The effect of T-2 toxin on human platelets

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

73,

2 10-2 17 (1984)

The Effect of T-2 Toxin on Human Platelets R. YAROM,*

R. MORE,*

A. ELDoR,t

AND B. YAGEN~

*Departments of Pathology and THaematology and the *Department of Natural Products, School of Pharmacy, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received

February

4, 1983; accepted

October

20, I983

The Effect of T-2 Toxin on Human Platelets. YAROM, R., MORE, R., ELDOR, A., ANDYAGEN, (1984). Toxicol. Appl. Pharmacol. 73, 2 10-2 17. Human platelets were incubated with T-2 toxin, the most toxic component of Fusarium fungi, at doses of 5 to 500 &lo9 platelets. A dose-related inhibition of platelet aggregation and release of dense bodies (these consist mainly of serotonin-containing granules) were observed. A change in membrane permeability in the absence of shape changes was also demonstrated by a heavy metal impregnation technique. There was no correlated inhibition of thromboxane synthesis or significant alterations in platelet calcium content. The microtubular system was also unaffected. Suppressed platelet aggregation may contribute to the lethal hemorrhagic phenomenon associated with intoxication by fiuarial toxms in man and animals. B.

T-2 toxin (3-d-hydroxy-4-8, 15diacetoxy-8cu-(3-methylbutyryloxy)- 12,13 epoxytrichothee-9-en) is a highly toxic metabolite (or mycotoxin) produced by Fusarium poae, F. sporotrichioides, and F. tricinctum. This mycotoxin is responsible for many signs found in human and animal intoxications (Ciegler, 1978; Hsu et al., 1972; Lutsky et al., 1978; Petrie et al., 1977; Rukmini et al., 1980). Alimentary toxic aleukia (ATA), a noninfectious, often fatal human disease, is related to ingestion of food derived from moldy gram (Lutsky, et al., 1978). ATA is well known in the Soviet Union and is characterized by hemorrhagic diathesis, bone marrow suppression, and susceptibility to sepsis. Experimental animals treated with T-2 toxin often develop clinical signs resembling those found in “moldy corn toxicosis” of cattle. The pathogenic mechanism of T-2 toxin induced disease is of special interest because of controversial reports that “yellow rain,” possibly used in warfare in Asia and elsewhere, contained this substance (Mirocha et al., 1982; Robinson, 1982). The hemorrhagic effects of T-2 have been attributed to vascular damage (Schoental et 0041-008X/84

$3.00

Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

210

al., 1979) or to thrombocytopenia (Lutsky et al., 1978; Petrie et al., 1977). The direct effect of T-2 toxin on platelet function and structure has not been studied. The work reported here deals with functional and ultrastructural responses of human platelets to incubation with varying doses of T-2 toxin in vitro. The results may contribute to a better understanding and treatment of the hemorrhagic phenomenon in Fusarium intoxication, and perhaps help elucidate the mode of action of T-2 on cell membranes. METHODS T-2 toxin was prepared as previously reported (Lutsky ef a/., 1978). An alcoholic stock solution of T-2 toxin was diluted with normal saline before use so that the alcohol content of the working solution was less than 0.1%. This alcohol concentration was shown in preliminary tests not to affect platelet structure and functions. Blood from 12 healthy volunteers, who had not taken any medication for the previous few weeks, was drawn into a 0.10~~ volume of 3.2% sodium citrate. Platelet rich plasma (PRP) was obtained by centrifuging the blood at 1OOgfor I5 min. The platelets were counted and their number was adjusted with platelet poor plasma (PPP) to about 250 X log/liter.

