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High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate the trichothecin mycotoxin, T-2 (4 beta,15-diacetoxy-3 aopha-hydroxy-8 alpha [3-methyl-butyryloxy]-12,13-epoxy delta 9-trichothecin), and its metabolites in plasma and urine samples from cynomolgus monkeys treated with the toxin. A 15-min gradient elution system was developed to separate and measure radiolabeled T-2 mycotoxin and its metabolites. The HPLC technique for separating T-2 and its metabolites was compared with thin-layer chromatography. Samples from the in vitro metabolism of T-2 by plasma and urine were included as controls and as a measure of the toxin's stability in biological samples. Within 5 min, 22% of the plasma radiolabeled T-2 toxin was detected as metabolites after an intravenous administration of [3H] T-2 toxin to cynomolgus monkeys. By 24 h post-exposure, there was no parent T-2 toxin detected in plasma or urine. T-2 tetraol was the major metabolite detected in the plasma and urine of monkeys. Other metabolites observed in urine up to 5 days after exposure were 3'OH-T-2 and 3'OH-HT-2. We conclude that T-2 toxin was rapidly metabolized to more polar metabolites, which were eliminated in urine.

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We used an in-vitro, inhibition of protein synthesis assay (PSI) to test a wide variety of drugs for possible therapeutic use against ricin, a toxic glycoprotein that causes death in animals by inhibiting protein synthesis. Selection of test drugs was based on possible interference with ricin activity at different stages of the toxic process. Most of the drugs tested had no effect on ricin-induced PSI, were toxic when tested alone, or enhanced the toxicity of ricin. The only ones showing protection were galactose, lactose, and several derivatives of these sugars, Brefeldin A (BFA), 3'-azido-3'-deoxythymidine (AZT), and a purine derivative (BM33203). THe sugar derivatives provided 50% protection against a PSI ED99 of ricin (0.1 micrograms/ml). Concentrations of BFA greater than 0.5 micro M caused about 50% PSI by itself, but blocked any further inhibitory effects of ricin. AZT, at optimum concentrations, reached a maximum protection level of about 40% in the presence of an ED99 dose of ricin, while the nucleoside derivative, BM33203 and AZT appeared to have an additive effect, showing up to 80% protection from an ED99 dose of ricin. Drugs showing protection in the PSI cell assay showed no protection from ricin in a cell-free translation assay used to determine if they would block ricin at the protein synthesis site.

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The toxicity of ricin in susceptible cells is well characterized biochemically, but the pathophysiological implications of its toxicity and the immune response to ricin challenge in the lung are unknown. Incubating macrophage cell line with ricin (1 pM-10 nM) for 4 hours markedly inhibited 3H-leucine incorporation (acid insoluble) into protein (> 95%, at 1 nM) without affecting the acid-soluble radioactivity. In spite of increased uptake of total thymidine (141 +/- 13.5%) and total uridine (135 +/- 17.2%), DNA synthesis in ricin-treated cells was progressively inhibited although RNA synthesis was not affected. Fluocinolone (an anti-inflammatory glucocorticoid) pretreatment increased the ricin-induced inhibition of protein synthesis. The synergistic effect of fluocinolone on ricin-induced protein synthesis inhibition was due to an increased binding (167%, p < 0.01) and internalization (134 +/- 12%, p < 0.025) of ricin. Partial protection from ricin-induced inhibition of protein synthesis by indomethacin (nonsteroidal, anti-inflammatory agent) was due to decreased binding and internalization of ricin. These results show that macrophages are sensitive to ricin and that pharmacologically active drugs may regulate ricin's toxicity, perhaps by controlling synthesis and release of certain mediators of fast death.

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Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca(2+)-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.

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Primary cultures of rat hepatocytes were used in a screen in vitro for agents which protect against the toxic effects of the hepatoxin microcystin-LR. Exposure of cells to microcystin-LR, a cyclic heptapeptide produced by blue-green algae, resulted in clustering of hepatocytes within 15 min of addition. This initial response was followed by disruption of cellular function (measured by a protein synthesis assay) and eventual loss of membrane integrity, resulting in leakage of cytosolic enzymes (measured by LDH release). The antibiotic rifampicin was effective in reducing LDH release and preventing cell clustering at concentrations as low as 2.0 mum. Cytochalasins provided some protection at 10 mum. However, microscopy revealed disruption of hepatocytes in the presence of cytochalasins alone, at or above this concentration. Bile acids, cholic acid and deoxycholate were protective at higher concentrations (0.1 mm), but when given alone caused some leakage of cytosolic enzymes. Di-naphthalene dyes, trypan blue and trypan red, had no detrimental effects on hepatocytes and showed some protection at 20 mum. Hepatic uptake of tritium-labelled microcystin-LR was suppressed by bile acids and rifampicin, providing further evidence that blocking or competing with bile acid uptake also prevents microcystin-LR association with, and toxicity in, isolated hepatocytes. Although all drugs tested worked to varying degrees, rifampicin was most effective in providing protection of hepatocytes from toxin at lower concentrations while having no detrimental effects on the cells when added alone.

