivatives such as T-2 toxin, neosolaniol, T-2 tetraol, and deacetyl T-2 toxin were minor metabolites, whereas unknown metabolites named TB-1 to TB-8 were more quantitatively significant metabolites in broiler chickens. Identification of T-2 metabolites in the chicken exereta. Because attempts at isolation and characterization of metabolites in radioactive excreta were unsuccessful, the excreta were collected from birds that were fed a ration containing 10,ig of nonradioactive T-2 toxin per g for 3 days to acquire appreciable amounts of metabolites for structural confirmation. Individual C08 eluates were analyzed by GC-MS (selected ion monitoring mode) after derivatization to TMS ethers. Concentrations of known metabolites, namely T-2 toxin (I), T-2 toxin (II), neosolaniol (IV), and T-2 tetraol (VI) were estimated as 5.5, 1.4, 1.25 and 0.2 ppm, respectively. To obtain samples suitable for full-scan mass spectra, the C18-20% MeO eluate fraction was purified on silica gel TLC plates developed in chloroform-methanol (5:1, vol/vol), and bands corresponding to T-2 tetraol and TB-6 were individually scraped and eluted. After derivatization with TMS and TFA reagents, the T-2 tetraol band revealed a peak having a similar retention time on GLC to that of the standard T-2 tetraol: 7.56 min (TMS ether) and 2.72 min (TFA ester). GC-MS of the peak (TMS ether) revealed the following diagnostic fragments (m/ e; 586 [M+], 494 [M-90], 365, 275, 193, 147, 103, and 73 [ Fig. 4A]). The fragmentation pattern of

5 1176 YOSIZAWA, SWANSON, AND MIROCA TABLE 1. Chromatographic separation of 3 residues recovered from excreta of broiler chickens administered 3-labeled T-2 toxin Fraction '-labeled T-2 stan- 4 hb 12 hb 48 hb darda dpm/administered (x 10O) ± ± ± ± 0.16 dpm/c3cn-extracted (x ± ± ± ± ) (% of administered) ( ± 3.70) (17.26 ± 10.90) (24.93 ± 6.84) (70.02 ± 17.19) dpm/c3cn-residue (x 107) 1.04 ± ± ± 2.50 (% of administered (2.05 ± 1.41) (5.23 ± 2.73) (10.17 ± 4.45) dpm/excreted, % of ± ± ± administered ± ± ± 6.66 Extraction efficiency (%) Recovery (%) of extracted dpm Petroleum ether extract 0.20 ± ± 0.14 C3CN distillate ± ± ± 0.13 C3CN concentrate ± ± ± ± XAD-20 eluate 8.49 ± ± ± ± 0.82 XAD-90% MeO eluate ± ± ± ± Florisil-CC13/MeO eluate ± ± ± ± Florisil-MeO eluate 0.42 ± ± ± ± 3.06 C,8-20 eluate 0.06 ± ± ± ± 0.05 C18-20% MeO eluate 0.16 ± ± ± ± 2.85 C18-50% MeO eluate 3.54 ± ± ± ± 8.38 C18-70% MeO eluate ± ± ± ± 1.53 C18-MeO eluate 5.77 ± ± ± ± 0.15 a Each value is the average of three replicate samples ± standard error. b Time after administration of 3-labeled T-2 toxin. Each value is the average of six replicate samples ± standard error. TABLE 2. APPL. ENVIRON. MICROBIOL. Thin-layer chromatographic quantification of metabolites in the excreta of broiler chickens administered 3-labeled T-2 toxin Concn (% of administered dpm) at: Metabolitesa 4hb 12hb 48h^ T-2 toxinc 0.02 ± ± ± 0.01 Neosolaniolc 0.07 ± ± ± 0.06 T-2 toxinc 0.76 ± ± ± 0.35 Deacetyl T ± ± ± 0.20 T-2 tetraolc 0.13 ± ± ± 0.25 TB ± ± ± 0.05 TB ± ± ± 0.12 TB ± ± ± 3.91 TB ± ± ± 2.44 TB ± ± ± 0.95 TB-6C 0.03 ± ± ± 0.32 TB ± ± ± 0.46 TB ± ± ± 0.35 (3.62 ± 1.95)d (4.66 ± 1.86)d (25.47 ± 2.93)d a Rf values of individual metabolites on TLC plates are shown in the text. btime after administration of 3-labeled T-2 toxin. Each value is the average of six replicate samples + standard error. C These metabolites were confirmed by GC-MS (SIM-monitoring) analysis after derivatization with TMS and TFA reagents. d Percent of administered dpm calculated by adding TB-8 to the Florisil-methanol eluate. the mass spectrum was identical to those of the shown in Fig. 4B, analysis by GC-MS of the standard T-2 tetraol. TMS ether gave a molecialar ion at m/e 556, The band corresponding to TB-6 gave a prom- indicating that TB-6 is a monoacetate of T-2 inent peak at a retention time of 9.33 min (TMS tetraol, and an ion at mle 466 (M+-90), indicating ether) and at 4.13 min (TFA ester) on GLC. As the elimination of a trimethylsilyloxy fragment

