Toxicology, 70 (1991) 133-140 Elsevier Scientific Publishers Ireland Ltd.
Acute effects of T-2 toxin on radioactive iron incorporation into circulating erythrocytes in mice G r a c i e l a C. Faifer* and H e c t o r M. G o d o y * * Lahoratorio de Micotoxinas y Toxicologia Animal, Instituto de Patobiologia. CICV. INTA, Castelar. Pcia. de Buenos Aires, (A~entina) Received May 22nd, 1991: accepted July 12th, 1991)
Summary The 24-h and 72-h incorporation of 59Fe into circulating erythrocytes in mice were strongly inhibited by a single subcutaneous dose of T-2 toxin given 1 h before the radioisotope. The system is extremely sensitive, since a significant effect was detected with T-2 toxin doses as low as 0.30 mg/kg, which is about one-tenth of the LDs0 in the BALB/c strain used for the present study. In the treated animals no initial changes were observed in the blood SgFe levels or in the rate of radioisotope clearance from plasma, indicating that the toxin does not interfere with iron absorption or transport. It is concluded that the inhibition observed reflects the damage produced by this toxin on reticulocytes and/or erythroblasts, and therefore this method could be of value as a very sensitive means of studying the risk of erythropoietic injury produced by dietary exposure to trichothecene mycotoxins. Key words." Trichothecenes; Mycotoxins: Bone marrow toxicity: Erythropoietic damage
Introduction The trichothecenes are a group of extremely potent toxins, which are produced as secondary metabolites by several fungal genera, such as Fusarium, Stachybotrys, Myrothecium or Cephalosporium . Some of these toxins, particularly those from Fusarium, are frequently found as contaminants of wheat, barley, corn and other cereal products in several areas of the world and have been implicated in outbreaks of human and animal disease . Acute symptomatology observed in the intoxication with trichothecenes is characterized by dermal irritation, hemorrhage, nausea, vomiting, refusal of feeds, diarrhea, abortions and neurological disturbances , whereas chronic low-level exCorrespondence to: Hector M. Godoy, Laboratorio de Toxicologia, lnstituto de Patobiologia, CICV INTA, CC 77, 1708 Moron, Pcia. de Buenos Aires, Argentina. Taken from a thesis to be submitted by G.C. Faifer to the University of Buenos Aires in partial fulfillment of the requirements for the degree of Doctor in Biology. *Recipient of a Research Fellowship from CONICET, Argentina. **Career investigator of the Comision de Investigaciones Cientificas, Provincia de Buenos Aires, Argentina. 0300-483X/91/$03,50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
134 posure most frequently involves hematological and/or immunological alterations . At the cellular level these toxins are known to damage the proliferating cells of tissues such as intestinal mucosa, spleen, thymus and bone marrow and these effects apparently derive from the potent activity of these toxins as inhibitors of protein biosynthesis . In order to assess the risk associated with the presence of low amounts of trichothecenes in human or animal diet, more sensitive methodology is required, to detect and quantitate the early alterations produced on the organism, particularly at the level of hemopoietic and lymphoid tissues, which appear to be the critical targets for these radiomimetic toxins. Radioactive iron uptake and turnover is routinely used as an index of erythropoietic activity . Among the several methods that have been described, one of the simplest is the measurement of the 59Fe incorporation into circulating erythrocytes, which has been used as a rapid and sensitive experimental approach to detect erythropoietic damage produced by several chemicals . In the present report we describe some work undertaken with the purpose of testing 59Fe uptake into mouse erythrocytes as an early index of erythropoietic damage produced by T-2 toxin, one of the most potent trichothecenes known. In this first series of experiments, a single subcutaneous injection of T-2 toxin was given 1 h before the administration of 59Fe and the radioactivity incorporated into the red blood cells was measured 24 or 72 h later. It has been previously shown that, in mice, the 24-h 59Fe utilization measures only the radioisotope taken up by reticulocytes, whereas the 72-h 59Fe utilization measures the overall uptake by all the iron-incorporating erythroid precursor cells: proerythroblasts, erythroblasts and reticulocytes . Materials and methods Chemicals
T-2 toxin was prepared in our laboratory by a modification of the method of Burmeister , using a toxigenic strain of Fusarium tricinctum, which was grown on a Czapeck-Dox/peptone medium at room temperature over a period of 14 days. The concentrated chloroform extract was purified through a column of silica gel (Merck, Art. 7734) developed with a linear gradient of toluene/ethyl acetate. Crude T-2 toxin was repeatedly crystallized from toluene/hexane until the melting point was 150-152°C. Radioactive iron was obtained as a sterile 59Fe citrate solution from the Comision Nacional de Energia Atomica, (Argentina). Dimethylsulfoxide (DMSO) was purchased from Sigma Chemical Co. (Missouri, U.S.A.). All other chemicals were of analytical grade or better. Animals and treatments
Male BALB/c mice (25-30 g) were obtained from the animal house of the Center of Veterinary Research (CICV), 1NTA, Castelar and maintained on a diet of pelleted chow and water ad libitum.
