Comparative acute and combinative toxicity of aflatoxin B1 and T-2 toxin in animals and immortalized human cell lines

Aflatoxin B1 (AFB1) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health and as potential biochemical weapons threats. In this study the acute and combinative toxicity of AFB1 and T-2 were tested in F-344 rats, mosquitofish (Gambusia affinis), immortalized human hepatoma cells (HepG2) and human bronchial epithelial cells (BEAS-2B). Preliminary experiments were conducted in order to assess the acute toxicity and to obtain LD50, LC50 and IC50 values for individual toxins in each model, respectively. This was followed by testing combinations of AFB1 and T-2 to obtain LD50, LC50 and IC50 values for the combination in each model. All models demonstrated a significant dose response in the observed parameters to treatment. The potency of the mixture was gauged through the determination of the interaction index metric. The results of this study demonstrate that these two toxins interacted to produce alterations in the toxic responses generally classifiable as additive; however, a synergistic interaction was noted in the case of BEAS-2B. It can be gathered that this combination may pose a significant threat to public health and further research needs to be completed addressing alterations in metabolism and detoxification that may influence the toxic manifestations in combination.     

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.     

Binding of T-2 toxin to eukaryotic cell ribosomes

The binding of radiolabeled T-2 to eukaryotic ribosomes was studied. The toxin bound to ribosomes in a time-, temperature- and concentration-dependent manner. The binding was saturable (0.3 nM), reversible at 37 degrees (half-time approximately 2.5 hr) and specific. The stoichiometry was one toxin molecule bound per ribosome. Binding of T-2 appeared to stablize the toxin recognition site to thermal degradation. A synthetically derived epimer of T-2 bound to the same ribosomal site as authentic T-2, but apparently with lower affinity. Two other trichothecene toxins tested blocked the binding of T-2 to ribosomes in a manner reflecting their protein synthesis inhibitory potencies. Anisomycin blocked the binding of T-2 to both isolated ribosomes and cells, whereas emetine blocked binding only to cells. Our data, together with that in the accompanying paper (Middlebrook JL and Leatherman DL, Biochem Pharmacol 38: 3093-3102, 1989), suggest that T-2 interaction with CHO cells is best viewed as a free, bidirectional movement of toxin across the plasma membrane and specific high-affinity binding to ribosomes.     

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.     

Mesenteric mast cell degranulation in acute T-2 toxin poisoning.

T-2 toxin-induced alterations in rat mesenteric mast cell granulation were measured by cytophotometric analyses of the metachromatic reaction of mast cell granules with azure B. Hypogranulation (diminution of metachromatic material) was observed 8 h following injections of T-2 toxin (0.5-1.5 LD50, i.p.). These data suggest that mast cell activation occurs during acute T-2 intoxication and raise the possibility that mast cell mediators may contribute to toxin-induced cardiovascular collapse.     

Metabolic pathways of T-2 toxin

Among the naturally-occurring trichothecenes found in food and feed, T-2 toxin is the most potent and toxic mycotoxin. After ingestion of T-2 toxin into the organism, it is processed and eliminated. Some metabolites of this trichothecene are equally toxic or slightly more toxic than T-2 itself, and therefore, the metabolic fate of T-2 toxin has been of great concern. The main reactions in trichothecene metabolism are hydrolysis, hydroxylation and deep oxidation. Typical metabolites of T-2 toxin in an organism are HT-2 toxin, T-2-triol, T-2-tetraol, 3'-hydroxy-T-2, and 3'-hydroxy-HT-2 toxin. There are significant differences in the metabolic pathways of T-2 toxin between ruminants and non-ruminants. Ruminants have been more resistant to the adverse effects of T-2 toxin due to microbial degradation within rumen microorganisms. Some plant species are resistant to T-2 toxin, while others are capable of its intake and metabolisation.     

