Pharmacokinetics of T-2 tetraol, a urinary metabolite of the trichothecene mycotoxin, T-2 toxin, in dog

1. The urinary metabolites of T-2 toxin were identified and analysed quantitatively after i.v. administration to dogs. 2. A new routine assay for T-2 tetraol was developed and a pharmacokinetic study was carried out on this final hydrolytic metabolite of T-2 toxin. T-2 tetraol was excreted in urine for 2-3 days. Its 'sigma minus' plot demonstrated a significantly longer apparent half-life than its precursors (T-2 toxin and HT-2 toxin). This fact was explained by extraplasma binding causing prolongation of the metabolism and excretion of T-2 toxin metabolites. 3. The urinary metabolites of T-2 toxin were: HT-2 toxin, T-2 triol and T-2 tetraol. The metabolites were excreted in free and conjugated forms. In two dogs T-2 toxin was found in the urine in an amount which accounts for 3.2 and 16% of the administered dose respectively. The cumulative amount of the identified metabolites and toxins formed in the urine ranged from 9.7 to 17.3% in four dogs and 44.7% in one dog.     

Pharmacokinetics of T-2 tetraol,a urinary metabolite of the trichothecene mycotoxin,T-2 toxin,in dog

1. The urinary metabolites of T-2 toxin were identified and analysed quantitatively after i.v. administration to dogs.

2. A new routine assay for T-2 tetraol was developed and a pharmacokinetic study was carried out on this final hydrolytic metabolite of T-2 toxin. T-2 tetraol was excreted in urine for 2–3 days. Its ‘sigma minus' plot demonstrated a significantly longer apparent half-life than its precursors (T-2 toxin and HT-2 toxin). This fact was explained by extraplasma binding causing prolongation of the metabolism and excretion of T-2 toxin metabolites.

3. The urinary metabolites of T-2 toxin were: HT-2 toxin, T-2 triol and T-2 tetraol. The metabolites were excreted in free and conjugated forms. In two dogs T-2 toxin was found in the urine in an amount which accounts for 3.2 and 16% of the administered dose respectively. The cumulative amount of the identified metabolites and toxins formed in the urine ranged from 9.7 to 17.3% in four dogs and 44.7% in one dog.     

Metabolism of T-2 mycotoxin by cultured cells

T-2 mycotoxin is a small (i.e. mol. wt 466), non-protein toxin. We studied its metabolism in Chinese hamster ovary (CHO) cells, African green monkey kidney (VERO) cells, human fibroblasts and mouse connective tissue cells (L-929). Confluent cells were exposed to [3H]-T-2(0.01 micrograms/ml) for 1 hr at 37 degrees C. The toxin was removed, cells rinsed, and unlabeled culture media added for 4 hr (37 degrees C). Cell monolayers were extracted and media and cell extracts were spotted on thin-layer chromatography plates with known standards. Thin-layer plates were developed and scanned for radioactivity, and metabolites were identified based on co-migration with known standards. CHO and VERO cells metabolized T-2 to a greater per cent and to a wider variety of metabolites than the other two cell types. In CHO, fibroblast and L-929 cells, the major metabolite was HT-2 toxin, while in VERO cells an unknown metabolite, more polar than T-2, was the major metabolite. Cell and media extracts of CHO and VERO cells revealed smaller amounts of T-2 triol, T-2 tetraol and several unknowns. In both cell types, metabolites were detected in labeled media by 1 hr and in increasing amounts in unlabeled media by 4 hr. Under the above conditions, 37-58% of the radioactivity remained as T-2 toxin after 4 hr in both cell types. The data suggest that some cultured cell lines possess enzyme systems capable of limited metabolism of T-2 mycotoxin to a variety of known and some as yet unidentified metabolites.     

Metabolism of T-2 toxin by rat brain homogenate

HT-2 toxin was the sole metabolite formed when T-2 toxin was treated with homogenate from brain without its blood content. Homogenate from brain with its full blood content produced--besides HT-2 toxin--T-2 triol, neosolaniol, 4-deacetylneosolaniol and T-2 tetraol, i.e. the same metabolites formed by incubation of T-2 toxin with whole rat blood.     