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Isolated rat livers were perfused for 60 min with either 0.3 or 0.5 microgram/ml (initial volume, 119 ml) of [3H]microcystin-LR at a constant flow of 10 ml/min in a recirculating system. During the 60-min exposure, toxin caused stimulation of glycogenolysis, liver engorgement, and cessation of bile flow. Electron micrographs of liver showed dilation of bile canaliculi and the space of Disse. loss of sinusoidal lining architecture, and decreased hepatocyte intercellular contacts. Although hepatocytes did not exhibit overt necrosis, mitochondria were hydropic, occasionally encircled by whorls of rough endoplasmic reticulum, and desmosomal tonofilaments were decreased on the plasma membrane lateral surface. Isolated mitochondria displayed inhibition of state 3 respiration and a 50-60% decrease in the respiratory control index, characteristic of hydropism. Distribution of radiolabel was 1.7% to bile, 79% to perfusate, and 16% to liver. Two to four percent was recovered in perfusate that leaked from the surface of the liver. Of the radiolabel found in bile and perfusate, 78 and 100% were associated with parent toxin, respectively. The radiolabel in liver, associated with the cytosolic fraction (S-100), corresponded to parent toxin (15%) and to a more-polar component(s) (85%). The elimination half-life from perfusate was 130 +/- 10 min (0.5 microgram/ml) and the hepatic extraction ratio 0.07 +/- 0.01. Although the calculated hepatic extraction ratio was low, there was a significant accumulation of microcystin in the liver. Many toxic effects of microcystin in the perfused liver mimicked those observed in the whole animal, suggesting that this model can be used as an alternative to whole animals for screening of potential therapeutic agents.

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Microcystin-LR (MCYST-LR), a cyclic peptide hepatotoxin, associates with high-molecular-weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 +/- 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high-performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time- and temperature-dependent and did not require endogenous molecules less than 6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST-LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration-dependent inhibition of [3H]MCYST-LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A.

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The LD50 (25 hr, i.p.) for microcystin-LR in fed rats (122 micrograms/kg) was significantly higher than that in fasted rats (72 micrograms/kg). At doses of 100, 150 and 200 micrograms of microcystin-LR per kg, the median times to death were 31.9, 18.2 and 11.2 hr for fed rats, and 1.8, 1.7 and 1.5 hr for fasted rats. A sublethal dose of microcystin (50 micrograms/kg) afforded protection to fasted, but not fed, rats against a subsequent lethal dose (200 micrograms/kg) challenge given 72 hr later. Biochemical and ultrastructural changes resulting from microcystin-LR (100 micrograms/kg, i.p.) were compared in fed and fasted rats 1 hr after injection. In both groups, liver weight and serum levels of sorbitol dehydrogenase and glucose significantly increased. Plasma membranes, isolated from livers of fed or fasted rats, exhibited similar toxin-induced changes in associated cytoskeletal elements. Liver mitochondria from toxin-treated, fasted rats exhibited complete inhibition of state 3 respiration, while those from toxin-treated, fed rats had ADP/O ratios and respiratory control indices comparable to control values. The primary event responsible for enhanced microcystin hepatotoxicity in the fasted state has not yet been identified. Depletion of glycogen stores and a decreased respiratory capacity may, however, play significant roles in this degenerative process.

