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.

Effect of Ochratoxin A on human haematopoietic progenitors proliferation and differentiation: an in vitro study.

Ochratoxin A (OTA) is a mycotoxin food and feed-contaminant known to induce nephro and hepatotoxicity in human and animal, and related to human Balkan Endemic Nephropathy. However, haematological troubles are also observed in case of acute OTA intoxication. These disorders observed in animals emphasise the necessity to determine if OTA exposure induce damage to haematopoietic system in human. The effect on haematopoiesis has been evaluated using in vitro clonogenic assays of the three lineages i.e., platelet, red and white blood cell progenitors. Human erythroblastic and granulomonocytic progenitor proliferation is decreased in the presence of 10(2) microM OTA. Platelet progenitors were destroyed at 10(2) microM OTA. For the lowest concentrations haematopoietic progenitor proliferation is not affected by OTA. Comparison with other mycotoxins known to be myelotoxic shows that OTA is less myelotoxic than trichothecenes.

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.

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.

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.