Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs.

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

In vitro effects of trichothecenes on human dendritic cells.

The aim of this work was to study the in vitro effects of trichothecenes on human dendritic cells. Trichothecenes are mycotoxins produced by fungi such as Fusarium, Myrothecium, and Stachybotrys. Two aspects have been explored in this work: the cytotoxicity of trichothecenes on immature dendritic cells to determine IC 50 (inhibition concentration), and the effects of trichothecenes on dendritic cell maturation process. Two mycotoxins (T-2 and DON) known to be immunotoxic have been tested on a model of monocyte-derived dendritic cells culture. Cytotoxic effects of T-2 toxin and DON on immature dendritic cells showed that DON is less potent than T-2 toxin. The exposure to trichothecenes during dendritic cell maturation upon addition of LPS or TNF-alpha markedly inhibited the up-regulation of maturation markers such as CD-86, HLA-DR and CCR7. Features of LPS or TNF-alpha -mediated maturation of dendritic cells, such as IL-10 and IL-12 secretions and endocytosis, were also impaired in response to trichothecenes treatment. These results suggest trichothecenes have adverse effects on dendritic cells and dendritic cell maturation process.

Challenges and Successes Using Nanomedicines for Aerosol Delivery to the Airways.

Numerous diseases affect the respiratory tract and the aerosol administration has been widely considered as an adapted and non-invasive method for local delivery. This pathway induces a lung concentration and thus also limits, systemic side effects. However, aerosol delivery of active pharmaceutical ingredients represents a real challenge, due to numerous obstacles such as the specific respiratory movement, the presence of mucus or surfactant, and the mucociliary clearance. Nanomedicines, such as liposomes, micelles or nanoparticles, offer the possibility to increase bioavailability and favor intracellular penetration of specific drugs into lung tissue. This review focuses on the description of aerosol formulations and cellular barriers including design, characteristics and progressive adaptation to airways anatomy. Then, aerosolized formulations currently clinically approved, or in clinical trial are summarized according to the encapsulated drug. In a final section, promising aerosol formulations in pre-clinical studies are detailed.

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.

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.

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.

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.

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.

Improvement of human dendritic cell culture for immunotoxicological investigations.

A toxic injury such as a decrease in the number of immature dendritic cells caused by a cytotoxic effect or a disturbance in their maturation process can be responsible for immunodepression. There is a need to improve in vitro assays on human dendritic cells used to detect and evaluate adverse effects of xenobiotics. Two aspects were explored in this work: cytotoxic effects of xenobiotics on immature dendritic cells, and the interference of xenobiotics with dendritic cell maturation. Dendritic cells of two different origins were tested. Dendritic cells obtained either from umbilical cord blood CD34(+) cells or, for the first time, from umbilical cord blood monocytes. The cytotoxicity assay on immature dendritic cells has been improved. For the study of the potential adverse effects of xenobiotics on the maturation process of dendritic cells, several parameters were selected such as expression of markers (CD86, CD83, HLA-DR), secretion of interleukins 10 and 12, and proliferation of autologous lymphocytes. The relevance and the efficiency of the protocol applied were tested using two mycotoxins, T-2 toxin and deoxynivalence, DON, which are known to be immunosuppressive, and one phycotoxin, domoic acid, which is known not to have any immunotoxic effect. Assays using umbilical cord monocyte dendritic cell cultures with the protocol defined in this work, which involves a cytotoxicity study followed by evaluation of several markers of adverse effects on the dendritic cell maturation process, revealed their usefulness for investigating xenobiotic immunotoxicity toward immune primary reactions.

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.