T-2 TOXIN

211

EFFECT ON PLATELETS

Morphological tests were carried out on 0.2- or OS-ml aliquots of PRP which were incubated at 37°C for 20 min with the following doses of T-2 toxin: 5, 10, 50, 100, 150, 300, and 500 &lo9 platelets (in constant volumes of solvent). In each set of experiments, platelets from the same donor, incubated with solvent only, served as the baseline control. The effectof T-2 toxin on the microtubular system.This systemis sensitive to temperature changes and causesshape changes in platelets at 0°C. With a return to 37”C, the normal disc shape is resumed. In ah experiments, platelets in the PRP were rested I5 min in an incubator at 37°C. Then 300 or 500 &lo9 platelets of T-2 toxin was added to some samples of PRP while solvent only was added to others. After 15 min at 37°C all the sampIes were placed in a refrigerator at 0°C for 15 min. The last step consisted of placing the PRP samples back in the incubator at 37°C for 25 min. Part of the platelets were examined by transmission electron microscopy after incubation at 37’C, after 15 min at 0°C and after 25 min of return to 37’C. Electron microscopy. A drop of PRP from each aliquot was placed on a Formvar coated E/M grid. After 30 set this drop was dried with ashless filter paper and viewed unstained in a transmission electron microscope (Philips 400). The platelets spread out on the support and were easily visible. The number of electron opaque bodies in each of 100 to 200 platelets was counted. The total number of electron dense bodies per 100 platelets, the average number per platelet, and the percentage of platelets with more than nine opaque bodies each were calculated. For ultrathin sectioning, 0.5-ml ahquots (treated with T-2 toxin and controls) were fixed with 0.2% of glutaraldehyde in Hanks’ buffered saline solution. After 30 min, the platelets were pelleted and further processed for transmission electron microscopy and/or impregnation with heavy metals at a pH of 3.5 (Yarom et al., 1982). Platelet aggregation. A platelet aggregation test was carried out on blood from five of the donors. In two cases, repeat examinations were done on the same person but with blood drawn on a different day. The results were reproducible. The PRP prepared as above was divided into three or more parts and incubated for 10 to 30 min at 37°C with different doses of T-2 toxin. For each donor, a PRP sample incubated with solvent only served as a control. The usual concentrations of T-2 toxin used were 50, 150, and 300 &lo9 cells; in several cases T-2 toxin amounts of 10, 100, and 500 llg/109cells were also tested. Aggregation tests of control samples were often repeated at the end of the maximum period (2 hr) allowed after blood withdrawal. Ifthis differed horn the previous reading, an average of the two was taken as the baseline. Aggregation was monitored in a Payton dual channel aggregometer. PRP samples of 0.5 ml were first stirred at 37°C for 1 mitt, then the various aggregating agents were added and the change in light transmission was recorded for 4 min. The deflection integral (i.e., the area beneath the recorded curve) was measured; the control sample

from each donor (with solvent only) served as a 100% baseline. The effectof the drug on aggregation with different activators was expressed as a percentage change from the control sample in each case. The effect of duration of exposure to T-2 on the aggregation changes was examined with epinephrine stimulation in one case. Radioimmunoassayfor thromboxane Bz (TXBJ. Samples of 0.1 ml were withdrawn from the aggregation cuvettes4 min aher addition of the aggregating agents. These were quenched immediately with 0.9 ml of 0.1 M phosphate buffered saline (pH 7.4) containing 0.1% gelatin and lo-’ M indomethacin. The samples were transferred from liquid nitrogen to a refrigerator at -70°C in which they were kept until assayed.’The specificity and cross-reactivity of the antibody were as previously described (Levin et al.. 1981). Radioimmunoamay was carried out according to the method of Bauminger ef al. (1973). Calcium concentration. Calcium concentration in platelets from 7 of the 12 donors was measured by atomic absorption spectrometry (Sweetman et al., 1980). The number of platelets in the PRP was counted in a Coulter Counter (Model S plus). One-milliliter aliquots were incubated either with T-2 toxin in doses as indicated in Table 1 or with solvent only for 30 min. Each sample was then centrifuged at 800 g for 20 min, and the pellet was washed twice with Tris buffer. The platelets were then lysed with 4 ml of a solution containing 5% trichloroacetic acid, 0.5% lanthanum oxide, and 0.3% NaCl. The lysate was centrifuged at 800 g for 15 min, and the supematant fraction was used for calcium estimations.

RESULTS In unstained whole mounts, the platelets spread out on the grid support allowing visualization of cell outline, intracellular vacuoles, and electron opaque bodies. The main change that occurred after incubation with various doses of T-2 toxin was in the number of eIectron opaque bodies. There was little or no effect with solvent only or with small doses of toxin. With 10 and 50 pg T-2/109 cells, there was a significant increase in number of opaque bodies per 100 platelets and of platelets ’ The aggregating agents in the final concentrations used on samples treated with T-2 toxin and on control samples were as follows: epinephrine, 5.5 mM (Gotham Pharmaceutical Co., Inc., Brooklyn, N.Y.), collagen, 0.8 4 ml (“Collagen reagent. Horn”, Hormon-Chemie, Munchen GMBH posthxh lOI), and arachidonic acid, 0.2 mM and adenosine diphosphate (ADO) 1.2 mM (both from Sigma Chemical Co., St. Louis, MO.).