6 VOL. 39, L n a ^ I S a- U- I- a IS iII L1dlll i r * am - - am w a - am ama ma 73~~~~~~~~~3 -~ a FIG. 4. Mass spectra of the trimethylsilyl ethersof T-2 tetraol (A) and TB-6 (B) isolated from excreta of broiler chickens administered T-2 toxin. The spectra were obtained on a combination GC-MS (LKB-9000) at 20 ev with a gas chromatographic column and conditions as described in the text. (A) Normalized to m/e 193, the base peak is m/e 73. (B) Nornalized to m/e 275, the base peak is m/e 193. from the C-8 position of the trichothecene nucleus. Ions which appeared at m/e 335, 275, 247, 193, 185, 157, and 117 were diagnostic fragments derived from a trichothecene nucleus. From this information, TB-6 should have an acetoxyl group at C-3, C-4, or C-15 position. Both C-3 and C-4 monoacetate (chemically synthesized), however, gave different retention times on GLC. Therefore, the C-15 monoacetate (V, Fig. 1) [15-acetoxy-3a,4f8,8a-trihydroxy-12,13-epoxytrichothec-9-ene, 4-deacetyl-neosolaniol] was tentatively proposed for the chemical structure of TB-6. This compound was assumed to be an intermediate metabolite in S5M the transfornation T-2 METABOLITES IN CICKEN EXCRETA 1177 of T-2 toxin into T-2 tetraol. Further study on the structural elucidation of T-2 metabolites in chicken excreta and edible tissue will be described in a subsequent paper. ACKNOWVLEDGMEENTS This research was supported by Food and Drug Administration Contract RFP LITERATURE CITD 1. Chi, M. S., T. S. Robison, C. J. Mirocha, and W. Shimoda Transmission radioactivity into eggs from laying hens (Gallus domesticus) adminiistered tritium labeled T-2 toxin. Poultry Sci. 57: Chi, M. S., T. S. Robison, C. J. Mirocha, S. P. Swanson, and W. Shimoda Excretion and tissue distribution of radioactivity from tritium-labeled T-2 toxin in chicks. Toxicol. Appl. Pharmacol. 45: Ellison, R. A., and F. N. Kotsonis In vitro metabolism of T-2 toxin. Appl. Microbiol. 27: Ghosal, S., K. Bi8was, and B. K. Chattopadhyay Differences in the chemical constituents of Mangiferaindica, infected with AspergiUus niger and Fusarium moniliforme. Phytochemistry 17: Ghosal, S., K. Biswas, R. S. Srivastava, D. K. Chakrabarti, and K. C. B. Chaudhary Toxic substances produced by Fusarium V: occurrence of zearalenone, diacetoxyscirpenol, and T-2 toxin in moldy corn infected with Fusarium moniliforme Sheld. J. Pharm. Sci. 67: su, I. C., E. B. Smalley, F. M. Strong, and W. E. Ribelin Identification of T-2 toxin in moldy corn Appl. associated with a lethal toxicosis in dairy cattle. Microbiol. 24: Joffe, A. S Alimentary toxic aleukia, p In S. Kadis, A. Ciegler, and S. J. Ajl (ed.), Microbial toxins, vol. 7. ademic Press, Inc., New York. 8. Kamimura,., M. Nishijima, K. Saito, S. Takahashi, A. Ibe, S. Ochiai, and Y. Naoi Studies on mycotoxins in foods (VIII). Analytical procedure of Food yg. trichothecene mycotoxins in cereals. Jpn. J. Soc. 19: Matsumoto,., T. Ito, and Y. Ueno Toxicological approaches to the metabolites of Fusaria. XII. Fate and distribution of T-2 toxin in mice. Jpn. J. Exp. Med. 48: Mirocha, C. J., S. V. Pathre, B. Schauerhamer, and C. M. Christensen Natural occurrence of Fusarium toxins in feedstuff. Appl. Environ. Microbiol. 32: Mirocha, C. J., B. Schauerhamer, C. M. Christensen, and T. Kommedahl Zearalenone, deoynivalenol and T-2 toxin associated with stalk rot in corn. Appl. Environ. Microbiol. 38: Puls, R., and J. A. Greenway Fusariotoxicosis from barley in British Columbia. II. Analysis and toxicity of suspected barley. Can. J. Comp. Med. 40: Robison, T. S., C. J. Mirocha,. J. Kurtz, J. C. Behrens, M. S. Chi, G. A. Weaver, and S. D. Nystrom Transmission of T-2 toxin into bovine and procine milk. J. Dairy Sci. 62: Rukmini, C., and R. V. Bhat Occurrence of T-2 toxin in Fusarium-infested sorghum from India. J. Agric. Food Chem. 26: Szathmary, C. I., C. J. Mirocha, M. Palyusik, and S. V. Pathre Identification of mycotoxins produced by species of Fusarium and Stachybotrys obtained from eastern Europe. Appl. Environ. Microbiol. 32: Ueno, Y., K. Ishii, K. Sakai, S. Kanaeda,. Tsunoda, T. Tanaka, and M. Enomoto Toxicological approaches to the metabolites of Fusaria. IV. Microbial survey on bean hulls poisoning of horses with toxic trichothecens, neosolaniol, and T-2 toxin of Fusarium solani. Jpn. J. Exp. Med. 42: Ueno, Y., N. Sate, K. Sakai, and M. Enomoto Toxicological approaches to the metabolites of Fusaria. V. Neosolaniol, T-2 toxin and butenolide, toxic metabolites of Fusarium sporotrichioides NRRL 3510 and Fusariumpoae Jpn. J. Exp. Med. 42: Vesonder, R. F., A. Ciegler, R. F. Rogers, K. A. Burbride, R. J. Bothast, and A.. Jensen Survey of 1977 crop year preharvest corn for vomitoxin. Appl. Environ. Microbiol. 36: Wallace, E. M., S. V. Pathre, C. J. Mirocha, T. S. Robison, and S. W. Fenton Synthesis of radiolabeled T-2 toxin. J. Agric. Food Chem. 25: Yoshizawa, T., and N. Morooka Trichothecenes from mold-infected cereals in Japan, p In J. V. Rodricks, C. W. esseltine, and M. A. Mehlman (ed.), Mycotoxins in human and animal health. M. A. Pathotox Publishing Inc., Park Forest South, Ill.