Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Fungal degradation of aflatoxin B1

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.     

Involvement of lignocellulolytic enzymes in the decomposition of leaf litter in a subtropical forest

The involvement of ligninolytic and cellulolytic enzymes, such as laccase, lignin peroxidase, manganese peroxidase, carboxymethylcellulase (CMCase), and filter paper activity (FPA), in the decomposition process of leaf litter driven by 6 soil-inhabiting fungi imperfecti was studied under solid-state fermentations. All the tested fungi exhibited varied production profiles of lignocellulolytic enzymes and each caused different losses in total organic matter (TOM) during decomposition. Based on the results, the 6 fungi could be divided into 2 functional groups: Group 1 includes Alternaria sp., Penicillium sp., Acremonium sp., and Trichoderma sp., and Group 2 includes Pestalotiopsis sp. and Aspergillus fumigatus. Group 1, with higher CMCase and FPA activities, showed a higher decomposition rate than the fungi of Group 2 over the first 16 d, and thereafter the cellulolytic activities and decomposition rate slowed down. Group 2 showed the maximum and significantly higher CMCase and FPA activities than those of the Group 1 fungi during the later days. This, combined with the much higher laccase activity, produced a synergistic reaction that led to a much faster average mass loss rate. These results suggest that the fungi of Group 1 are efficient decomposers of cellulose and that the fungi of Group 2 are efficient decomposers of lignocellulose. During cultivation, Pestalotiopsis sp. produced an appreciable amount of laccase activity (0.56+/-0.09 U/ml) without the addition of inducers and caused a loss in TOM of 38.2%+/-3.0%, suggesting that it has high potential to be a new efficient laccase-producing fungus.     

Diversity in antifungal activity of strains of <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> from the Brazilian Amazon

Chromobacterium violaceum is a free-living Gram-negative bacterium found in soil and aquatic habitats; abundantly present in the Brazilian Amazon, it is an important example of exploitable microbial diversity of the tropics. In this study, 24 strains from the Brazilian Amazon and ATCC 12472(T) were investigated for biocontrol potential of seven fungi pathogenic to soybean [Glycine max (L.) Merril] seed. Both cells and the supernatants of two Brazilian strains, 07-1 and 27-1, together with ATCC 12472(T) were strongly antagonistic to six out of the seven fungi. The antifungal activity of the Brazilian strains to Fusarium sp., Phomopsis sp. and Cercospora kikuchi was consistently stronger than that of ATCC 12472(T). In addition, the two Brazilian strains, but not ATCC 12472(T), were effective against Corynespora sp., and all three strains and their supernatants were equally effective against Aspergillus sp. and Colletotrichum sp. None of the strains had antifungal activity against Botroyodiplodia sp. Three potential mechanisms related to the antibiosis were investigated: violacein toxicity, cyanide production and chitinolytic activity; however, it was not possible to associate any of them with the antifungal activity. The results highlight the biotechnological potential still to be explored within the poorly characterized microbial biodiversity of the tropics.     

Identification of lactic acid bacteria from fermented tea leaves (miang) in Thailand and proposals of Lactobacillus thailandensis sp. nov., Lactobacillus camelliae sp. nov., and Pediococcus siamensis sp. nov

Eighteen rod-shaped homofermentatives, six heterofermentatives, and a coccal homofermentative lactic acid bacteria were isolated from fermented tea leaves (miang) produced in the northern part of Thailand. The isolates were placed in a monophyletic cluster consisting of Lactobacillus and Pediococcus species. They were divided into seven groups by phenotypic and chemotaxonomic characteristics, DNA-DNA similarity, and 16S rRNA gene sequences. Groups I to VI belonged to Lactobacillus and Group VII to Pediococcus. All of the strains tested produced DL-lactic acid but those in Group IV produced L-lactic acid. The strains tested in Groups I, II and V had meso-diaminopimelic acid in the cell wall. Six strains in Group I were identified as Lactobacillus pantheris; five strains in Group II as Lactobacillus pentosus; and four strains in Group V as Lactobacillus suebicus. Two strains in Group VI showed high DNA-DNA similarity for each other and MCH4-2 was closest to Lactobacillus fermentum CECT 562(T) with 99.5% of 16S rRNA gene sequence similarity. Five strains in Group III are proposed as Lactobacillus thailandensis sp. nov., and MCH5-2(T) (BCC 21235(T)=JCM 13996(T)=NRIC 0671(T)=PCU 272(T)) is the type strain which has 49 mol% G+C of DNA. Two strains in Group IV are proposed as Lactobacillus camelliae sp. nov., and the type strain is MCH3-1(T) (BCC 21233(T)=JCM 13995(T)=NRIC 0672(T)=PCU 273(T)) which has 51.9 mol% G+C of DNA. One strain in Group VII is proposed as Pediococcus siamensis sp. nov., and MCH3-2(T) (BCC 21234(T)=JCM 13997(T)=NRIC 0675(T)=PCU 274(T)) is the type strain which has 42 mol% G+C of DNA.     

