Dietary exposure to aflatoxins, ochratoxin A and deoxynivalenol from a total diet study in an adult urban Lebanese population.

Exposure to mycotoxins may be associated with carcinogenic, immunosuppressant and estrogenic effects. In the Middle-East, studies investigating food contamination and dietary exposure to mycotoxins are particularly scarce. This study aims at evaluating the dietary exposure of an adult Lebanese urban population to four mycotoxins (AFB1, AFM1, OTA, DON) classified as priority food contaminants by the WHO. Dietary exposure assessment was performed by means of the total diet study approach. Average and excessive consumer exposure estimates (p95) were calculated and compared with appropriate toxicological reference values (TRVs). Average dietary exposure levels to OTA and DON represented 29.9% and 156.8% of the respective TRVs, with the p95 exposure estimates approaching or exceeding the TRVs for these mycotoxins (95.1% and 355.8%, respectively). Based on the mean dietary exposure level to AFB1, cancer risk was estimated at 0.0527-0.0545cases/100,000persons/year, while mean exposure to AFM1 was associated with a population risk of 0.0018-0.0027cases/100,000persons/year. The study's findings place Lebanon among countries that are highly exposed to mycotoxins through the diet and call for larger-scale studies aiming at providing a comprehensive assessment of the dietary exposure of the Lebanese population to mycotoxins as well as to other food contaminants.

Effects of beauvericin, enniatin b and moniliformin on human dendritic cells and macrophages: an in vitro study.

The aim of this study was to assess the in vitro effects of emerging mycotoxins beauvericin, enniatin B and moniliformin on human dendritic cells and macrophages. Beauvericin and enniatin B were cytotoxic on these cells. IC50 were equal to 1.0 µM, 2.9 µM and 2.5 µM beauvericin for immature dendritic cells, mature dendritic cells and macrophages, respectively. IC50 were equal to 1.6 µM, 2.6 µM and 2.5 µM for immature dendritic cells, mature dendritic cells and macrophages exposed to enniatin B, respectively. Effects on the differentiation process of monocytes into macrophages or into immature dendritic cells as well as effects on dendritic cells maturation have been studied. The differentiation process of monocytes into immature dendritic cells was not disturbed in the presence of beauvericin. Dendritic cells exposed to beauvericin during the maturation process presented a decrease of CCR7 expression and an increase of IL-10 secretion. Monocytes exposed to beauvericin during the differentiation process into macrophages presented a decrease of endocytosis ability. The differentiation process of monocytes into immature dendritic cells was not disturbed in the presence of enniatin B. Dendritic cells exposed to enniatin B during the maturation process presented a decrease of expression of the maturation makers CD80, CD86 and CCR7 and an increase of IL-10 secretion. Monocytes exposed to enniatin B during the differentiation process into macrophages presented a decrease of endocytosis ability and an increase of CD71. CD1a expression and endocytosis capacity were decreased on immature dendritic cells exposed to moniliformin. Monocytes-derived macrophages exposed to moniliformin during the differentiation process presented a decrease of endocytosis ability, and a decrease of CD71 and HLA-DR expression. According to these results, immunological disorders could be observed on human after ingestion of these alimentary toxins.

Co-exposure of Fusarium mycotoxins: in vitro myelotoxicity assessment on human hematopoietic progenitors.

Mycotoxins such as beauvericin (BEA), deoxynivalenol (DON), enniatin B (ENB), fumonisin B1 (FB1), T-2 toxin and zearalenone (ZEA) can co-occur in food commodities. This aim of this study was to assess the myelotoxicity of these mycotoxins in couple using in vitro human granulo-monocytic (Colony Forming Unit-Granulocyte and Macrophage, CFU-GM) hematopoietic progenitors. Clonogenic assays have been performed in the presence of the following couples of fusariotoxins: DON + BEA, DON + FB1, DON + T-2, DON + ZEA, T-2 + ZEA and BEA + ENB. Co-exposure of human CFU-GM to DON + BEA resulted in synergic myelotoxic effects. The combination of DON + T-2 presented additive or synergic myelotoxic effects. The couples DON + ZEA, T-2 + ZEA and BEA + ENB had additive myelotoxic effects, while the combination of DON + FB1 showed antagonist myelotoxic effects. These in vitro results suggested that the simultaneous presence of mycotoxins in food commodities and diet may be more myelotoxic than the presence of one mycotoxin alone. Diminution of hematopoietic progenitors could give rise to a decrease number of mature blood cells, inducing agranulocytosis and/or thrombocytopenia and in severe cases aplastic anemia.

