Femoral Bone Marrow Cells Research Articles

Evaluation of the genotoxic effect of nonylphenol applied in different doses on the bone marrow

Nonylphenols are endocrine disrupting toxic compounds and are widely used. For this reason, our study aimed to investigate the genotoxic effect of nonylphenols applied at different doses on mouse bone marrow cells. Groups were classified as control group, the sham administered only group, low dose, medium dose and high dose nonylphenol administered group. After euthanasia, polychromatic erythrocyte/Normochromatic erythrocyte ratio and micronuclear polychromatic erythrocyte count were determined in femur bone marrow cells and analyzed to detect DNA damage. While polychromatic erythrocyte/Normochromatic erythrocyte ratio was found to be lower in the groups treated with different doses of nonylphenol, especially in the group treated with high-dose nonylphenol, this decrease was not statistically significant. When the micronucleated polychromatic erythrocyte values were examined, it was observed that there was a statistically significant increase, especially in the medium and high dose groups compared to the control. High-dose nonylphenol has a toxic effect and may have a long-term genotoxic effect such as cancer development by interacting with genotoxic agents.

The Antiapoptotic Effect of Danggui Buxue Tang and Its Main Components on Hematopoietic Cells in Mice with Bone Marrow Suppression

To explore the hematopoiesis protection effect of Danggui Buxue Tang (DBT) and its main components Angelica polysaccharide (APS) and Astragalus polysaccharide (ASPS) on myelosuppression mice, and the mechanism of anti-apoptosis of Meg-01 cells. Mice were radiated with 4 Gy of 137Csγ ray to establish the model of radiation-induced myelosuppression. DBT, APS or ASPS (10 mg/kg) were injected into irradiated mice. Peripheral blood cell counts were performed on mice before radiation (day 0) and day 7, 14 and 21 after radiation. On the 21st day, poor plasma platelets were collected from mice to detect TPO concentration and then the mice were sacrificed. The femoral bone marrow cells were cultured for colony cell forming units (CFU). Meg-01 cells were cultured without FBS for 24 h to induce apoptosis, and then treated with DBT/APS/ASPS for 72 h. Flow cytometry (FCM) was used to detect early apoptosis (Annexin V), mitochondrial membrane potential (JC-1) and the expression of Caspase-3 to analyze the effect of DBT/APS/ASPS on cell apoptosis. DBT can stimulate the recovery of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) of myelosuppression mice, especially for WBC and PLT (P<0.01, P<0.05). Compared with the control group, the number of BFU-E, CFU-MK and CFU-GM increased after adding DBT (BFU-E & CFU-GM: P<0.05； CFU-MK: P<0.01). The effect of DBT on blood TPO concentration in mice was not obvious (P=0.89). RBC, WBC and PLT were increased in APS group compared with control group (P<0.05). WBC increased after the treatment of ASPS (P<0.05). APS stimulated the formation of CFU-F, CFU-MK and CFU-GM (P<0.05). Only CFU-GM increased in ASPS group(P<0.05). Besides, DBT decreased the apoptosis of Meg-01 cells (P<0.05). The early apoptosis rate and total death rate in APS (100 μg/ml) group were lower than that of control group (P<0.01, P<0.05). The early apoptosis rate of ASPS (100 μg/ml) group was lower than that of control group (P<0.05). JC-1 and Caspase-3 showed that APS (100 μg/ml) significantly reduced apoptosis rate (P<0.01, P<0.05). DBT has protective effect on hematopoietic system, especially WBC and PLT, and has anti-apoptotic effect on Meg-01. It was found that the above effects of DBT were mainly caused by APS, and its anti-apoptosis mechanism was carried out mainly through JC-1 and Caspase-3 pathways.