Immunoperoxidase localization of T-2 toxin

Antibody against T-2 toxin was used for monitoring the fate of T-2 toxin in mice given a single po dose of 11 mg/kg by the peroxidase-antiperoxidase (PAP) method. T-2 toxin was demonstrable in the esophagus from 5 min to about 24 hr postdosing. In the stomach, T-2 toxin was detected within the cytoplasm of intact and injured epithelial cells. In the duodenum, T-2 toxin was primarily localized within the surface epithelium and phagocytic elements (macrophages and neutrophils) of the duodenal lamina propria, especially toward the tips of the villi. Following sloughing of duodenal villous tips, the recovering villous tip epithelial cells frequently showed both cytoplasmic and nuclear T-2 toxin. The jejunum showed weak T-2 toxin within the cytoplasm of villous tip epithelial cells only. The ileum never demonstrated T-2 toxin. Tissue response in the gastrointestinal (GI) tract was characterized by transient edema, marked cytolysis and sloughing, and a subsequent leukocytic invasion of the stomach and proximal small intestine. Evidence of severe gastric and less severe duodenal bleeding was apparent and associated with a marked loss of gastric epithelium and intestinal villous tips. The kidney medulla contained the majority of T-2 toxin stain. T-2 toxin was noted within the distal tubular cells, the cells of the collecting tubules, and the epithelium covering the papilla. T-2 toxin was never demonstrated in any of the hepatic tissue examined.     

T-2 toxin effect on cultured myocardial cells

Beat rate, contractility and viability of cultured myocardial cells perfused with solutions containing various concentrations of T-2 toxin were studied. While doses below 50 micrograms/ml had no immediate effect, those above 250 micrograms/ml decreased beat rate and amplitude. After 10-30 min of perfusion most cells stopped beating and did not restart after withdrawal of toxin. Nevertheless, most cells remained viable as judged by morphology and trypan blue exclusion. A 24-h exposure to doses of 5 or 2.5 micrograms/ml of toxin decreased the beat rate and inotropic responses of the myocytes. After 48 h cell death ensued. Thus T-2 toxin has some direct toxicity to myocardial cells but the lethal dose seems too high to make this the cause of cardiovascular failure.     

Uptake of the naturally occurring 3-alpha-hydroxy isomer of T-2 toxin by a murine B cell hybridoma

The uptake of the naturally occurring 3-alpha-hydroxy isomer of T-2 toxin (alpha-T-2 toxin) was investigated in a murine B cell hybridoma as a model for trichothecene-lymphocyte interactions. alpha-[3H]T-2 toxin was prepared by oxidation of T-2 toxin and reduction with [3H]NaBH4 followed by normal phase and reverse phase high-performance liquid chromatography. Uptake of alpha-[3H]T-2 toxin by hybridoma cells was both time- and concentration-dependent. The antibiotic anisomycin inhibited uptake of alpha-[3H]T-2 toxin by hybridoma cells, which suggests ribosomal involvement in the uptake mechanism. Uptake of alpha-[3H]T-2 toxin was also inhibited by verrucarin A, roridin A and deoxynivalenol, and the inhibition followed a trichothecene structure-activity rank similar to that established for protein synthesis inhibition and in vivo toxicity. The characteristics of uptake of alpha-[3H]T-2 toxin by isolated splenocytes were qualitatively similar to those of the hybridoma but accumulation at equilibrium was less. Accumulation of alpha-[3H]T-2 toxin by erythrocytes, cells lacking ribosomes, did not increase with time and was not affected by the presence of unlabelled toxin. The results suggested that specific accumulation and uptake of alpha-[3H]T-2 toxin by the murine B cell hybridoma and spleen cells were highly consistent with a model based on intracellular binding of T-2 toxin to ribosomes.     

The in vitro penetration and distribution of T-2 toxin through human skin

The penetration and distribution of T-2 toxin in excised human abdominal skin has been determined for a dose range of 1.0-2.6 micrograms/cm2 skin using an ethanol vehicle and a saline receptor solution. In all cases the overall percentage penetration of T-2 after 48 h was low, the greatest amounts of toxin being present in the stratum corneum with less in the epidermis and relatively little in the dermis. Vehicle: skin partition coefficients support this finding. Neither penetration nor distribution were changed by a rabbit serum receptor solution. Electron micrographs showed that at 1.8 micrograms/cm2 and above the contents of the intercellular space are leached out to leave the integument as a porous membrane. The distribution of T-2 within the skin after 48 h would suggest that for doses up to 2.6 micrograms/cm2 the irritative and inflammatory effects on the skin would be of more immediate concern than would systemic toxicity.     