In vitro myelotoxicity assessment of the emerging mycotoxins Beauvericin, Enniatin b and Moniliformin on human hematopoietic progenitors.

The aim of this study was to screen potential myelotoxicity of the emerging mycotoxins Beauvericin, Enniatin b and Moniliformin using human hematopoietic progenitor clonogenic assays. Depending on mycotoxins, inhibitory effects on proliferation of white blood cells progenitors (CFU-GM), platelet progenitors (CFU-MK) and red blood cells progenitors (BFU-E) have been detected at various concentrations. Beauvericin was cytotoxic at 32µM, 3.2µM and 6.4µM, had no effect on proliferation in the presence of 0.032µM, 0.16µM and 0.064µM, and the IC(50) was equal to 3.4µM, 0.7µM and 3.7µM for CFU-GM, CFU-MK and BFU-E, respectively. Enniatin b was cytotoxic at 6µM, 1.8µM and 5µM, had no effect on proliferation in the presence of 1µM, 1.1µM and 1.2µM and the IC(50) was equal to 4.4µM, 1.3µM and 3.3µM for CFU-GM, CFU-MK and BFU-E, respectively. Moniliformin was not cytotoxic at tested concentrations for CFU-GM and CFU-MK and cytotoxic at 10µM for BFU-E, had no effect on proliferation in the presence of 5µM, 0.1µM and 0.1µM and the IC(50) was equal to 31µM, 39µM and 4.1µM for CFU-GM, CFU-MK and BFU-E, respectively. Inhibition of the BFU-E differentiation has been observed in the presence of Enniatin b or Moniliformin. For the three mycotoxins, variation of distribution of CFU-MK colonies according to their size has been observed. These in vitro effects may be responsible for in vivo hematological troubles in case of consumption of contaminated commodities. In vivo studies have to be performed to test this hypothesis.

Stem cells in myelotoxicity.

Myelotoxicity describes bone marrow failure due to adverse effect of xenobiotic on hematopoiesis. Hematopoiesis is a complex system in which pluripotent hematopoietic stem cells (PHSCs) differentiate into many highly specialized circulating blood cells involving the interaction of many cell types as well as the interaction of local and systemic growth factors. With respect to blood cell formation, two functional systems must be considered: the hematopoietic stem cells (PHSCs) and the progenitor cells, on one hand, and the stromal cells, which constitute the hematopoietic environment niche, on the other hand. There are three types of assays for hematopoietic progenitor clonogenic assays useable in myelotoxicology: CFU-GM assay for Colony Forming Unit Granulocyte and Macrophage, BFU-E assay for Burst Forming Unit Erythroid, and CFU-MK assay for Colony Forming Unit Megakaryocyte from several species as well as from murine as from mammalian and human. Clonogenic assays have been used to detect myelotoxicity induced by chemicals, drug, food and environmental contaminants. Designs and applications are described in this review.

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-alpha 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-alpha 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.

Dietary exposure to lead, cadmium, mercury and radionuclides of an adult urban population in Lebanon: a total diet study approach.

Human exposure to toxic chemicals is suspected of being responsible for a wide range of human health disorders. This study is the first in Lebanon to evaluate the dietary exposure of an adult urban population to three heavy metals (lead, cadmium and mercury) and to radionuclides. Exposure assessment was performed by means of the total diet study approach as recommended by the Word Health Organization. Five 'total diets' were collected during 2003-04. Average and maximal consumer exposure estimates to heavy metals were calculated and compared with appropriate reference values and with intakes reported from other countries. The average dietary intakes of lead, cadmium and mercury represented 7, 17 and 5.6%, respectively, of the appropriate provisional tolerable weekly intakes (PTWI). The mean dietary intake of methylmercury represented 17.5% of the appropriate PTWI. Cs-134 and I-131 were not detected in any of the food samples. Traces of Cs-137 were only found in five food samples. The exposure assessment conducted places Lebanon among countries least exposed to heavy metals through the diet and it highlights the safety of the food supply from radioactive contamination.