The activities of mycotoxins derived from Fusarium and related substances in a short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells)

Cell transformation assays using BALB/3T3 cells can mimic the two-stage process of chemical carcinogenesis in experimental animals. A short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), which was developed by Ohmori et al. and modified by Asada et al., has been reported to detect both tumor initiators and promoters as transformation initiators and promoters, respectively, with their differences based on their protocols. In this new short-term assay, we examined mycotoxins derived from Fusarium and related substances for the initiation and promotion activities of the transformation. The tested substances included deoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, fumonisin B(1), fumonisin B(2), zearalenone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol. Fumonisin B(1) and T-2 toxin were positive for promoting activity in the assay. Especially, T-2 toxin was active at concentrations as low as 0.001-0.002microg/mL in the culture medium. From a comparison between the results of this study and published carcinogenicity assay data, it was expected that the Bhas 42 cell transformation assay had a good correlation with the two-stage carcinogenicity tests using experimental animals for estimation of the tumor-promoting activity.     

Microbial acetyl conjugation of T-2 toxin and its derivatives.

The acetyl conjugation of T-2 toxin and its derivatives, the 12,13-epoxytrichothecene mycotoxins, was studied by using mycelia of trichothecene-producing strains of Fusarium graminearum, F. nivale, Calonectria nivalis, and F. sporotrichoides, T-2 toxin was efficiently converted into acetyl T-2 toxin by all strains except a T-2 toxin-producing strain of F. sporotrichoides, which hydrolyzed the substrate to HT-2-toxin and neosolaniol. HT-2 toxin was conjugated to 3-acetyl HT-2 toxin as an only product by mycelia of F. graminearum and C. nivalis, but was also resistant to conjugation by both F. nivale and F. sporotrichoides. Neosolaniol was also biotransformed selectively into 3-acetyl neosolaniol by F. graminearum. However, 3-acetyl HT-2 toxin was not acetylated by any of the strains under the conditions employed, but was hydrolyzed to HT-2 toxin by F. graminearum and F. nivale. This is the first report on the biological 3 alpha-O-acetyl conjugation of T-2 toxin and its derivatives.     

Metabolism of T-2 toxin in Curtobacterium sp. strain 114-2.

The metabolic pathway of T-2 toxin in Curtobacterium sp. strain 114, one of the T-2 toxin-assimilating soil bacteria, was investigated by thin-layer and gas-liquid chromatographic analyses. T-2 toxin added to the basal medium as a single carbon and energy source was biotransformed into HT-2 toxin and an unknown metabolite. Infrared, mass spectrum, proton magnetic resonance, and other physico-chemical analyses identified this new metabolite as T-2 triol. T-2 toxin was first deacetylated by the bacterium into HT-2 toxin, and this metabolite was then biotransformed into T-2 triol without formation of neosolaniol and T-2 tetraol. No trichothecenes remained in the culture medium after prolonged culture. Some properties of T-2 toxin-hydrolyzing enzymes were observed with whole cells, the cell-free soluble fraction, and the culture filtrate. Besides T-2 toxin, trichothecenes such as diacetoxyscirpenol, neosolaniol, nivalenol, and fusarenon-X were also assimilated by this bacterium.     

Biological Assays for Two Mycotoxins Produced by Fusarium tricinctum

A survey was made to detect microorganisms useful for assaying butenolide [4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone] and T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12,13-epoxytricothec -9-en-3alpha-ol]. These mycotoxins produced by strains of Fusarium tricinctum have been implicated in mycotoxicosis of livestock. Although butenolide proved to be a very weak antibiotic, assay discs containing 100 mug of this toxin inhibited Sprillum serpens NRRL B-2052, Vibrio tyrogenus NRRL B-1033, and Xanthomonas campestris NRRL B-1459. T-2 toxin had no effect on 54 bacterial strains but inhibited 6 of 11 fungi. Growth of Rhodotorula rubra NRRL Y-7222 and Penicillium digitatum NRRL 1202 was retarded by assay discs containing 4 mug of T-2 toxin. Solutions with less than 1 mug of T-2 per ml toxin were readily detected by a pea seed germination test. Germination was reduced more than 50% when seeds imbibed solutions of 0.5 mug of T-2 toxin per ml. Butenolide had no effect on pea seed germination at concentrations as high as 200 mug/ml.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid.