T-2 toxin effect on bacterial infection and leukocyte functions

The effects of T-2 toxin on bacterial infection and leukocyte function and structure were examined in vivo and in vitro. Rats were innoculated with staphylococci after pretreatment with or without T-2 toxin. The T-2 pretreated rats failed to mount a cellular response to the bacteria. Blood and bone marrow cells were markedly suppressed by the T-2 toxin, the myeloid series being the most affected. In vitro studies with human leukocytes showed that small, nonkilling doses of T-2 toxin inhibited chemotaxis, chemiluminescence stimulated by bacteria, and phagocytosis of bacteria. It was concluded that this inhibition may contribute towards sepsis and rapid onset of death in T-2 toxin poisoning.     

Effects of concurrent prenatal exposure to rubratoxin B and T-2 toxin in the mouse

T-2 toxin and rubratoxin B are toxic mold metabolites that are known mammalian teratogens. These mycotoxins were given individually or together by ip injection to pregnant CD-1 mice at doses of 0.5 (T-2 toxin) and 0.4 (rubratoxin B) mg/kg on day 10 of gestation. The combination resulted in an increased adverse effect on both fetal weight and mortality of the conceptus in comparison with either treatment given alone at the doses used. Only T-2 toxin resulted in gross malformations, and these were not increased in incidence by addition of rubratoxin B.     

T-2 toxin inhibits the differentiation of human monocytes into dendritic cells and macrophages

The aim of this work was to study the in vitro effect of T-2 toxin on human monocyte differentiation into macrophages and dendritic cells. Cytotoxicity of T-2 toxin on monocytes, on monocytes in differentiation process into macrophages or dendritic cells, and on immature dendritic cells and macrophages was evaluated to determine IC50. Monocytes are more sensitive to T-2 toxin than to differentiate cells. IC50 were equal to 0.11 nM for monocyte, to 45 and 30 nM for monocyte during differentiation process for 24 and 48 h of incubation, respectively, to 38 and 20 nM for immature dendritic cells after 24 and 48 h of incubation, and to 22 and 20 nM for macrophages after 24 and 48 h of incubation. T-2 toxin effects on monocyte differentiation process into macrophages have been explored: according to phenotypic expressions (CD71, CD14, CD11a, CD80, CD86, HLA-DR and CD64), endocytic capacity, phagocytosis, burst respiratory activity and TNF-α secretion. In the presence of 10 nM of T-2 toxin (no cytotoxic concentration), CD71 expression is downregulated compared to control. Endocytosis and phagocytosis capacities are less effective as burst respiratory activity and TNF-α secretion. Monocyte differentiation process into dendritic cells in the presence of 10 nM T-2 toxin is also markedly disturbed. Expression of CD1a (specific dendritic cells marker) is downregulated while that of CD14 (specific monocyte marker) is upregulated. CD11a, CD80, CD86, HLA-DR and CD64 expressions did not change. These results show that T-2 toxin disturbs human monocytes differentiation process into macrophages and dendritic cells. These results could significantly contribute to immunosuppressive properties of this alimentary toxin.     

Toxicological features of T-2 toxin and related trichothecenes

Toxicological characteristics of T-2 toxin and related trichothecenes, mycotoxins produced by Fusarium, Trichoderma, Verrucaria, and others, were investigated in regard to LD50 values, dermal toxicity, hematological changes, and tumorigenicity. The LD50 values (mg/kg) of T-2 toxin in adult male mice were po 10.5, ip 5.2, sc 2.1, and iv 4.2, and those of nivalenol were ip 4.1 and iv 6.3. These data showed that the lethal toxicity of T-2 toxin and nivalenol was about 10 times higher than deoxynivalenol. Newborn and immature animals were much more susceptible than adults. Inhalation experiments with T-2 toxin revealed that 33 ppb T-2 toxin for 160-min and 140 ppb T-2 toxin for 30-min exposure were enough to cause death in mice within several days. The dermal toxicity of T-2 toxin and macrocyclic trichothecenes (verrucarin A and roridin A) was significantly higher than the other trichothecenes, and the induction of edema and other dermal toxicities is caused by direct attack of the trichothecenes on the capillary vessels. No tumorigenicity of fusarenon-X to dermal tissues was shown in mice. Pretreatments of mice with SH-compounds, prednisolon, phenobarbital, and 3-methylcholanthrene did not change the LD50 value of T-2 toxin.     