Myelotoxicity of trichothecenes and apoptosis: an in vitro study on human cord blood CD34+ hematopoietic progenitor.

Previous studies have revealed that hematological disorders associated with trichothecenes intoxication in humans could result from hematopoiesis inhibition. The most frequent and potent trichothecene mycotoxins are T-2 toxin and deoxynivalenol (DON), respectively. Apoptosis induction by these two toxins was investigated in vitro on human hematopoietic progenitors (CD34+ cells). Hoechst coloration, DNA fragmentation and annexin-V/PI labeling in flow cytometry showed that T-2 toxin, in contrast to DON, induced apoptosis in CD34+ cells. T-2 toxin effect was dose- and time-dependent with a significant increase of apoptotic cells as early as 3h after incubation at 10(-7) M and a maximum reached at 12 h. This observation evidenced the high sensitivity of hematopoietic progenitors to T-2 toxin. The inhibition of T-2 toxin-induced apoptosis by a pan-caspase inhibitor (Z-VAD-fmk) suggested the involvement of caspases. The proportional increase of caspase-3 specific activity (DEVDase) with T-2 toxin concentration confirmed its role in the process. After incubation of CD34+ cells with T-2 toxin, in conditions that induced apoptosis, clonal expansion of granulo-monocytes, erythrocytes and megakaryocytes precursors was dose-dependently inhibited. The hematological effects observed in T-2 toxin mycotoxicosis could then be assigned to hematopoiesis inhibition by apoptosis. Different mechanisms that need to be further elucidated are involved in DON myelotoxicity.

Inhibition of CFU-E/BFU-E and CFU-GM colony growth by cyclophosphamide, 5-fluorouracil and taxol: development of a high-throughput in vitro method.

The major side effect of anticancer drugs is damage to the hematopoietic system. These compounds may interfere with cell proliferation and differentiation in different blood lineages causing many diseases such as neutropenia, aplastic anaemia or trombocytopenia. The clonogenic assays are useful in vitro tools for evaluating and predicting acute xenobiotics myelotoxicity. A miniaturisation of these assays, in order to reduce costs and increase the number of compounds that could be tested, is under investigation. The in vitro sensitivity of human burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) to three anticancer drugs: cyclophosphamide (CTX), 5-fluorouracil (5-FU) and taxol (TAX) was evaluated both in 35 mm plate and 96-well plate systems and the dose-response curves, IC50 values and IC90 values were compared. The correlation between in vitro data and clinical plasma levels confirms that severe hematotoxicity is the primary adverse effect of these drugs with an evident selectivity on erythroid progenitors for cyclophosphamide. IC50 and IC90 values, calculated on the basis of results obtained with the traditional assay, correlate with those obtained in microplate, as well as the dose-response curves, indicating that the 96 well plate assay could be a useful and reliable tool for high-throughput screening in early stages of drug development.

Do trichothecenes reduce viability of circulating blood cells and modify haemostasis parameters?

This manuscript describes the results of experiments conducted using human blood cells to determine the ability of T-2 toxin and DON to cause changes in clotting time, platelet aggregation, red blood cell haemolysis, RBC glucose content, lactate release, glutathione depletion, as well as white blood cell viability. In vitro results showed that haemostasis parameters and erythrocytes were not affected at concentrations able to induce inhibition of haematopoietic progenitor proliferation. In the presence of 10(-8) M and 10(-6) M T-2, the leucocyte number decreased at 24 h by 30% and 50% respectively. A 50% decrease in leucocyte number was observed for 10(-5) M DON. Results were compared with haematopoietic progenitor sensitivities. Due to the differences in sensitivities between mature blood cells and haematopoietic progenitors, haematological problems associated with trichothecene intoxication could be attributed to haematopoiesis inhibition.

Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

In vitro myelotoxicity of propanil and 3,4-dichloroaniline on murine and human CFU-E/BFU-E progenitors.

Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice, but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study, an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E, CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors, has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M, and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand, human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and >500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors, as was found for the murine cells, emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose.

Food contamination by metals and pesticides in the European Union. Should we worry?

The estimation of the risk associated with dietary intakes of heavy metals and pesticide residues by the consumer is a vital and integral part of regulatory processes. The exposure of the consumer is compared directly to the acceptable daily intake (ADI) for pesticides and to the tolerable daily intake (TDI) for heavy metals. The exposure is obtained using the basic equation: Exposure (mg/kg b.w./day)=Consumption (mg/kg b.w./day) x Residue (mg/kg). The establishment of the ADI and the TDI is based on the results of toxicological studies that involve the determination of the lowest-no-observed-adverse-effect level/10 (SF1) x10 (SF2), where SF corresponds to 'Safety Factor'. SF1 and SF2 account for interspecies and intraspecies variability, respectively. In order to evaluate the risk for the consumer, that is associated to the presence of heavy metals and pesticides in food, a review of the level of contamination in European countries has been made. The exposure of European consumers to lead, cadmium, arsenic and mercury is superior to the TDI. For pesticides, the first step is to compare the detected amount of residues of a specific pesticide to the maximum residue level (MRL) authorized in foodstuffs. If the residue level in food exceeds the MRL, the theoretical maximum daily intakes and the ADI have to be taken into account in order to assess the risk for the consumer.

Improvement of megakaryocytic progenitor culture for toxicological investigations.

The aim of this work was to obtain an in vitro test for the evaluation of xenobiotic toxicity on the proliferation and on the differentiation of megakaryocyte progenitors. The rapid rate of blood cell renewal makes the hematopoietic system a susceptible target for xenobiotic toxicity. Hematotoxic molecules can affect one or more hematopoietic lineages leading to blood disorders. Megakaryocytopoiesis in vitro models applied to toxicological investigations needs to be accurate, precise, reproducible, sensitive and specific. Human hematopoietic progenitors from umbilical cord blood were seeded in a collagen medium. Three solvents have been selected (ethanol, methanol, acetone), and one (dimethyl sulfoxide; DMSO) has been eliminated due to its cytotoxicity at tested concentrations. Cryopreservation did not affect the sensitivity of CFU-MK to xenobiotics. An overnight incubation of cell suspensions as cell suspension enrichment before plating gave better cloning efficiency than CD34(+) cells negative selection. Comparison between different parameters allowed us to propose a protocol suitable for an in vitro megakaryocytopoiesis model in toxicological investigations. The effects of three toxins were studied on CFU-MK development in order to verify the efficiency of this clonogenic assays for toxicity testing. The CFU-MK culture conditions defined revealed their usefulness for investigating drug cytoxicity towards megakaryocytic progenitors and disturbance of their proliferation.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

In vitro effect of lead acetate on human erythropoietic progenitors.

Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis. The aim of the present work was to study human erythropoiesis in vitro in the presence of lead. Human erythroblastic progenitors, burst-forming units-erythroid (BFU-E), were exposed to lead acetate at increasing concentrations during 14 days of culture. Hematotoxicity was evaluated in vitro according to proliferation and differentiation of cell colonies arising from BFU-E development. The ability of cells to synthesize proteins, porphyrins, and hemoglobin was measured by spectrophotometric tests and by high-pressure liquid chromatography (HPLC). Results showed that in the presence of 10(-3) mol/L lead acetate, no hemoglobinized cells were observed in culture and no fluorescent porphyrins were detected in cells. Up to 10(-3) mol/L, lead acetate is not cytotoxic, i.e., it does not induce cell destruction. The present work demonstrates that lead acetate interferes with the porphyrin synthesis of human erythroblastic progenitors in vitro. The decrease of porphyrin content with 10(-5) mol/L lead acetate suggest that delta-aminolevulinic acid dehydratase can be inhibited by lead acetate during in vitro erythropoiesis. In vivo erythropoiesis occurs in the bone marrow. As about 95% of the body burden of lead in adults is located in the bones with a biological half-life of some years, the concentration of lead acetate found to block porphyrin synthesis in vitro has to be compared with in situ bone marrow lead concentrations.