The Hematopoietic Protective Effect of PDGF-BB on Radiation-Induced Myelosuppression Model Mice

To investigate the hematopoietic protective effect of platelet-derived growth factor (PDGF)-BB on radiation-induced myelosuppression model mice and effect of anti-apoptosis of megakaryocyte line Meg-01 cells, and its possible mechanism. Mice were radiated with 4 Gy of 137Csγ ray to establish the model of myelosuppression. Mice were weighed and peripheral blood cell were counted before radiation (day 0) and day 7, 14 and 21 after radiation. On the 21 st day, the mice were killed. The sternal tissues of the mice were taken for morphological observation, and the femoral bone marrow cells were cultured for the assay of colony cell forming units (CFU). Meg-01 cells were cultured without FBS for 24 h to induce apoptosis, and then treated with PDGF-BB for 48 h. The effects of PDGF-BB on the proliferation were investigated by cell counting. Flow cytometry was used to detect early apoptosis (Annexin V), mitochondrial membrane potential (JC-1) and the expression of caspase-3. Peripheral blood cell counts of mice showed that PDGF-BB stimulated the recovery of white blood cells, red blood cells and platelets after radiation (P<0.05), especially for white blood cells. Morphological examination showed bone marrow hyperplasia in PDGF-BB group, the numbers of megakaryocytes and their progenitor cells were higher than those in the control group. PDGF-BB significantly stimulated the formation of CFU-MK, CFU-GM, BFU-E and CFU-F. PDGF-BB showed a strong proliferation effect in the concentration range of 5-50 ng/ml (P<0.001). PDGF-BB (50 ng/ml) significantly reduced the positive expression of Annexin V (P<0.01). The mitochondrial membrane potential in the control group was decreased when compared with PDGF-BB group, which indicated that the number of apoptotic cells was increased (P<0.01). Besides, the expression of caspase-3 in PDGF-BB group was significantly lower than that in control group (P<0.05). PDGF-BB has a protective effect on the hematopoietic system of myelosuppression model mice, especially megakaryocytes and their progenitor cells. PDGF-BB has pro-proliferative and anti-apoptotic effects on Meg-01 cells, and the mechanism may be mediated through JC-1 and caspase-3 pathway.

Evaluation of Genotoxic and Cytotoxic Activity of Ganoderma Lucidum In Rat Bone Marrow Induced By Lipopolysaccharide

Objective: It was aimed to investigate the antigenotoxic effect of Ganoderma lucidum against the damage caused by lipopolysaccharide (LPS) in rat bone marrow cells by micronucleus test and comet analysis.&#x0D; Method: In the experiment, 28 Wistar albino female rats were used and 4 groups were formed. Only physiological saline was applied to the control group. A single dose of 7.5 mg/kg LPS was administered intraperitoneally to the LPS group. LPS+ Ganoderma lucidum (GL): Ganoderma lucidum was given by gavage at a dose of 50 mg/kg per day for one week. Then, on the 8th day, LPS was applied intraperitoneally. Ganoderma lucidum group: GL was given by gavage at 50 mg/kg per day for 1 week. After 24 hours, the rats were euthanized by cervical dislocation under anesthesia. Analyzes were performed for percentage of micronucleated polychromatic erythrocytes (MNPCE) and Polychromatic erythrocytes/Normochromatic erythrocytes (PCE/NCE) ratio and DNA damage in rat femur bone marrow cells.&#x0D; Results: When the percentage of micronucleus and PCE/NCE ratio were examined, no statistical significance was observed when the groups were compared both with the control and among themselves. Compared to the control group, it was observed that the comet parameters of the experimental groups (excluding the head DNA) increased significantly. Length tail (L tail), tail DNA, olive tail moment (OTM) values were significantly different both compared to the control and among themselves. While there was a significant difference between LPS+GL group and LPS and GL groups in Tail Moment (TM) value and Length comet (L comet) value, no difference was observed between LPS and GL groups.&#x0D; Conclusion: When all of the findings is considered, Ganoderma lucidum cannot be classified as a genotoxic agent. We can say, however, that it reverses the genotoxic damage in issue.