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The distribution, excretion and hepatic metabolism of [3H]microcystin-LR (sublethal i.v.) were measured in mice. Plasma elimination was biexponential with alpha- and beta-phase half-lives of 0.8 and 6.9 min, respectively. At 60 min, liver contained 67 +/- 4% of dose. Through the 6-day study the amount of hepatic radioactivity did not change whereas 23.7 +/- 1.7% of the dose was excreted; 9.2 +/- 1.0% in urine and 14.5 +/- 1.1% in feces. Approximately 60% of the urine and fecal radiolabel 6 and 12 hr postinjection was the parent toxin. Hepatic cytosol, which contained 70 +/- 2% of the hepatic radiolabel (1 hr through 6 days), was prepared for high-performance liquid chromatography analysis by heat denaturation, pronase digestion and C18 Sep Pak extraction. At 1 hr, 35 +/- 2% of the radiolabel was insoluble or C18 Sep Pak-bound; 43 +/- 3% was associated with a peak of retention time (rt) 6.6 min, and 16 +/- 3% with the parent toxin (rt 9.4 min). After 6 days, 8 +/- 1% was C18 Sep Pak-bound or insoluble; 5 +/- 0% occurred at rt 6.6 min, 17 +/- 1% with parent and 60 +/- 2% was associated with rt 8.1 min. Two other peaks, rt 4.9 and 5.6 min, appeared transiently. Analysis of hepatic cytosol by desalting chromatography under nondenaturing and denaturing conditions revealed that all of the radiolabel was associated with cytosolic components, and 83 +/- 5% was bound covalently through 1 day. By day 6 the amount of covalently bound isotope decreased to 42 +/- 11%. This is the first study to describe the long-term hepatic retention of microcystin toxin and documents putative detoxication products.

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The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with [3H]T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%, perfusate 38% and liver 7%. Livers were fractionated into mitochondria, endoplasmic reticulum (smooth and rough), plasma membrane and nuclei. Plasma membrane fractions contained 38% of the radiolabel within 5 min, decreasing to less than 1% at the end of the 120-min perfusion. Smooth endoplasmic reticulum contained 27% of the radiolabel by 5 min and increased to 43% over the 120-min perfusion. The mitochondrial fraction contained 3% of the radiolabel by 30 min and increased to 10% after 120-min perfusion. Label in the nuclear fraction remained constant at 7% from 30 to 120 min. By 15 min, only the parent toxin was detected in the mitochondrial fraction. In the other fractions, radiolabel was associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distribution of radiolabel in liver suggested an immediate association of [3H]T-2 with plasma membranes and a subsequent association of toxin and metabolites with endoplasmic reticulum, mitochondria and nuclei, the known sites of action of this toxin.

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The toxic effects of microcystin-LR, a cyclic heptapeptide isolated from the cyanobacterium Microcystis aeruginosa, were studied in the fasted rat model and in subcellular fractions from fasted, toxin-treated and control rats. Hepatotoxic effects of a lethal dose (100 micrograms/kg) were examined 15-90 min post-injection. Elevations of serum enzymes, particularly sorbitol dehydrogenase, specific for liver mitochondria, correlated with hepatic damage. Electron micrographs showed progressive cellular disruption, including dilation of rough endoplasmic reticulum, incorporation of cellular components into cytolysosomes, hydropic mitochondria devoid of electron-opaque deposits, loss of desmosome-associated intermediate filaments, disruption of sinusoidal architecture and, ultimately, lysis of hepatocytes. The appearance of hydropic mitochondria correlated with loss of coupled electron transport. Changes in plasma membrane-associated cytoskeletal filaments correlated with loss of desmosome tonofilaments. In contrast to in vivo exposure to microcystin-LR, in vitro exposure to toxin had no effect on mitochondria or cytoskeletal filaments, suggesting that the toxic effects observed in vivo were indirect and may be dependent on bioactivation of the toxin or a cascade of events not supported in in vitro models.

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Chemically tritiated microcystin-LR (spec. act. 194 mCi/mmol), purified to greater than 95% by C-18 reverse-phase high performance liquid chromatography, exhibited the same retention time and ultraviolet absorption profile as unlabeled toxin. Acid-hydrolyzed [3H]-toxin yielded tritiated glutamate and beta-methylasparate. Stability of the nonexchangeable [3H]-toxin in saline and urine was greater than 93% after 42 days stored at 22 degrees, 4 degrees or -20 degrees C. In blood, the breakdown of toxin was temperature- and time-dependent (63% at 22 degrees C, 28 days). Unlabeled toxin was stable for greater than 42 days stored at either 4 degrees or -20 degrees C in saline. The LD50 (mouse, i.p.) of [3H]-microcystin-LR and unlabeled toxin was the same [75 micrograms/kg (65-90) and 65 micrograms/kg (53-80), respectively]. From 3 to 90 min after i.p. injection of 70 micrograms/kg [3H]-microcystin-LR there was a slow absorption of toxin from the peritoneal cavity and efficient accumulation in liver. The elimination half-life of the plasma concentration curve was 29 min. Tritium distribution in tissue at death or 6 hr post injection was similar for all doses (13-101 micrograms/kg). At 101 micrograms/kg, liver contained 56 +/- 1%, intestine 7 +/- 1%, kidney 0.9 +/- 0.2% and carcass 10 +/- 1% of the injected dose. Heart, spleen, lung and skeletal muscle contained less than 1% of the radiolabel.