Relevance of clonogenic assays in hematotoxicology.

Clonogenic assays have been established in hematology for 30 years. They have been widely used in fundamental studies on hematopoiesis and they are also routinely used in clinical hematology to confirm diagnosis or to predict time to recovery in cases of bone marrow failure. Their use in toxicological studies is more recent. Adverse effects of xenobiotics can induce hematological problems and pathologies such as neutropenia, thrombocytopenia, anemia, and aplastic anemia. Three clonogenic assays are proposed for granulopoiesis, megakaryopoieisis and erythropoieisis. Hematopoietic progenitors from murine or human origin can be cultured in the presence of xenobiotics using validated protocols to complete standard animal toxicological studies. These clonogenic assays can help to predict adverse effects of drugs or toxicants. Clonogenic assays using white blood cell progenitors (CFU-GM culture) have recently been validated by ECVAM and can be used routinely. Megakaryocyte progenitor (CFU-MK) culture is under development and prevalidation in toxicological studies supported by ECVAM. Red blood cells progenitor culture (BFU-E) has been proposed but needs international validation to be recognized.

Trichothecene toxicity on human megakaryocyte progenitors (CFU-MK).

Trichothecenes are mycotoxins produced by various species of fungi, which can occur on various agricultural products. Among these compounds, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) are the most naturally encountered and the most potent trichothecenes. Consumption of trichothecene contaminated foods by farm animals and humans leads to mycotoxicosis. Trichothecenes are known to induce haematological disorders such as neutropenia, aplastic anemia and thrombocytopenia in humans and animals. Four trichothecenes, T-2 toxin, HT-2 toxin, DAS and DON have been tested on human platelet progenitors (CFU-MK) using a culture model of CFU-MK optimized for toxicological studies. Trichothecenes cause, at low concentrations, cytotoxic effects in megakaryocyte progenitors, which could induce thrombocytopenia. Sensitivity of human CFU-MK is compared to respective sensitivities of human red blood cell progenitors (BFU-E) and white blood cell progenitors (CF-U-GM) that were described in previous works.

Simulation of automatic measurement of cell distribution inhomogeneity in clonogenic assays.

The use of hematopoietic progenitors in toxicology often requires scoring colonies, micro- and macroclusters, and evaluation of their distribution while taking into account their shape. Unfortunately, this evaluation is long and tedious, and classification errors may occur. Image analysis can improve the method if there is an automatic way of performing the classification analysis. It has been previously demonstrated that cell conglomerates can be automatically detected. The present paper describes three image analysis algorithms and reports on their capabilities to analyze cell heterogeneity in culture. The comparative tests are made on computer-generated images representing different cell distributions. Combining two of these three algorithms gives fairly good results on complex images created by adding various simple distributions.

In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors.

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DON has been detected in agricultural products worldwide and persists in products after processing. In humans as well as in animals, DON has been shown to induce both alimentary and hematological toxicities. Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood from rat bone marrow were cultured in the presence of DON (from 10(-6) to 10(-8) mol/L) for 14 days. DON rapidly inhibits human and rat CFU-GM in a concentration-dependent manner between 10(-6) and 2.5 x 10(-7) mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 x 10(-8), 2.9 x 10(-8), 3.9 x 10(-8) mol/L for human CFU-GM and 2.6 x 10(-7), 1.5 x 10(-7), and 1.6 x 10(-7) mol/L for rat CFU-GM. The present study defines the cytotoxic and inhibitory DON concentrations for rat and human CFU-GM and provides a system for further investigation of cellular DON targets and elucidation of the mechanism of trichothecene hematotoxicity. Moreover, we propose one of the trichothecenes tested in our studies as a reference molecule for in vitro studies, since one mycotoxin seems to be the most potent myelotoxic inhibitor of CFU-GM detected to date.