212

YAROM TABLE

1

PLATELET CALCIUM CONCENTRATION“ T-2 toxin Donor No.

2 4 5 6

Control

sample 225 224 145 262

50 /Lg./ 109 cells

100 or 150 &lo9 cells

197 135 153 250 190 229 123 182 f 44

10

150

11 12

380 197

235 147 133 290 192 326 126

226 + 74

207 f 73

.T

A

SD

’ In nmol/109 cells.

with numerous such bodies. This number decreased again to control values with higher doses (Figs. 1, 2, and 3). In conventionally prepared ultrathin sections, the T-2 treated platelets were, like the controls, discoid in shape with little evidence of activation. The cell membrane, microtubules, and other intracellular structures showed no obvious morphological abnormalities (Fig. 4). On exposure to 0°C the experimental, like the control, platelets underwent a shape change (Fig. 5). In both cases a

ET AL.

return to normal shape occurred after 25 min at 37°C (Fig. 6). In ultrathin sections of platelets treated by the heavy metal impregnation technique, no change from normal occurred with smaller doses of T-2 toxin. The platelets continued to show differentiation into the three groups previously described (Yarom et al., 1982): type 1 platelets with dark granules and a prominent reticular network, type 2 platelets with densely stained cytoplasm; and type 3 platelets with microvesicles and little other staining (Fig. 7). With larger doses of toxin (100 or more Mg/ lo9 cells), the stain penetration became very poor, and pale cells of normal shape, but indistinct internal structure, were the rule (Fig. 8). The aggregation results are shown in Figs. 9 and 10. There was little change from control when the platelets were incubated with a dose of 10 pg T-2 toxin/ lo9 platelets. With 50, 100, or 150 pg T-2 toxin/ 1O9 cells, there was inhibition of aggregation by epinephrine, arachidonic acid, and collagen. ADP aggregation was, as a rule, slightly enhanced by these doses of T-2 toxin. Aggregation induced by all the activators, including ADP, was decreased or abolished when the dose of T-2 toxin was 300 &lo9 platelets or higher. The synthesis of TXB2 showed much variability. In most cases, however, there was a

FIGS. 1 and 2. Electron micrograph of whole, air-dried platelet (unfixed, unstained) showing clearly the electron opaque bodies (DB) of different sizes. Figure 1 is from an untreated control while Fig. 2 is from a sample incubated with 50 pg T-2 toxin/lo9 platelets. There is an increased number of dense bodies in this platelet.

T-2 TOXIN

0” x E % 50 y ii

40

0

“0 30

I

-’ r t 0

2

5

DOSE

OF

10 T-Z

1 r 8

50

loo

20

213

EFFECT ON PLATELETS

IE 0 % ? I i

IN pg/logPLATELETS

FIG. 3. Graph illustrating the biphasic change in the number of electron opaque bodies per 100 platelets and proportion of cells with numerous such bodies after various doses of T-2 toxin.

marked decrease in TXBz production whenever aggregation was severely inhibited by larger doses of T-2 toxin. With moderate doses

FIG. 4. Electron micrograph of platelet treated with T2 toxin (150 &109) showing preservation of disc shape and of microtubules (X75,000).

of T-2 toxin, the inhibition of aggregation after stimulation with epinephrine or collagen was not accompanied by a corresponding decrease in TXBl synthesis (Fig. 10). When arachidonic acid (a percursor of prostaglandin synthesis) was the activator, there seemed to be a correlated suppression of TXB2 synthesis and aggregation. The effect of T-2 toxin on the platelet calcium concentration is shown in Table 1. There was much individual variation among the donors. Although in most cases there seemed to be a decrease of calcium content after incubation with T-2 toxin, the overall figures (when averaged) showed no statistical difference from controls. DISCUSSION The results of this study shows that T-2 toxin affected platelets in vitro. The doses used were within the range of doses reported to be

FIG. 5. Electron micrograph of platelet incubated with T-2 toxin (600 &IO9 cells) and kept for 15 min at a temperature of 0°C. Like controls, the treated cells underwent a shape change (X 12,000).

214

YAROM

B?