CD4+ T-cell activation of bone marrow causes bone fragility and insulin resistance in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) causes an increased risk of bone fractures. However, the pathophysiology of diabetic bone fragility is not completely understood. It has been proposed that an inflammatory microenvironment in bone could be a major mechanism by inducing uncontrolled bone resorption, inadequate bone formation and consequently more porous bones. We propose that activated T-cells in the bone marrow cause a pro-inflammatory microenvironment in bone, and cause bone fragility in T2DM. We induced T2DM in C57BL/6 male mice through a hypercaloric diet rich in carbohydrates and low doses of streptozocin. In T2DM mice we inhibited systemic activation of T-cells with a fusion protein between the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 and the Fc domain of human immunoglobulin G (CTLA4-Ig). We analysed the effects of T2DM or CTLA4-Ig in lymphocyte cell subsets and antigen-presenting cells in peripheral blood and femoral bone marrow; and their effect on the metabolic phenotype, blood and bone cytokine concentration, femoral bone microarchitecture and biomechanical properties, and the number of osteoblast-like cells in the femoral endosteum. We performed a Pearson multiple correlation analysis between all variables in order to understand the global mechanism. Results demonstrated that CTLA4-Ig decreased the number of activated CD4+ T-cells in the femoral bone marrow and consequently decreased TNF-α and RANK-L concentration in bone, notably improved femoral bone microarchitecture and biomechanical properties, increased the number of osteoblast-like cells, and reduces osteoclastic activity compared to T2DM mice that did not receive the inhibitor. Interestingly, we observed that blood glucose levels and insulin resistance may be related to the increase in activated CD4+ T-cells in the bone marrow. We conclude that bone marrow activated CD4+ T-cells cause poor bone quality and insulin resistance in T2DM.

Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.

Here, we studied the metabolic function of LAMTOR1 from macrophages using LAMTOR1 macrophage-specific knockout (MKO) mice. LAMTOR1 MKO mice showed resistance to high-fat diet (HFD)-induced obesity, lipid steatosis, and glucose metabolic disorders, with elevated levels of pro-inflammatory cytokines. The energy expenditure, oxygen consumption, and CO2 production increased significantly in HFD-fed MKO vs. wild-type (WT) mice. HE and immunohistochemistry staining showed a remarkable CD68+ Kupffer cell accumulation in the liver. Additionally, flow cytometry revealed that the proportion of macrophages and monocytes increased significantly in the liver of MKO mice. Of note, these macrophages were probably derived from the bone marrow since the proportion of CD11b+ cells as well as the proliferative activity was also increased in the context of femoral bone marrow cells. In addition, the Kupffer cells of both WT and KO mice were double-positive for the M1 (CD86) and M2 (CD206) markers. However, the expression of both M1 and M2 macrophage-related genes was increased in the liver of HFD-fed KO mice. Murine primary hepatocytes and Kupffer cells were further isolated and incubated with oleic acid for 24 h. The glucose output of primary hepatocytes from MKO mice was not affected. However, decreased lipid tolerance was observed in LAMTOR1-deficient Kupffer cells. Overall, our results suggest that LAMTOR1 deficiency in macrophages prevents obesity and metabolic disorders via the accumulation of Kupffer cells in the liver and the consequent hyper-inflammation and increased energy expenditure. Therefore, our results provide a new perspective for macrophage-derived LAMTOR1 in the context of systemic metabolism.

Characteristic Distribution of Hematopoietic Cells in Bone Marrow of Xenopus Laevis.

Bone marrow is the principal site of hematopoiesis in mammals. Amphibians were the first phylogenetic group in vertebrates to acquire bone marrow, but the distribution of hematopoietic cells in the bone marrow of the primitive frog, Xenopus laevis (X. laevis) has not been well documented. The purpose of this study was to perform a histological investigation of the distribution of hematopoietic cells in femoral bone marrow at various stages of development in X. laevis. Hematopoietic cells showed preferential distribution on the endosteal surface of cortical bone throughout all stages of development, from tadpole to aged frog. In mature frogs, hematopoietic cells appeared at the boundary between the epiphysis and the bone marrow. The distribution of hematopoietic cells around the blood vessels was limited to a small number of vessels in the bone marrow. Abundant adipose tissue was observed in the bone marrow cavity from the tadpole stage to the mature frog stage. Hematopoietic cells showed preferential distribution in a belt-like fashion on the surface of newly-formed bones in a bone regeneration model in the diaphysis of X. laevis. These results indicate that the distribution of hematopoietic cells in bone marrow in X. laevis differs from that in mammals, and that the bone marrow of X. laevis constitutes a useful model for exploring the mechanism underlying the phylogenetic differentiation of bone marrow hematopoiesis.

Gamma Tocotrienol Protects Mice From Targeted Thoracic Radiation Injury.