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The stabilities of tritium-labeled T-2, HT-2, and T-2 tetraol were studied in blood and urine at -70 degrees, 4 degrees, and 23 degrees C for 6 months in the presence of EDTA or NaF. Samples were counted with a radiochromatographic scanner and results indicated the stability of T-2 tetraol greater than T-2 greater than HT-2. Toxins were most stable when stored at -70 degrees C, in the presence of NaF, and in urine (pH 6). They were less stable in saline (control, pH 7) and least stable in blood (pH 8). These results suggest that urine and T-2 tetraol are the biological fluid and metabolite of choice for diagnostic purposes.

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We investigated the effect of T-2 toxin on rat liver mitochondrial protein synthesis. Isolated rat liver mitochondria were supplemented with an S-100 supernatant from rat liver and an external ATP-generating system. We used an in vitro assay employing cycloheximide, an inhibitor of cytoplasmic protein synthesis, and chloramphenicol, an inhibitor of mitochondrial protein synthesis, to distinguish mitochondrial protein synthesis from the cytoplasmic process. Amino acid incorporation into mitochondria was dependent on the concentration of mitochondria and was inhibited by chloramphenicol. The rate of uptake of [3H]leucine into mitochondrial protein was unaffected by the addition of T-2 toxin and was not a rate-limiting step in incorporation. However, 0.02 micrograms/ml of T-2 toxin decreased the rate of protein synthesis by isolated mitochondria by 50%. The degree of protein synthesis inhibition correlated with the amount of T-2 toxin taken up by the mitochondria. While T-2 toxin is known to inhibit eukaryotic protein synthesis, this is the first time T-2 was shown to inhibit mitochondrial protein synthesis.

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A standard radioimmunoassay was compared with radiochromatography for the ability to detect unlabeled T-2 mycotoxin in organs from exposed animals. When 10% of HT-2, the only known metabolite that cross-reacts with T-2, was included and expressed as T-2 equivalents in the radiochromatographic detection, correlation between toxin detection in liver, spleen, and kidney by the 2 techniques was r = 0.98. An unknown metabolite was detected in heart extract by radiochromatography. Inclusion of this material in the T-2 equivalents detected by radiochromatography indicated a near-perfect correlation (r = 0.95; p greater than 0.05; slope = 0.82; y = intercept = 72) among all 4 tissues.

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Discs of abdominal skin (obtained from humans and hybrid monkeys at autopsy) were mounted on diffusion cells. The epidermal surfaces were dosed with [3H]T-2 dissolved in dimethyl sulfoxide (DMSO). The rate of [3H]T-2 penetration (expressed as ng/cm2/hr) through human skin was 0.38 +/- 0.10 and 3.85 +/- 0.96 (means +/- 95% confidence limit) when dosed with 74 and 582 ng/cm2, respectively. [3H]T-2 penetrated through monkey skin at the rate of 0.37 +/- 0.14, 0.80 +/- 0.43, 4.13 +/- 1.71, and 6.55 +/- 3.45 when dosed with 70, 155, 555 and 1063 ng/cm2, respectively. Analysis of the receptor fluid bathing human skin revealed 15% of the radioactivity was associated with T-2, 71% with HT-2 toxin (HT-2), and 6.3% with an unknown metabolite more polar than HT-2. The radioactivity in the receptor fluid bathing monkey skin was associated with T-2 (87%) and HT-2 (1.0%). The results are consistent with the hypothesis that metabolism of T-2 occurred during penetration through the excised skin and did not occur in the receptor fluid due to enzymes leaching out of the skin. These findings indicate that excised monkey skin is a good model for T-2 penetration through human skin when DMSO is the vehicle, but that dermal metabolism of T-2 is different in these two species.