Cerebral toxicity of the trichothecene toxin T-2, of the products of its hydrolysis and of some related toxins

T-2 toxin and its metabolites (resulting from enzymatic hydrolysis by rat brain homogenate) were applied to the midbrain of albino rats, either in solid form or dissolved in dimethyl sulfoxide (DMSO). Solid implants of HT-2 toxin and of T-2 triol were lethal in the range of 10-20 micrograms per rat, i.e. similar to the effect of T-2 toxin itself. For four further trichothecenes, the following decreasing order of toxicities was found: T-2 tetraol = iso-T-2 toxin greater than T-2 tetraol tetraacetate greater than T-2 toxin acetate. Implants of the last compound were the least toxic in the present series of trichothecenes; its LD50 value was nearly ten times higher than that of T-2 toxin. A similar gradation of toxicity was observed upon intracerebral injection of the compounds dissolved in DMSO. Here the only exception was the markedly reduced toxicity of T-2 toxin itself. From these data, the role of free 3 alpha- and 4 beta-hydroxyl groups has been evaluated. For subcutaneous applications, the largest ratio of LD50 values was 5, i.e. for the pair T-2 triol-T-2 tetraol tetraacetate. Among the signs of central intoxication, convulsions, adipsia and aphagia were marked. Pathological changes in the brain tissue, mainly involving necrotic, hemorrhagic and inflammatory lesions at the sites of application, were similar for all trichothecenes tested in this study.     

Pharmacokinetics and protein binding of trichothecene mycotoxins, T-2 toxin and HT-2 toxin, in dogs

The pharmacokinetics of T-2 toxin, following i.m. and i.v. administration (0.4 mg/kg), were investigated in five dogs. Following i.m. administration, the mean pharmacokinetic parameters for T-2 and HT-2 toxins were, respectively: apparent half-life 21 +/- 5 and 73 +/- 7 min; peak plasma concentration 182 +/- 42 and 74 +/- 16 ng/ml; time to reach peak plasma concentration 9.4 +/- 6.4 and 49 +/- 11 min. Mean residence time calculation, using moment analysis, showed that the terminal slope of T-2 toxin plasma levels following i.m. administration corresponds to the absorption rate constant of the toxin due to the flip-flop phenomenon. T-2 toxin was completely absorbed following i.m. administration and its absolute bioavailability was 1.17 +/- 0.25. A plasma protein binding study showed that in a concentration range of 70-500 ng/ml, T-2 and HT-2 toxins have a mean free fraction of 30.6 +/- 3.1% and 32.6 +/- 3.6% with no concentration dependency. At physiological conditions (temperature and pH), both T-2 and HT-2 toxins were unstable in whole blood and their in vitro stability half-lives were 6.9 and 0.84 hr, respectively. However, under similar conditions, these toxins were stable in plasma for 7 hr. Their instability in whole blood, therefore, may be related to enzymes present in the blood cells.     

Fate and distribution of 3H-labeled T-2 mycotoxin in guinea pigs

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.     

Metabolism and Clearance of T-2 Mycotoxin in Perfused Rat Livers

Metabolism and Clearance of T-2 Mycotoxin in Perfused Rat Livers.PACE, J.G. (1986). Fundam. Appl. Toxicol. 7, 424-433. Isolatedperfused rat livers were used to study the metabolism and clearanceof T-2 mycotoxin, a nonprotein Fusarium metabolite known tocause illness or death on contact or by ingestion. To evaluatethe in vitro hepatic metabolism, clearance, and rate of biliaryexcretion of T-2 toxin, [³H]T-2 toxin was delivered under constantperfusate flow (8 ml/min, 33.9 µg T-2/min) in a single-passexperiment. Steady-state conditions were achieved within 10min as indicated by a constant exit rate of radiolabel in theeffluent. At steady state, 93 ± 4% of the delivered [³H]T-2was extracted and metabolized by the liver, while 4.6 ±0.3% remained unmetabolized in the effluent perfusate. The excretionrate of metabolites and conjugates into bile was constant aftera 10-min perfusion. Radioactivity measured in bile accountedfor 55% of the total radiolabel delivered during the perfusionexperiment (1 hr). T-2 toxin was metabolized and eliminatedas 3Tiydroxy HT-2, 3Tiydroxy T-2 triol, 4-deacetylneosolaniol,T-2 tetraol, and glucuronide conjugates of HT-2, 3Tiydroxy HT-2,and T-2 tetraol. Approximately 7% of the administered radiolabelremained 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-2and its metabolites equaled 97.6% (bile, 52.5%; perfusate, 38.0%;liver, 7.1%). Approximately 3% of the biliary radiolabel wasnot identified. These studies describe the use of a perfusedorgan system to determine the rate of formation of T-2 metabolitesand their elimination into bile.     