Species-specific hemolysis of erythrocytes by T-2 toxin

The tricothecene mycotoxin, T-2 toxin interacts differently with mammalian erythrocytes. Pig, man, rabbit, guinea pig, horse, dog, rat, and mouse erythrocytes are all lysed to a varying degree by T-2 toxin. But cow, sheep, goat, buffalo, and deer erythrocytes are all resistant to hemolysis by T-2 toxin. Since erythrocytes from ruminant animals contain little or no phosphatidylcholine, perhaps the presence of phosphatidylcholine in the membrane is required for the hemolytic action of T-2 toxin. Sheep erythrocytes were used to encapsulate T-2 toxin further confirming the resistance of erythrocytes from animals with ruminant physiology to T-2 toxin lysis.     

Penetration of [3H]T-2 toxin through excised human and guinea-pig skin during exposure to [3H]T-2 toxin adsorbed to corn dust

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.     

In vitro effects of T-2 toxin on the mitogen responsiveness and antibody-producing ability of human lymphocytes

The effects of T-2 toxin on the in vitro mitogen responses and the antibody-producing ability of human peripheral blood lymphocytes were evaluated. T-2 toxin inhibited the mitogen response to concanavalin A (ConA) at a lower concentration (1.6 ng/ml) as compared to phytohemagglutinin (2.4 ng/ml) and pokeweed mitogen (2.4 ng/ml). Maximum inhibition was observed when the toxin was present during the first 8 h; however, the cultures were not refractory to inhibition until 48 h after culture initiation. The antibody-producing ability was inhibited by T-2 toxin concentrations of greater than or equal to 3.2 ng/ml. T-2 toxin did not induce or interfere with the generation of suppressor cells by ConA. The results of this study indicate that various lymphocyte subpopulations have different susceptibilities to T-2 toxin. The activation process associated with lymphocyte proliferation appears to be one of the most sensitive time periods.     

Induction of differentiation in human myeloid leukemic cells by T-2 toxin and other trichothecenes

Trichothecenes are sesquiterpen mycotoxins characterized by the tetracyclic 12,13-epoxytrichothec-9-ene skeleton. We determined the effect of these mycotoxins on the growth and differentiation of the human acute promyelocytic leukemia (HL-60) cell line. Sixteen natural and semisynthetic trichothecenes were tested at concentrations of 0.2-60,000 ng/ml. The cytotoxicity exerted by these compounds varied: e.g., roridin A was found to be toxic at 1 ng/ml, whereas T-2 palmitate was not toxic even at 1 microgram/ml. These compounds varied also in their potential to induce differentiation: 9,10-epoxy T-2 toxin and T-2 toxin induced differentiation at concentrations of 2-5 ng/ml, while 9,10-dihydro T-2 toxin was effective only at 100 ng/ml. Other trichothecenes (e.g., verrucarin A and verrucarol) did not induce differentiation at either subtoxic or toxic concentrations. Cell differentiation was always associated with cytotoxicity; optimal concentrations for induction of differentiation were usually 30-60% of the toxic concentrations. The HL-60 cell population was found to be heterogenous with respect to the ability to differentiate in response to trichothecenes, while in some clones up to 70% of the cells underwent differentiation, and other clones were completely resistant. The latter clones could, however, be induced to differentiate by other agents such as retinoic acid, dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate. Some of the inducible clones differentiated into neutrophilic granulocytes while others into mature macrophages. Thus, a single trichothecene could induce differentiation into either cell types, depending on the clone used. This study presents a new group of differentiation inducers. Further investigation is required to evaluate their possible therapeutic application.     

Cytochrome P450 (CYP) expression in human myeloblastic and lymphoid cell lines

To explore the physiological roles of cytochrome P450 (CYP) in peripheral blood cells, we examined which isoforms of CYP families were expressed in human myeloid leukemia cell lines (U937, HL-60 and K562) and lymphoid cell lines (BALL-1, MOLT-4 and Jurkat) by RT-PCR. We observed relatively high expression of CYP1A1, CYP1B1, CYP2A6, CYP2A7, CYP2D6, and CYP2E1 in all cell types, but CYP2A13 and CYP2C9 expression was not detected. Expressions of aryl hydrocarbon (Ah) receptor and Ah receptor nuclear translocator (ARNT), which mediate induction of the CYP1 family, were also detected in all cell types. Cell-type specific expression of CYP3A4 and CYP3A5 was observed in MOLT-4 and K562 cells. Weak, but significant, expression of CYP3A7 was detected in K562 cells. The profile of CYP expression in the culture cells reported here provides information that furthers our understanding of the physiological roles of CYP enzymes in human blood cells.