I, 4

ET AL.

mal number of these bodies with sodium citrate as the anticoagulant is about 650/100 cells. The dose-related augmentation in numbers of electron opaque bodies may be due either to an increase in number of nucleoids or to an inhibition of spontaneous (or accidental) release of the dense bodies. Preservation of the disc shape in treated platelets and their return to normal shape after cold-induced deformation show that T-2 toxin does not damage the microtubules or the cytoskeleton. As suggested by the heavy metal impregnation technique, larger doses of T-2 toxin induce a fundamental change in platelet cell membranes. At pH 3.5 (the isoelectric point of platelets), permeability to cations decreased sufficiently to prevent cellular staining. T-2 toxin is lipophilic (Ciegler, 1978; Schoental et al., 1979). It apparently enters

FIG. 6. Same sample as in Fig. 5 but brought back to a temperature of 37°C for 25 min. The cells returned to a disc shape indicating preservation of the cytoskeleton. With this high dose of T-2 there was an increase in vacuolation (X 12,000).

toxic for mammals. The LD50 is usually given as 0.5 to 5 mg/kg (Ciegler, 1978; Lutsky et a/., 1978). A person weighing 50 kg has approximately 5 liters of blood and therefore around 1012 platelets (5 X 200 X log/liter) in circulation. T-2 toxin enters the blood rapidly since alter a single dose, by any route, it is found in most organs within 4 hr (Ciegler, 1978). Exposure to as little as 50 mg of toxin could soon give a level of 10 mg/liter, and this concentration could mean 50 fig/l O9 platelets, a dose shown here to affect platelets in vitro. The electron opaque bodies seen in the whole, air-dried cells were influenced by T-2 toxin. Their identity is not clear, but- they probably include the serotonin-containing delta granules usually called “dense bodies,” as well as the nucleoids of the alpha granules whose high calcium content renders them electron opaque (Sat0 et al., 1975). The nor-

FIG. 7. Electron micrograph of platelets treated with T-2 toxin (10 &log) impregnated with heavy metals. With this small dose, the appearance of the cells was like in controls, three cell types being recognimble--(a) type 1, reticular cells with dark alpha granules, (b) type 2, dark, metahophilic cells, and (c) type 3, poorly staining cells with microvesicles (~12,000).

T-2 TOXIN

215

EFFECT ON PLATELETS

FIG. 8. Electron micrograph of platelets incubated with a larger dose of T-2 toxin ( 150 pg/ 109) impregnated with heavy metals. There is little penetration of the metallic cations and no division into the three cell types. The cells are all pate but their shape and ultrastructure appear to be well preserved (X 12,000).

that T-2 toxin does not affect the enzymes of the cyclooxygenase-mediated arachidonic acid cascade. What is possible, is that T-2 toxin acts in a manner resembling detergents and other membrane active agents (Pestronk et al., 1982) by bringing about a change in the lipid composition of the plasma membrane proper. Whatever the explanation of the platelet changes produced by the toxin, their clinical implications are paramount. Death is rapid after exposure to Fusarium toxins and is usually accompanied by severe hemorrhages, mainly from the gastointestinal tract. Furthermore, in experimental animals, hemorrhages are found in many organs. The hemorrhagic diathesis, although said to be due to damaged blood vessels (Schoental et al., 1979), may be caused by a direct, rapid, and dose-related effect of T-2 toxin on platelet function. With smaller doses and the enhancement of ADP aggregation, hemostasis may be

1 COLLAGEN CONTROL

cells easily, and in platelets, it may rapidly alter surface-related functions. Of these, the most pertinent to the clinical picture of intoxication is the aggregation abnormality. At higher concentrations of T-2 toxin, it seems as if the platelets become paralyzed by a mechanism which is still unclear. The persistence of temperature-induced shape changes and the insignificant alterations in platelet calcium content negate the possibility that chelation or blockage of inward calcium fluxes is responsible for the inhibitory changes. Since stimulation by arachidonic acid is also inhibited by T-2 toxin, its effect is probably not receptor mediated. Platelets which are not capable of aggregating (as in thrombasthenia) may still produce thromboxanes (Hardisty, 1977). Here too, TX& synthesis was not inhibited in parallel with depressed aggregations. This result shows

T-2 SOfig

T-2 lOO/.lg

MINS.

4 ARACHIOONIC

ACID CONTROL

FIG. 9. Light transmission records of platelets from one patient stimulated with different aggregating agents. ADPinduced aggregation is affected in two ways--aggrekation is enhanced by smaller doses of T-2 toxin but inhibited by higher ones. T-2 toxin (all dose levels) inhibited two platelet aggregations induced by all the other agents.

216

YAROM

ET AL.