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Cytophotometric assessment of granulosa cell protein and nucleic acid levels during the estrous cycle in rats treated with T-2 toxin

Female Sprague-Dawley rats (160-180g.) with normal estrous cyclicity established by vaginal smears, were injected intraperitoneally with 0.45 mg/kg (low dose) or 0.68 mg/kg (high dose) of T-2 toxin, a trichothecene with potent protein inhibitory abilities. Control animals were injected with only the 100% ethanol vehicle. All animals were decapitated at 8 hours post-exposure, and their ovaries removed and processed for paraffin sectioning. Coomassie, Feulgen, and azure B/DNase staining procedures were used to show granulosa cell protein levels, F-DNA stainability, and basophilia/RNA levels, respectively. Quantification of these parameters was accomplished using scanning-integrating microdensitometry. T-2 toxin treatment groups had granulosa cell protein levels significantly lower than those of the control animals. However, rats exposed to the lower dose of T-2 toxin generally showed a more marked suppression of protein levels than the high dose group, regardless of the stage of the estrous cycle. In addition, rats that received lower doses of T-2 toxin had impaired translation and template activity in response to injury, when compared with the rats in the high dose group. These results are attributed to the lesser degree of circulatory impairment in the low T-2 toxin dosage group, which allows a higher amount of T-2 toxin to interact with the cells.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Isolation and characterization of acid- and Al-tolerant microorganisms

Acid- and aluminum (Al)-tolerant microorganisms were isolated from tea fields, from which six strains were selected and identified as Cryptococcus humicola, Rhodotorula glutinis, Aspergillus flavus Link, Penicillium sp., Penicillium janthinellum Biourge and Trichoderma asperellum. They were tolerant to Al up to 100-200 mM and could grow at low pH, 2.5-2.2. In a glucose medium (pH 3.5) the pH of the spent medium decreased to below 3.0. The toxic inorganic monomeric Al in the spent medium decreased with three strains (A. flavus F-6b, Penicillium sp. F-8b and P. janthinellum F-13), but the total Al remained constant for all strains. In a soil extract medium (pH 3.5), the pH of the spent medium of all strains increased to around 6.0-7. 2 and total Al in the spent medium was removed by precipitation due to pH increase. Thus, different tolerance mechanisms were suggested in glucose and soil extract media.     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

Ovarian consequences of low dose peroral Fusarium (T-2) toxin in a ewe and heifer model

The effect of low dose peroral Fusarium produced T-2 toxin intake upon the ovarian function was evaluated in ewes (n = 30; Trial 1) and heifers (n = 7; Trial 2). Half of the ewes and all of the heifers were fed rich, acidosis-inducing concentrate. The 30 ewes were divided into 6 groups of 5 animals each. They were given 0, 0.3 or 0.9 mg/day (0, 5 or 15 ug/kg) purified T-2 toxin per os for 21 days (3x2 factorial design). Four of the 7 heifers were fed 9 mg/day (25 ug/kg) of the same purified T-2 toxin for 20 days while 3 remained untreated. The estrus cycles in all animals were synchronized prior to the trials and the T-2 exposure was started in the mid-luteal phase. The acidic condition in the rumen was estimated by the determination of urinary net acid-base excretion. The ovarian activity was followed with blood sampling for progesterone on alternate days (Trial 1) or with ultrasonography and sampling for progesterone daily (Trial 2). All of the heifers and concentrate-fed ewes showed a compensated acidosis, during first two thirds of T-2 exposure. In Trial 1, ovarian malfunction manifested as lower P4 peak concentration in the midluteal phase, shortening of the CL lifespan and prolonged follicular phases. These malfunctions were detected in 3 and 3 ewes fed concentrate and 0.3 mg and 0.9 mg T-2 toxin. Lower P4 peak concentration was observed in 1 ewe fed regular diet and 0.9 mg T-2 toxin. None of the control and acidotic groups (0 mg T-2), or ewes fed regular diet with 0.3 mg T-2 showed any ovarian malfunction. In Trial 2, after PGF2, administration the ovulation occured later and the plasma progesterone level remained low (< 3 nmol/l) for a longer period in T-2 treated heifers, than their untreated control mates (5.0+/-0.7 vs 3.7+/-0.5 d, P<0.05 and 8.3+/-0.4 vs 6.3+/-0.9 d, P<0.01, respectively). These results show that the peroral T-2 intake can significantly retard the folliculus maturation and ovulation and perhaps the subsequent luteinisation also in ruminants kept on concentrate-rich diet.     

Correlation of Biological to Chromatographic Data for Two Mycotoxins Elaborated by Fusarium

Thirty-seven identified strains of Fusarium, most of them isolated from fescue grass, were tested for their ability to elaborate mycotoxins in laboratory culture. The presence of the toxins was determined by infrared light, thin-layer chromatography, mouse toxicity, fungistatic effects, and phytotoxic properties. A good correlation was demonstrated between T-2 toxin detection by thin-layer chromatography and inhibition of Rhodotorula rubra by culture extracts. All of the strains producing either butenolide or T-2 toxin were toxic to mice with but one exception; those producing T-2 toxin inhibited growth of the yeast.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.