High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin.

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and ß-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

Arsenite and monomethylarsonous acid disrupt erythropoiesis through combined effects on differentiation and survival pathways in early erythroid progenitors.

Strong epidemiological evidence demonstrates an association between chronic arsenic exposure and anemia. We recently found that As+3 impairs erythropoiesis by disrupting the function of GATA-1; however the downstream pathways impacted by the loss of GATA-1 function have not been evaluated. Additionally, our previous findings indicate that the predominant arsenical in the bone marrow of mice exposed to As+3 in their drinking water for 30 days was MMA+3, but the impacts of this arsenical on erythorpoisis also remain largely unknown. The goal of this study was to address these critical knowledge gaps by evaluating the comparative effects of arsenite (As+3) and the As+3 metabolite, monomethyarsonous acid (MMA+3) on two critical regulatory pathways that control the differentiation and survival of early erythroid progenitor cells. We found that 500 nM As+3 and 100 and 500 nM MMA+3 suppress erythropoiesis by impairing the differentiation of early stage erythroid progenitors. The suppression of early erythroid progenitor cell development was attributed to combined effects on differentiation and survival pathways mediated by disruption of GATA-1 and STAT5. Our results show that As+3 primarily disrupted GATA-1 function; whereas, MMA+3 suppressed both GATA-1 and STAT5 activity. Collectively, these findings provide novel mechanistic insights into arsenic-induced dyserythropoiesis and suggest that MMA+3 may be more toxic than As+3 to early developing erythroid cells.

Design, synthesis, and optimization of a series of 2-azaspiro[3.3]heptane derivatives as orally bioavailable fetal hemoglobin inducers.

Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (HbF) is a promising approach for the management of ß-thalassemia and sickle cell disease (SCD). We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules that could induce HbF, which resulted in identification of the hit compound 1. Exploration of structure-activity relationships and optimization of ADME properties led to 2-azaspiro[3.3]heptane derivative 18, which is more rigid and has a unique structure. In vivo using cynomolgus monkeys, compound 18 induced a significant dose-dependent increase in globin switching, with developable properties. Moreover, compound 18 showed no genotoxic effects and was much safer than hydroxyurea. These findings could facilitate the development of effective new therapies for the treatment of ß-hemoglobinopathies, including SCD.

UNC0638 induces high levels of fetal hemoglobin expression in ß-thalassemia/HbE erythroid progenitor cells.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of ß-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from ß-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of ß-thalassemia.

Erythropoietin Ameliorates Ischemia/Reperfusion-Induced Acute Kidney Injury via Inflammasome Suppression in Mice.

Acute kidney injury (AKI) is the most common condition in hospitalized patients. As ischemia/reperfusion-induced AKI (IR-AKI) is as a major contributor to end-stage disease, an effective therapeutic intervention for IR-AKI is imperative. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Here, we investigated the renoprotective effects of EPO in an IR-AKI mouse model. Mice were assigned to sham, EPO only, and IR only groups, and the IR group was treated with EPO prior to injury. EPO was administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. The serum was harvested for renal functional measurements. The kidneys were subjected to histological evaluation, and the biochemical changes associated with renal injury were assessed. EPO significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPO-treated mice. Macrophage infiltration and expression of ICAM-1 and MCP-1 were also significantly reduced in EPO-treated mice. Furthermore, the expression of inflammasome-related factors (NLRP1, NLRP3, and caspase-1 cleavage), via the activation of the COX-2 and NF-B signaling pathways were significantly reduced following EPO treatment. To our knowledge, this is the first study to demonstrate that inflammasome-mediated inflammation might be a potential target of EPO as a treatment for ischemic AKI.

Dangguibuxue decoction abolishes abnormal accumulation of erythroid progenitor cells induced by melanoma.

ETHNOPHARMACOLOGIC RELEVANCE: Dangguibuxue decoction (DGBX), is a well-known traditional Chinese medicine that contains two types of materials used to treat anemia. In this study, we aimed to explore the effect and mechanism of DGBX on abolishing erythroid progenitor cell (Ter119+CD71+) accumulation induced by melanoma. MATERIALS AND METHODS: B16/F10 melanoma cells were used to establish transplanted and metastatic melanoma models. DGBX or normal saline were administered intragastrically daily after the models were established. Tumor sizes and metastatic nodules were observed after tumor cell inoculation. To further test the function of DGBX on erythroid progenitor cell (EPC) accumulation and immunosuppressive abilities, the percentage of EPCs in the blood, and spleen were quantified with flow cytometry. The proportion of CD8+ T cells and related functional mediators, IFN-γ and TNF-α,were also quantified with flow cytometry. To further strengthen our in vivo observations, DGBX serum was prepared from the rats three days after DGBX was administered. Liquid chromatography-mass spectrometry was carried out to control the quality of the experiments. B16/F10 melanomacells were cultured with DGBX serum, and proliferation and apoptosis were observed with the CCK8 assay and AnnexinV/7AAD staining, respectively. EPCs were isolated from B16/F10-bearing mice and cultured under erythroid differentiation conditions. EPCs were treated with DGBX serum, and mature red cell proportions and cell denucleations were tested with flow cytometry and Giemsa staining of the cultured EPCs. Flow cytometry and qPCR were used to analyze the effects of DGBX on the expression of key molecules involved in erythroid development and to explore the mechanism by which DGBX relieves abnormal EPC accumulation. RESULTS: DGBX treatments significantly reduced B16 melanoma tumor sizes and metastatic nodules. Most importantly, our study strongly suggested that DGBX could alleviate anemia, and systematically enhance anti-tumor immune responses by reducing abnormal EPC accumulation. Moreover, DGBX serum treatments had no direct effect on tumor cell proliferation and apoptosis, but could promote EPCs to differentiate into mature red blood cells, in vitro. Mechanistically, at least in part, DGBX relieved abnormal EPC accumulation by altering the "master switch" transcription factors, Pu.1 and Gata-1. CONCLUSIONS: DGBX significantly alleviates abnormal tumor-induced EPC accumulation, inhibits B16 melanoma progression, and enhances anti-tumor immune responses.