Fate and distribution of H-labeled T-2 mycotoxin in guinea pigs

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 radioacivity 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.     

Human Biomonitoring of T-2 Toxin,T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples (n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22–6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3′-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.     

Comparison of in vivo and in vitro percutaneous absorption of T-2 toxin in guinea pigs

The fate and distribution of T-2 were examined in 6 guinea pigs. T-2 (1.2 micrograms/cm2), in methanol or DMSO, was painted onto the shaved backs of guinea pigs, a screen barrier was applied, urine and feces were collected daily and the guinea pigs were killed after 48 hr. Disks of skin (lateral to the in vivo site of application) were excised from the guinea pigs and used for in vitro penetration studies with static diffusion cells. Skin excised from 6 additional guinea pigs was used for penetration studies with flow-through diffusion cells. For in vitro studies, T-2 dissolved in methanol or DMSO was applied to the epidermal surfaces and the appearance of penetrant in receptor fluid bathing the dermal surfaces was monitored for 48 hr. Metabolism of T-2 was measured by using thin layer radiochromatography to identify metabolites. In the in vivo study, mean cutaneous absorption (n = 3) after 48 hr (expressed as per cent dose) was 22.5 and 51.9 for the methanol and DMSO groups, respectively. In vitro cutaneous penetration for static diffusion cells was 3.9 and 38.4 for the methanol and DMSO groups. For flow-through diffusion cells, mean penetration (n = 9) was 14.6 and 42.6 for the methanol and DMSO groups. Urinary metabolites of T-2 were T-2 triol, 3' OH-HT-2, T-2 tetraol, the glucuronide conjugate of HT-2 and several more polar metabolites. The main metabolite of T-2 in the receptor fluid bathing the dermal surfaces of excised skin was HT-2.     

Pharmacokinetics of T-2 toxin and its metabolite HT-2 toxin, after intravenous administration in dogs

The pharmacokinetics of T-2 toxin and HT-2 toxin were investigated comparatively in five dogs, after iv administration of the toxins (0.4 mg/kg). T-2 toxin was very rapidly and almost completely biotransformed to HT-2 toxin (fm = 83.6 +/- 3.9%). The following mean pharmacokinetic parameters were determined in this study for T-2 toxin and HT-2 toxin, respectively: half-life 5.3 +/- 2.1 and 19.6 +/- 4.7 min, clearance 0.107 +/- 0.056 and 0.167 +/- 0.074 liters/min/kg, and volume of distribution 0.86 +/- 0.63 and 4.47 +/- 1.38 liters/kg. The high clearance values suggest that the metabolism of T-2 toxin and HT-2 toxin is carried out in blood and/or through diffusion by nonspecific carboxyesterases. The results of this study suggest the possibility of a sustenance of T-2 toxin metabolism by its binding to blood cells.     