Radiation injury will result in multiorgan dysfuntion leading to multiorgan failure. In addition to many factors such as radiation dose, dose rate, the severity of the injury will also depend on organ systems which are exposed. Here, we report the protective property of gamma tocotrienol (GT3) in total as well as partial body irradiation (PBI) model in C3H/HeN male mice. We have carried out PBI by targeting thoracic region (lung-PBI) using Small Animal Radiation Research Platform, an X-ray irradiator with capabilities of an image guided irradiation with a variable collimator with minimized exposure to non-targeted tissues and organs. Precise and accurate irradiation of lungs was carried out at either 14 or 16 Gy at an approximate dose rate of 2.6 Gy/min. Though a low throughput model, it is amenable to change the field size on the spot. No damage to other non-targeted organs was observed in histopathological evaluation. There was no significant change in peripheral blood counts of irradiated mice in comparison to naïve mice. Femoral bone marrow cells had no damage in irradiated mice. As expected, damage to the targeted tissue was observed in the histopathological evaluation and non-targeted tissue was found normal. Regeneration and increase of cellularity and megakaryocytes on GT3 treatment was compared to significant loss of cellularity in saline group. Peak alveolitis was observed on day 14 post-PBI and protection from alveolitis by GT3 was noted. In irradiated lung tissue, thirty proteins were found to be differentially expressed but modulated by GT3 to reverse the effects of irradiation. We propose that possible mode of action of GT3 could be Angiopoietin 2-Tie2 pathway leading to AKT/ERK pathways resulting in disruption in cell survival/angiogenesis.

Genotoxic effects of drospirenone and ethinylestradiol in human breast cells (in vitro) and bone marrow cells of female mice (in vivo)

Estrogen and progesterone congeners as found in various oral contraceptive formulations have been implicated as the cause of cancer in sex and tissue-specific targets. The mechanism of carcinogenesis by sex steroids is still debatable. In this study, we evaluated the genotoxicity induced by two components of one of the commonly used oral contraceptive formulation; drospirenone and ethinylestradiol in human breast cells (MCF-7) in vitro and in bone marrow cells of female mice in vivo. DNA damage was assessed by alkaline comet assay. Both of the drugs produced DNA damage in human breast cells at exposure concentrations which are about 100-fold and above than normally found in human blood after their lowest recommended doses. The DNA damage was produced only after metabolic activation by mice liver S-9 fraction in both cases. The co-exposure with both the compounds at median exposure levels resulted in potentiation of DNA damage. In bone marrow cells of adult female mice, both the compounds produced DNA damage at human equivalent doses after exposure was carried out repeatedly for approximately one estrus cycle (5 days). The co-administration with the compounds resulted in potentiation of DNA damage as indicated by percent tail DNA in comet assay. Thus it is concluded that drospirenone and ethinylestradiol cause DNA damage in certain target specific tissue (mammary epithelial cells) and in female bone marrow cells. The co-exposure with drospirenone and ethinylestradiol results in potentiation of genotoxicity which may pose a threat of cancer development in women taking these drugs for long periods.

Evaluation of genotoxicity after acute and chronic exposure to 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) in rodents using machine learning algorithms.

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides in the world, but its mutagenic and carcinogenic potential is still controversial. We simulated environmental exposure to 2,4-D, with the objective of evaluating the genotoxic effect of acute and chronic exposure to 2,4-D in rodents. We also evaluated the performance of machine learning algorithms in detecting differences in exposure groups through recognition performed from genotoxic characteristics. In the acute phase, 88 Swiss mice were used, distributed in five groups and exposed to nebulizations at different time intervals (24, 48, 72 and 192 hr). In the chronic phase, 88 Wistar rats were used, distributed in two groups (inhaled and oral) and exposed for six months. Femoral bone marrow cells were collected for a micronucleus test and comet assay. Data were evaluated by pattern recognition algorithms. In acute exposure, medium and high concentrations induced DNA damage in the comet assay, but these concentrations did not increase micronucleated cells. In the chronic exposure, there was an increase in micronuclei and DNA damage in the comet assay in all exposed groups regardless of the exposure route. The data showed a robust pattern of distinction between exposed and nonexposed groups to 2,4-D. Our data showed that both acute inhalation exposure and chronic oral and inhalation exposure to 2,4-D can cause genotoxic effects regardless of concentration. Machine learning showed a clear distinction between the control groups and those exposed to 2,4-D, and the effects of exposure are not concentration-dependent.