The fraction of CD117/c-KIT-expressing erythroid precursors predicts ESA response in low-risk myelodysplastic syndromes.

BACKGROUND: Compelling evidence has emerged for the relevance of flow cytometry (FC) in the diagnostic work-up of myelodysplastic syndromes (MDS) but due to technical issues, the erythroid lineage has been under investigated, specifically in the therapeutic context. METHODS: Using the "no red cell lysis" method developed to set up the RED-score, we specifically quantified the fraction of CD117/c-KIT-expressing erythroid precursors in a cohort of 144 MDS patients and studied the correlation with response to erythropoiesis-stimulating agents (ESA) in a sub cohort of 63 low-risk MDS patients. RESULTS: We confirmed the previously reported increase in CD117/c-KIT-expressing erythroid precursors in a subset of MDS patients and demonstrated a strong association between a cut off of CD117/c-KIT-expressing erythroid precursors ≥3% and ESA response (P = 0.001), independent of red blood cell requirement. From our observations, we hypothesized that a decrease in CD117/c-KIT-expressing erythroid precursors could be a mechanism of ESA failure. Moreover, the fraction of CD117/c-KIT-expressing erythroid precursors was correlated with progression-free survival in low-risk MDS patients (P = 0.018). In vitro, we demonstrated in an EPO dependent cell line that CD117/c-KIT expression is necessary for cell survival under EPO stimulation. CONCLUSIONS: The quantification of the CD117/c-KIT-expressing erythroid precursors could be proposed as a new theranostic and prognostic marker in MDS treated by ESA. Future studies will be required to determine whether modulating CD117/c-KIT expression and signaling could be used to improve anemia in MDS. © 2019 International Clinical Cytometry Society.

Calibration, Selection and Identifiability Analysis of a Mathematical Model of the in vitro Erythropoiesis in Normal and Perturbed Contexts.

The in vivo erythropoiesis, which is the generation of mature red blood cells in the bone marrow of whole organisms, has been described by a variety of mathematical models in the past decades. However, the in vitro erythropoiesis, which produces red blood cells in cultures, has received much less attention from the modelling community. In this paper, we propose the first mathematical model of in vitro erythropoiesis. We start by formulating different models and select the best one at fitting experimental data of in vitro erythropoietic differentiation obtained from chicken erythroid progenitor cells. It is based on a set of linear ODE, describing 3 hypothetical populations of cells at different stages of differentiation. We then compute confidence intervals for all of its parameters estimates, and conclude that our model is fully identifiable. Finally, we use this model to compute the effect of a chemical drug called Rapamycin, which affects all states of differentiation in the culture, and relate these effects to specific parameter variations. We provide the first model for the kinetics of in vitro cellular differentiation which is proven to be identifiable. It will serve as a basis for a model which will better account for the variability which is inherent to the experimental protocol used for the model calibration.

Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors.

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.

Erythropoietin induces bone marrow and plasma fibroblast growth factor 23 during acute kidney injury.

It is accepted that osteoblasts/osteocytes are the major source for circulating fibroblast growth factor 23 (FGF23). However, erythropoietic cells of bone marrow also express FGF23. The modulation of FGF23 expression in bone marrow and potential contribution to circulating FGF23 has not been well studied. Moreover, recent studies show that plasma FGF23 may increase early during acute kidney injury (AKI). Erythropoietin, a kidney-derived hormone that targets erythropoietic cells, increases in AKI. Here we tested whether an acute increase of plasma erythropoietin induces FGF23 expression in erythropoietic cells of bone marrow thereby contributing to the increase of circulating FGF23 in AKI. We found that erythroid progenitor cells of bone marrow express FGF23. Erythropoietin increased FGF23 expression in vivo and in bone marrow cell cultures via the homodimeric erythropoietin receptor. In experimental AKI secondary to hemorrhagic shock or sepsis in rodents, there was a rapid increase of plasma erythropoietin, and an induction of bone marrow FGF23 expression together with a rapid increase of circulating FGF23. Blockade of the erythropoietin receptor fully prevented the induction of bone marrow FGF23 and partially suppressed the increase of circulating FGF23. Finally, there was an early increase of both circulating FGF23 and erythropoietin in a cohort of patients with severe sepsis who developed AKI within 48 hours of admission. Thus, increases in plasma erythropoietin and erythropoietin receptor activation are mechanisms implicated in the increase of plasma FGF23 in AKI.

Population snapshots predict early haematopoietic and erythroid hierarchies.

The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.

Developmental differences between neonatal and adult human erythropoiesis.

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)- and peripheral blood (PB)-HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB-derived cells proliferated 4-fold more than PB-derived cells. However, CB-derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony-forming unit (CFU-E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R- GPA- CD34+ CD36+ cells generated from PB- than CB-HSPCs. This subset was found to generate both burst-forming unit (BFU-E) and CFU-E colonies in colony-forming assays. To further understand the differences between CB- and PB-HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle- and autophagy-related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays.

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.

Clostridium perfringens α-toxin impairs erythropoiesis by inhibition of erythroid differentiation.

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71-TER119+ cell populations but not the CD71+TER119- cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.