Metabolism of T-2 toxin in vascularly autoperfused jejunal loops of rats

The intestinal metabolism of T-2 toxin, a major trichothecene mycotoxin, was investigated in rats using the method of the vascularly autoperfused jejunal loop in situ. Tritium-labeled T-2 toxin was injected into the tied-off intestinal segments at a dose of 5 or 500 nmol, respectively. T-2 toxin and its metabolites in the blood draining from the jejunal loops, in the intestinal lumen, and in the intestinal tissue were determined by HPLC and GLC-MS. There was an extensive metabolic degradation of T-2 toxin, the metabolite pattern being similar for the two dosage levels. During the experimental period of 50 min only some 2% of the total dose appeared in the effluent plasma as unchanged T-2 toxin. Likewise at the end of the experiments unchanged T-2 toxin in the intestinal lumen and tissue was present in minute amounts only (less than 1% of the dose). HT-2 toxin was the main metabolite. About 25% of the total radioactivity administered appeared in the effluent plasma as HT-2 toxin, 18% in the lumen and 10% in the tissue. 3'-OH-HT-2 toxin accounted for 4-7% (effluent plasma), 5% (lumen), and 2% (tissue) of the total dose. Furthermore small amounts (less than 2% of the dose) of 3'-OH-T-2 toxin, T-2 tetraol, and 4-deacetylneosolaniol were found. No glucuronide or sulfate conjugates could be detected. In the jejunal segments which had been exposed to the 5-nmol dose only minimal morphological alterations were observed. On the other hand, in jejunal segments exposed to the high dose marked tissue damage was present. Nevertheless the gut tissue retained its ability to metabolize T-2 toxin. From the present results it is concluded that T-2 toxin is subject to a marked presystemic first pass effect after oral ingestion in vivo.     

Enterohepatic circulation of T-2 toxin metabolites in the rat

The enterohepatic circulation of T-2 toxin and its conjugated metabolites was examined in bile duct-cannulated male rats. Rats administered tritiated T-2 toxin intraduodenally (id) eliminated 44.65% and 57.25% of the administered dose in the bile within 4 and 8 hr post-dosing, respectively. TLC profiles of the T-2 metabolites were similar after intravascular and id administration. The major metabolites detected were 3'-OH-hydroxytryptamine-2 (HT-2), glucuronic acid conjugates, T-2 tetraol (TOL), 4-deacetylneosolaniol (4-DN), and HT-2. Tritium-labeled glucuronides obtained from the bile of rats administered [3H]T-2 toxin intravascularly were extracted and purified using C-18 and silica column chromatography. Enzymatic hydrolysis followed by TLC and GC/MS indicated that the aglycone portion of the glucuronides were composed of 3'-OH HT-2, HT-2, 4-DN, and TOL. After id administration of the glucuronides the rats eliminated 6.01% (4 hr) and 11.86% (8 hr) of the dose in the bile. No free metabolites of T-2 toxin were detected in the bile of any animals administered the purified glucuronides. Oral treatment of the rats with the beta-glucuronidase inhibitor, saccharolactone, did not produce a significant decline in the amount of radioactivity recovered in the bile following administration of the tritium-labeled glucuronides. These studies substantiate the enterohepatic circulation of T-2 toxin metabolites.     

Trichothecene mycotoxins inhibit phosphoinositide hydrolysis in bovine platelets stimulated with platelet activating factor.

The effects of the trichothecene mycotoxins, acetyl T-2 toxin, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and T-2 tetraol on phospholipid turnover were determined in bovine platelets prelabelled with [1-14C]arachidonic acid (AA). In resting, non-stimulated platelets exposed to acetyl T-2 toxin, a marked decrease in [1-14C]phosphatidylinositol (PI) along with a marked increase in [1-14C]phosphatidic acid (PA) were observed, whereas T-2 toxin, and HT-2 toxin only induced a significant increase in [1-14C]PA. In contrast, in platelet activating factor (PAF)-stimulated platelets, the mycotoxins were found to suppress both the agonist-induced loss of [1-14C]PI and the appearance of [1-14C]PA with acetyl T-2 toxin being the most effective and T-2 toxin, HT-2 toxin, and DAS essentially equally effective. T-2 tetraol and DON did not affect phospholipid metabolism either in unstimulated or PAF stimulated platelets. The alterations in [1-14C]PI and [1-14C]PA suggest that the inhibitory toxins may activate a specific phospholipase C (PLC) in the unstimulated platelets and then impede further PLC activation in PAF-stimulated platelets.     

Inhibition of bovine platelet function by T-2 toxin, HT-2 toxin, diacetoxyscirpenol and deoxynivalenol

The aggregation of bovine platelets suspended in homologous plasma is inhibited in the presence of T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) or deoxynivalenol (DON) when either collagen or ADP is used as the stimulatory agent for aggregation. For each of the mycotoxins the degree of inhibition is dependent on the amount of trichothecene present in the platelet suspension but is not dependent on the time of exposure of the platelets to the toxin. For both ADP- and collagen-stimulated platelets, the order of potency of inhibition is T-2 toxin greater than HT-2 toxin greater than DAS greater than DON. A significant (P less than 0.01) dose-dependent decrease was also observed in the amount of the thromboxane B2 released from collagen-stimulated platelets in the presence of each of the mycotoxins.     