GCMS analysis of sadagura (smokeless tobacco), its enhanced genomic instability causing potential due to arsenic co-exposure, and vitamin-C supplementation as a possible remedial measure: a study involving multiple model test systems

North-eastern states of India, including Assam, have a high prevalence of head and neck cancer cases. In these regions, Sadagura is a unique form of smokeless tobacco (SLT). There are fewer reports regarding the effects of simultaneous sadagura and arsenic co-exposure. Analysis of chemical compounds present in sadagura aqueous extract was done using gas chromatography–mass spectrometry. Estimation of arsenic contamination in groundwater and bioaccumulation in human tissues was performed by using atomic absorption spectroscopy. Buccal micronucleus cytome (BMCyt) assay and analysis of various peripheral blood parameters were performed among study volunteers. Chronic exposure (90 days) experiments were performed in mice test system in vivo to determine any possible protective potential of vitamin C (Vit-C) supplementation against sadagura and arsenic co-exposure. BMCyt assay results revealed a higher incidence of micronucleated cells (p < 0.001), and cell death biomarker among sadagura consumers residing in arsenic affected areas. Comet assay of mice femur bone marrow cells following chronic exposure of the test substances revealed a reduction in DNA damage due to Vit-C supplementation. Histological examination of the hepatic and renal tissues revealed marked improvement due to Vit-C supplementation in mice against sadagura and arsenic chronic co-exposure. Indiscriminate consumption, presence of various harmful compounds in sadagura along with arsenic co-exposure might be a vital link for the higher incidence of oral cancer in the region. Chronic Vit-C supplementation study results in mice show its effective remedial potential against combined sadagura and arsenic co-mediated genotoxicity and ultrastructural changes in major organs.

Effect of nutritional status on arsenic and smokeless tobacco induced genotoxicity, sperm abnormality and oxidative stress in mice in vivo.

Recently, high concentrations of arsenic have been documented in ground waters of Southern Assam, India. Indiscriminate smokeless tobacco consumption is a common practice in this region. Correlation between nutritional status and arsenic and smokeless tobacco-induced health effects has not been taken up in humans or other test systems. Mice were divided into groups based on protein (casein) content in the diet: High protein (40%), optimum protein (20%), and low protein (5%). Simultaneous chronic exposure (90 days) to arsenic and smokeless tobacco (sadagura) orally was given to evaluate the extent of the cytological and genotoxicological damage. Micronucleus assay and Comet assay of the femur bone marrow cells were conducted. Germ cell toxicity was evaluated by recording the sperm head abnormalities and total sperm count. Cell cycle analysis was performed in femur bone marrow cells using flow cytometer. Hepatic, renal, and intestinal tissues were analyzed for various oxidative stress evaluations. Histological examination of liver and kidney was performed. Notably, high protein diet groups had lower arsenic and sadagura induced genotoxicity, germ cell abnormalities and oxidative stress as compared to optimum protein and low protein diet counterparts. Our study indicates that sufficient levels of dietary protein appear to reduce the long-term arsenic and smokeless tobacco-induced toxicity in mice test system, as compared to lower or deficient amount of protein in the diet. This observation has implications and invites further studies especially epidemiological studies in the human population exposed to arsenic in South East Asian countries. Environ. Mol. Mutagen. 59:386-400, 2018. © 2018 Wiley Periodicals, Inc.

Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition.

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.

VEGF treatment promotes bone marrow-derived CXCR4+ mesenchymal stromal stem cell differentiation into vessel endothelial cells.