EPINEPHAINE

1050 TOXIN

T-2

150 IN “g/log

300 PLATELETS

-6

25-

-

AGGREGATIONS

-3

*.oTXB2

FIG. 10. Changes in platelet aggregation and in TX&, synthesis following various doses of T-2 toxin. The activating substances are indicated. In the aggregations the values at each point are averagesof percentage change from four to six samples f SD. In each case a sample from the same donor incubated with solvent only served as a control in which the light deflection integral was taken as a 100% aggregation.

maintained. With more severe intoxication. membrane changes become important and may lead to bleeding. Other intra- and extracellular factors also contribute to the Fusarium poisoning syndrome, but in any clinical treatment the role of platelet dysfunction must not be overlooked. ACKNOWLEDGMENTS This work was partially supported by a grant (to A.E.) from the Juzrykowski Foundation, New York, New York. Antisera against TXBr were a kind gilt of Dr. B. B. Weksler, Cornell Medical College, New York. Our thanks to Dr. A. 2. Joffe for growing the F. sporotrichiodes used for isolation of the toxin. We also thank Ms. S. Jackson, Ms. E. Hyman, Ms. H. Orgal, and Ms. Y. Havivi for their excellent technical assistance.

REFERENCES BAUMINGER, S., ZOR, U., AND LINDER, H. R. (1973). Radioimmunological assayof prostaglandin synthetase activity. Prostaglandins 4, 3 13-324.

CIEGLER, A. ( 1978). Trichothecenes; Occurrence and toxicosis. J. Food Prot. 41, 399-403. HARDISTY, R. M. (1977). Disorders of platelet function. Brit. Med. Bull. 33, 207-2 12. Hsu, I. C., SMALLEY, E. B., STRONG, F. M., AND RIBELIN, W. E. (1972). Identification of T-2 toxin in mouldy corn associated with a lethal toxicosis in cattle. Appl. Microbial. 24, 684-690. LEVIN, R. J., JAFFE, E. A., WEKSLER, B. B., AND TAKGOLDMAN, A. (1981). Nitroglycerine stimulates synthesis of prostacyclin by cultured human endothelial cells. J. Clin. Invest. 67, 762-768. LUTSKY, I., MOR, N., YAGEN, B., AND JOFFE,A. Z. (1978). The role of T-2 toxin in experimental alimentary toxic ale&a: A toxicity study in cats. Toxicol. Appl. Pharmacol. 43, 11 l-124. MIROCI.IA, C. J., WATSON, S. A., AND HAYES, A. W. (1982). Occurrence of trichothecenes in samples from Southeast Asia associated with “yellow rain.” Proceedings of the Fifth International IUPAC Symposium on Mycotoxins and Phycotoxins. Vienna, September, pp. 130-133. PESTRONK, A., PARHAD, L. M., DRAEHMAN, D. B., AND PRICE, D. L. (1982). Membrane myopathy; Morpho-

T-2 TOXIN

EFFECT ON PLATELETS

logical similarities to Duchenne muscular dystrophy. Muscle & Nerve 5, 209-2 14. PETRIE, L., ROBB, J., AND STEWART, A. F. (1977). The identification of T-2 toxin and its association with haemorrhagic syndrome in cattle. Vet. Rec. 101,326-327. ROBINSON,J. P. (1982). Chemical warfare: Some events of the past year and their implications. Pugwash Newsletter 19, 157-164. RUKMINI, C., PRASAS, J. S., AND RAO, K. (1980). Effect of feeding T-2 toxin to rats and monkeys. Food. Cosmet. Toxicol. 18, 267-269. SATO, T., HERMAN, L., CHANDLER, J. A., STRACHER,A., AND DETWILER, T. C. ( 1975). Localization of a throm-

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bin sensitive calcium pool in platelets. J. Histochem. Cytochem. 23, 103-106. S~HOENTAL, R., JOFFE, A. Z., AND YAGEN, B. (1979). Cardiovascular lesions and various tumors in rats given T-2 toxin, a trichothecene metabolite in Fusarium. Cancer Res. 39, 2 179-2 189. SWEETMAN, H. E., COGTA,J. L., VECCHRRUF,J. J., VALERI, C. R., AND SHEPRO,D. (1980). Dense bodies and total calcium in human platelets following aspirin ingestion for a period of two weeks. Thrombos. Res. 17,55-61. YAROM, R., MORE, R., HAVIVI, Y., LIJOVETSKY, G., AND MEYER, S. (1982). Studies of platelets with heavy metal impregnation techniques. Histochem. J. 14, 73-86.