In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues

Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies.     

Uptake and metabolism of T-2 toxin in relation to its cytotoxicity in lymphoid cells

The sensitivity of lymphoid cells to the cytotoxic effects of T-2 toxin (T-2) varies according to their degree of differentiation. To understand the mechanisms of these variations, the uptake and the metabolism of T-2 in susceptible (human lymphoma Daudi and phytohaemagglutinin-stimulated murine lymphocytes) and resistant (human leukaemia KE37 and REH) cells were studied in culture. When cells were incubated with [3H]T-2 a significant increase in the quantity of T-2 associated with the cell occurred during the first 30 min, this increased further from 10-16 hr, and decreased after 24 hr. Daudi and REH cells took up 20 and 3% of the T-2 present in the medium, respectively. Metabolites, extracted from the culture medium and from cells, were analysed by the thin-layer chromatography. The products were identified by comparison with standards for T-2 tetraol, T-2 triol, HT-2 toxin, neosolaniol and T-2. Qualitatively, similar metabolic pathways were found in all cells examined. The presence of these metabolites demonstrated that T-2 was taken up by these cells. A correlation existed between the relative sensitivities of the cells toward T-2 and the amount of intracellular T-2 and/or metabolites. It is thought that differences in the kinetics of uptake and processing of T-2 account for the known differences in cellular sensitivities to the toxin.     

Metabolism of T-2 toxin by blood cell carboxylesterases

Human and rat blood hydrolysed T-2 toxin along two different pathways giving HT-2 toxin and neosolaniol as primary metabolites, respectively. Neosolaniol represents a metabolic pathway different from that obtained by liver. Rat erythrocytes formed neosolaniol as a primary metabolite whereas white blood cells hydrolysed T-2 toxin to HT-2 toxin. Human erythrocytes formed both HT-2 toxin and neosolaniol whereas all human white cells produced only HT-2 as the primary metabolite. The enzymes responsible for hydrolysis of T-2 toxin to HT-2 toxin in white blood cells and T-2 toxin to neosolaniol in red blood cells were all identified as carboxylesterases by use of specific inhibitors. The ratio between trichothecene hydrolysis and 4-nitrophenyl butyrate hydrolysis varied among the different cell fractions indicating that specific isoenzymes are involved.     

Effects of sodium fluoride on uptake of T-2 mycotoxin in cultured cells

We examined the effect of sodium fluoride on uptake of tritium-labeled T-2 toxin (molecules of toxin/cell) in Chinese hamster ovary (CHO) and African green monkey kidney (VERO) cells. Correlations were made to temperature (22 and 37 degrees C) and toxin concentration (0.001 and 0.01 microgram/ml) over time (0-180 min). As expected, toxin uptake increased in both cell types with increasing time and temperature. VERO cells exhibited significant (P less than 0.05) increases in the rate (i.e. slope) of toxin uptake under all parameters, while the rate of toxin uptake in both cell types was generally greater at 37 degrees C compared to 22 degrees C. The rate of equilibrium was affected by both temperature and sodium fluoride. At 37 degrees C toxin uptake plateaued by 30 min in the presence of sodium fluoride. At 22 degrees C the rate of toxin uptake was slower, with or without sodium fluoride present. Statistical analysis of individual time points along the curve demonstrated that sodium fluoride significantly increased cell-associated toxin at most time points. Analysis of the slopes of uptake curves from 0 to 20 min indicated significant (P less than 0.05) differences in the rates of T-2 uptake in both cell types and toxin doses in the presence of sodium fluoride. The increase in toxin uptake in the presence of sodium fluoride was not due to altered cell membrane permeability caused by sodium fluoride. This study demonstrates that sodium fluoride significantly increases cell-associated T-2 toxin and the rate of toxin uptake in two cultured cell lines.