Stem/progenitor cells serve an important role in the process of blood vessel repair. However, the mechanism of vascular repair mediated by C-X-C chemokine receptor type 4-positive (CXCR4+) bone marrow-derived mesenchymal stem cells (BMSCs) following myocardial infarction remains unclear. The aim of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on vessel endothelial differentiation from BMSCs. CXCR4+ BMSCs were isolated from the femoral bone marrow of 2-month-old mice and the cells were treated with VEGF. Expression of endothelial cell markers and the functional properties were assessed by reverse transcription-quantitative polymerase chain reaction, flow cytometry and vascular formation analyses. The results indicated that the CXCR4+ BMSCs from femoral bone marrow cells expressed putative cell surface markers of mesenchymal stem cells. Treatment with VEGF induced platelet/endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) expression at the transcriptional and translational levels, compared with untreated controls. Moreover, VEGF treatment induced CXCR4+ BMSCs to form hollow tube-like structures on Matrigel, suggesting that the differentiated endothelial cells had the functional properties of blood vessels. The results demonstrate that the CXCR4+ BMSCs were able to differentiate into vessel endothelial cells following VEGF treatment. For cell transplantation in vascular disease, it may be concluded that CXCR4+ BMSCs are a novel source of endothelial progenitor cells with high potential for application in vascular repair.

Increased plasma VEGF levels following ischemic preconditioning are associated with downregulation of miRNA-762 and miR-3072-5p.

Ischemic preconditioning (IPC) has protective effects against ischemia-perfusion injury of organs. In the present study, we investigated the associated mechanisms after performing remote IPC (rIPC) of lower limbs by clamping abdominal aorta in mice. Subsequent experiments showed decreased damage and paralysis of lower limbs following spinal cord injury (SCI). Concomitantly, plasma vascular endothelial growth factor (VEGF) levels were increased 24 h after rIPC compared with those in sham-operated animals. In subsequent microRNA analyses, thirteen microRNAs were downregulated in exosomes 24 h after rIPC. Further studies of femoral CD34-positive bone marrow (BM) cells confirmed downregulation of these seven microRNAs 24 h after rIPC compared with those in sham-operated controls. Subsequent algorithm-based database searches suggested that two of the seven microRNAs bind to the 3′ UTR of VEGF mRNA, and following transfection into CD34-positive BM cells, anti-miR-762, and anti-miR-3072-5p inhibitors led to increased VEGF concentrations. The present data suggest that rIPC transiently increases plasma VEGF levels by downregulating miR-762 and miR-3072-5p in CD34-positive BM cells, leading to protection against organ ischemia.

CXCR4 is required for progression of T cell acute lymphoblastic leukemia

Abstract Of the most common childhood cancer, acute lymphoblastic leukemia (ALL), 15% of cases are caused by transformation of T cell progenitors (T-ALL). While survival rates have vastly improved for newly diagnosed T-ALL, substantial challenges remain in the treatment of relapsed disease and in the minimization of side-effects from central nervous system prophylaxis. Hence, the search for more effective, less toxic treatments continues. Homing receptors play multiple roles in cancer progression, yet little is known about the trafficking of T-ALL. Upon profiling homing receptor expression, we found high levels of the chemokine receptor CXCR4 on the surface of mouse and primary human T-ALL cells. Furthermore, T-ALL cells localized in close proximity to CXCL12-producing stromal cells in mouse femoral bone marrow. Deletion of Cxcl12 in vascular endothelial cells limited leukemia progression, suggesting a vascular niche for T-ALL. In parallel, we found CXCR4 expression to be essential for T-ALL maintenance and progression. Deletion of Cxcr4 in mouse T-ALL after disease onset led to disease remission and prolonged survival, and CXCR4 antagonism using the small molecule inhibitor AMD3465 suppressed human disease in a patient-derived xenograft model. Loss of CXCR4 signaling in T-ALL cells reduced Myc abundance, which was previously reported to regulate leukemia-initiating cell activity. In line with this, CXCR4-deficient T-ALL cells failed to establish disease when adoptively transferred into secondary hosts. Together, our findings highlight an important contribution of microenvironment in regulating T-ALL pathogenesis, and suggest targeting CXCL12:CXCR4 signaling as a powerful new strategy for treating this aggressive disease.

Evaluation of sesamol-induced histopathological, biochemical, haematological and genomic alteration after acute oral toxicity in female C57BL/6 mice.