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Isolated perfused rat livers were used to study the metabolism and clearance of T-2 mycotoxin, a nonprotein Fusarium metabolite known to cause illness or death on contact or by ingestion. To evaluate the in vitro hepatic metabolism, clearance, and rate of biliary excretion of T-2 toxin, [3H]T-2 toxin was delivered under constant perfusate flow (8 ml/min, 33.9 micrograms T-2/min) in a single-pass experiment. Steady-state conditions were achieved within 10 min as indicated by a constant exit rate of radiolabel in the effluent. At steady state, 93 +/- 4% of the delivered [3H]T-2 was extracted and metabolized by the liver, while 4.6 +/- 0.3% remained unmetabolized in the effluent perfusate. The excretion rate of metabolites and conjugates into bile was constant after a 10-min perfusion. Radioactivity measured in bile accounted for 55% of the total radiolabel delivered during the perfusion experiment (1 hr). T-2 toxin was metabolized and eliminated as 3'hydroxy HT-2, 3'hydroxy T-2 triol, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates of HT-2, 3'hydroxy HT-2, and T-2 tetraol. Approximately 7% of the administered radiolabel remained in the liver and was identified as 4-deacetylneosolaniol (18%), T-2 tetraol (41%), and conjugated metabolites (41%). Total recovery of administered radiolabel associated with T-2 and its metabolites equaled 97.6% (bile, 52.5%; perfusate, 38.0%; liver, 7.1%). Approximately 3% of the biliary radiolabel was not identified. These studies describe the use of a perfused organ system to determine the rate of formation of T-2 metabolites and their elimination into bile.

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Penetration of [3H]T-2 toxin through excised human and monkey skin stored at -60 degrees C was faster than through human and monkey skin stored at 4 degrees C, respectively. The permeability of refrigerated human skin was 34% of the permeability of refrigerated monkey skin. Increasing the concentration of [3H]T-2 toxin applied to the refrigerated monkey skin increased the amount of [3H]T-2 toxin penetrating the skin and enhanced the efficiency of penetration. Metabolites of [3H]T-2 toxin were identified in the receptor fluid bathing the dermal side of the excised human and monkey skin.

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T-2 toxin is a potent cytotoxic metabolite produced by the Fusarium species. The fate and distribution of 3H-labeled T-2 toxin were examined in male guinea pigs. Radioactivity was detected in all body tissues within 30 min after an im injection of an LD50 dose (1.04 mg/kg) of T-2 toxin. The plasma concentration of trichothecene molar equivalents versus time was multiphasic, with an initial absorption half-life equal to or less than 30 min. Bile contained a large amount of radioactivity which was identified as HT-2, 4-deacetylneosolaniol, 3'-hydroxy HT-2, 3'-hydroxy T-2 triol, and several more-polar unknowns. These T-2 metabolites are excreted from liver via bile into the intestine. Within 5 days, 75% of the total radioactivity was excreted in urine and feces at a ratio of 4 to 1. The appearance of radioactivity in the excreta was biphasic. Metabolic derivatives of T-2 excreted in urine were T-2 tetraol, 4-deacetylneosolaniol, 3'-hydroxy HT-2, and several unknowns. These studies showed a rapid appearance in and subsequent loss of radioactivity from tissues and body fluids. Only 0.01% of the total administered radioactivity was still detectable in tissues at 28 days. The distribution patterns and excretion rates suggest that liver and kidney are the principal organs of detoxication and excretion of T-2 toxin and its metabolites.

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An in vitro test system was used to measure the penetration of [3H]T-2 toxin through human epidermis, human whole skin (isolated dermis and epidermis), and through guinea-pig whole skin. To simulate the conditions which occur when agricultural workers are exposed to corn dust contaminated with T-2 toxin, the epidermal surface of each skin preparation was dosed with [3H]T-2 toxin adsorbed onto corn dust. The applied dose was 3.27 to 4.75 mg of corn dust containing 18.2 ppm [3H]T-2 toxin. The rate of percutaneous penetration was determined by measuring the accumulation of radioactivity in the receptor fluid bathing the dermal side of the excised skin. The total penetrations (expressed as percentage dose) through isolated human epidermis, human whole skin and through isolated guinea-pig whole skin were 1.12 +/- 0.26% (mean +/- standard deviation), 0.33 +/- 0.07%, and 0.13 +/- 0.07% respectively. The radioactive compounds in the receptor fluid bathing the human whole skin corresponded to T-2 toxin (69%) and HT-2 toxin (25%), as determined by thin-layer chromatography. Thus T-2 toxin adsorbed onto corn dust can partition into and penetrate through excised human and guinea-pig skin.

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