135 59Fe was injected intraperitoneally at a dose of 0.5 tzCi/animal, dissolved in physiological NaC1 saline . T-2 toxin was dissolved in DMSO and injected subcutaneously at different times before the administration of 59Fe. Control animals received the same amount of DMSO. All injections were made at 9:00 h to 10:00 h.
Methods of Analysis. Blood was withdrawn 24 h or 72 h after 59Fe injection, under ethyl ether anesthesia from the inferior cava vein and transferred into heparinized tubes. When appropriate, blood was centrifuged for 20 min at 2500 rev./min, plasma was aspirated with a Pasteur pipette and erythrocytes were repeatedly washed (three times) with NaCI isotonic saline. Whole blood (0.2 ml), or the corresponding amount of erythrocytes or plasma, reconstituted to 0.2 ml with physiological saline, were transferred to 0.5 ml polyethylene Beckman microtubes fitted with a cap, and the whole microtube was dipped into a glass vial filled with 20 ml of PPO/toluene (5 g/l) and counted in a Packard liquid scintillation spectrometer (model 2650). For standardization of the assay the amount of 59Fe corresponding to 100% incorporation (calculated assuming a blood volume of 6% of total body wt) was mixed with 0.2 ml of blood and counted as above.
Statistical evaluations The mean and the standard error of the mean were calculated. A two-tailed Student's t-test was used for comparison of two means. The level of significance was set at P _< 0.05. Results and Discussion
An approximate LD50 of T-2 toxin, injected subcutaneously, was determined in the BALB/c strain of mice used for the present study and found to be in the range 2.6-2.8 mg/kg. This compares well with the 2.1 mg/kg reported by Ueno [I0], working with 6-week-old ddYS mice. The time-course of the accumulation of radioactivity into erythrocytes after an i.p. injection of 59Fe is depicted in Fig. 1. It can be appreciated that the incorporation rises steadily up to 72 h, reaching about 30% of the injected dose, and it remains nearly constant thereafter. In agreement with the data reported by previous authors, it was found that about two-thirds of the maximal uptake occurred in the first 24 h, during which only the 59Fe uptake corresponding to reticulocytes would be detected in circulating blood . Following the experimental model described by Lee et al.  several doses of T-2 toxin were administered to the animals 1 h before the injection of the radioisotope and the radioactivity incorporated into erythrocytes was measured 24 and 72 h later. The results, shown in Fig. 2, reveal that the toxin produced a striking dosedependent inhibition of the 59Fe uptake into red blood cells. When expressed as a percentage of the respective controls, the inhibition curves corresponding to 24-h and 72-h 59Fe uptake were quite similar, with minor non-significant differences, ex-
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Fig. I. Time course of 59Fe incorporation into mouse circulating erythrocytes. 0.5 ~tCi of sterile 59Fe citrate were injected i.p. into adult (25 g) male BALB/c mice. At the times indicated blood was withdrawn and radioactivity measured in the erythrocyte fraction as indicated in Materials and methods• Each point is the mean of data from 10 animals. Vertical bars correspond to ± S.E.
cept with a T-2 toxin dose of 0.30 mg/kg, where a statistically significant inhibition was observed with 72-h, but not with 24-h 59Fe incorporation experiments. In order to test whether the inhibitory effects observed could be ascribed to an action of the toxin on the early phase of iron metabolism, the kinetics of the iron translocation into the vascular compartment and the distribution of the radioisotope between plasma and red cells were compared in control and T-2 toxin-treated mice. As shown in Table I, no significant differences in the plasma levels of 59Fe were seen in the intoxicated as compared to control mice. At 12 h and thereafter the radioactivity was totally absent from plasma in both experimental groups, indicating that the toxin does not affect the rate of 59Fe plasma clearance. Erythrocyte-bound radioactivity, on the other hand, was considerably reduced in the T-2 toxin-treated mice at 6 h and afterwards. These results suggest that the inhibition produced by T-2 toxin is not due to an impairment of iron absorption or transport, nor to an interference with iron uptake by the erythropoietic tissues, since the plasma 59Fe half life is not lengthened, at least in the early stage of the intoxication. Thus it appears very likely that the observed effects of the toxin are due to a direct action on the erythropoietic cells, and this could be related to its potent activity as inhibitor of protein synthesis , or to its cytotoxicity on proliferating cells , or both. In principle, it could be expected that the observed inhibition of the 24-h 59Fe uptake would express the action of T-2 toxin on hemoglobin synthesis in reticulocytes, whereas the 72-h uptake could reflect, in addition, the cytotoxic activi-
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()30 0".44 066 T-2 toxin dose (mg/kg)
Fig. 2. Inhibition of 24-h and 72-h 59Fe incorporation into erythrocytesby different doses of T-2 toxin (subcutaneously 1 h before the radioisotope). Results are expressed as % of solvent-treated controls. Vertical bars correspond to ± S.E. (8-10 animals were used for each experimental point). (*) Significantly different from controls (P