The objective of this study was to evaluate organ-wise toxicological effects of sesamol and determine the LD50 cut-off value and GHS category following acute oral toxicity method OECD 423. An acute oral toxicity study was carried out in female C57BL/6 mice. Observations for physical behaviour and measurements on haematology, biochemistry, histology of vital organs were performed. In addition, genotoxicity assessment using comet and micronuclei assays was also performed. Acute toxicological effects were observed at 2000 mg/kg, while no adverse effects observed at 300 mg/kg. The effects of 2000 mg/kg were manifested as severe histopathological changes in all organs (femur, spleen, gastrointestine, lungs, heart, kidney, liver, stomach and brain) and excessive DNA strands breaks occurred in femoral bone marrow cells and splenocytes. A single dose of sesamol (2000 mg/kg, body weight) caused the death of two mice (out of three) within 2 h. Hence, sesamol is in GHS category 4 (>300–2000) with LD50 cut-off value of 500 mg/kg body weight. In contrast, this study is correlated with the obtained GHS category 4 and LD50 cut-off value 580 mg/kg body weight by ProTox. In conclusions, the present study has classified sesamol toxicity and assessed organ-wise acute oral toxicity of sesamol in female C57BL/6 mice. Therefore, these findings may be useful for the selection of dosages for further pre-clinical evaluation and potential drug developmental of sesamol.

Involvement of Prolyl Hydroxylase Domain Protein in the Rosiglitazone-Induced Suppression of Osteoblast Differentiation.

Rosiglitazone is a well-known anti-diabetic drug that increases insulin sensitivity via peroxisome proliferator-activated receptor γ (PPARγ) activation, but unfortunately it causes bone loss in animals and humans. A previous study showed that prolyl hydroxylase domain protein (PHD) plays a role in rosiglitazone-induced adipocyte differentiation. Based on the inverse relationship between adipocyte and osteoblast differentiation, we investigated whether PHD is involved in the effects of rosiglitazone on osteoblast differentiation. Rosiglitazone inhibited osteoblast differentiation in a concentration-dependent manner, and in parallel induced three PHD isoforms (PHD1, 2, and 3). PHD inhibitors and knockdown of each isoform prevented the inhibitory effects of rosiglitazone on osteoblast differentiation and increased the expression of Runx2, a transcription factor essential for osteoblastogenesis. MG-132, a proteasomal inhibitor also prevented the rosiglitazone-induced degradation of Runx2. Furthermore, both increased PHD isoform expressions and reduced osteoblast differentiation by rosiglitazone were prevented by PPARγ antagonists, indicating these effects were mediated via PPARγ activation. In vivo oral administration of rosiglitazone to female ICR mice for 8 weeks reduced bone mineral densities and plasma alkaline phosphatase (ALP) activity, and increased PHD expression in femoral primary bone marrow cells and the ubiquitination of Runx2. Together, this suggests that the rosiglitazone-induced suppression of osteoblast differentiation is at least partly induced via PPARγ-mediated PHD induction and subsequent promotion of the ubiquitination and degradation of Runx2.

Mutagenicity and genotoxicity studies of arruva, an R,R-monatin salt isomer

Arruva, the R,R-isomer of monatin (sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate), an intense sweetener originally identified in root bark of the South African shrub Schlerochitin ilicifolius, was examined for its mutagenic and genotoxic potential via bacterial reverse mutation, mouse lymphoma and in vivo mouse micronucleus assays, all accomplished in the presence and absence of S9 metabolic activation. In the bacterial reverse mutation assay, arruva was determined to not cause reverse mutations in four Salmonella typhimurium strains and one Escherichia coli strain at concentrations up-cells did not exhibit concentration-related increases in mutant frequency at test concentrations up to 3200μg/ml. In the in vivo micronucleus test, arruva was administered to male mice via single gavage doses at 500, 1000 or 2000mg/kg bw. At 24 or 48h post-dose, the mice were euthanized and femoral bone marrow cells were collected for evaluation of micronucleated polychromatic erythrocyte (MPCE) presence. No statistically significant increases of MPEs were observed relative to the respective vehicle control groups. Under the conditions of these studies, arruva was concluded to be negative in all three assays, thereby indicating the absence of its potential mutagenicity or genotoxicity under the conditions tested.

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow

AimsBone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors. MethodsDiabetes was induced by 65mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis. ResultsThe size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by (1) lower (∼35%) fraction of adherent stromal cells (at 24h of culture); (2) lower (20–25%) alkaline phosphatase activity at 10 days of culture; and (3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals. ConclusionsExperimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.