losartan-potassium and phenylhydrazine

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1β. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Topics: Anemia; Animals; Cytokines; Erythrocyte Count; Erythropoiesis; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation; Goldfish; Interferon-gamma; LIM Domain Proteins; Parasitemia; Phenylhydrazines; Receptors, Erythropoietin; RNA, Messenger; Trypanosoma; Trypanosomiasis; Tumor Necrosis Factor-alpha

Neonatal unconjugated hyperbilirubinemia might cause severe bilirubin neurotoxicity in especially hemolytic conditions. The study aimed to elucidate the potential neuroprotective effects of erythropoietin (EPO) in hemolysis-induced hyperbilirubinemia. In newborn rats, hyperbilirubinemia secondary to hemolysis was induced by injecting with phenylhydrazine hydrochloride (PHZ) and rats were injected with either vehicle or EPO. At 54th hour of the PHZ injection, rats were decapitated. Serum levels of TNF-α, IL-1β, IL-10, brain-derived neurotrophic factor (BDNF) and S100-B and brain malondialdehyde, glutathione levels and myeloperoxidase activities were measured. TUNEL staining and NF-κB expression were evaluated. As compared to control pups, in vehicle-treated PHZ group, TNF-α and IL-1β levels, malondialdehyde level and myeloperoxidase activity were increased with concomitant decreases in IL-10 and glutathione. All EPO regimens reversed PHZ-induced alterations in IL-10, TNF-α, malondialdehyde and glutathione levels. Three-day-treatment abolished increases in myeloperoxidase activity and IL-1β levels, while BDNF and S100-B were elevated. Increased TUNEL (+) cells and NF-κB expressions in the brain of PHZ group were reduced in the 3-day-treated group. EPO exerted anti-inflammatory effects on PHZ-induced neural damage in newborn rats, while the neuroprotection was more obvious when the treatments were repeated successively. The results suggest that EPO treatment may have a therapeutic potential in supporting neuroplasticity in the hyperbilirubinemic neonates.

Topics: Animals; Animals, Newborn; Erythropoietin; Female; Hemolysis; Hyperbilirubinemia; Male; Neuroprotective Agents; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Treatment Outcome

Iron is crucial for many biological functions, but quantitatively the most important use of iron is in the production of hemoglobin in red blood cell precursors. The amount of iron in the plasma, and hence its availability for hemoglobin synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When the iron supply to erythroid precursors is limited, as often occurs during stimulated erythropoiesis, these cells produce signals to inhibit hepatic hepcidin production, thereby increasing the amount of iron that enters the plasma. How stimulated erythropoiesis suppresses hepcidin production is incompletely understood, but erythroferrone, Gdf15 and Twsg1 have emerged as candidate regulatory molecules. To further examine the relationship between erythropoiesis and the candidate erythroid regulators, we have studied five mouse models of anemia, including two models of β-thalassemia (Hbbth3/+ and RBC14), the hemoglobin deficit mouse (hbd), dietary iron deficient mice and mice treated with phenylhydrazine to induce acute hemolysis. Hematological parameters, iron status and the expression of Erfe (the gene encoding erythroferrone), Gdf15 and Twsg1 in the bone marrow and spleen were examined. Erfe expression was the most consistently upregulated of the candidate erythroid regulators in all of the mouse models examined. Gene expression was particularly high in the bone marrow and spleen of iron deficient animals, making erythroferrone an ideal candidate erythroid regulator, as its influence is strongest when iron supply to developing erythroid cells is limited. Gdf15 expression was also upregulated in most of the anemia models studied although the magnitude of the increase was generally less than that of Erfe. In contrast, very little regulation of Twsg1 was observed. These results support the prevailing hypothesis that erythroferrone is a promising erythroid regulator and demonstrate that Erfe expression is stimulated most strongly when the iron supply to developing erythroid cells is compromised.

Topics: Anemia; Animals; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Hepcidins; Iron; Iron Deficiencies; Liver; Mice, Inbred C57BL; Models, Genetic; Phenylhydrazines; Receptors, Transferrin

Hemolytic anemia is a common form of anemia due to hemolysis, resulting in disordered iron homeostasis. In this study, a dose of 40mg/kg phenylhydrazine (PHZ) was injected into mice to successfully establish a pronounced anemia animal model, which resulted in stress erythropoiesis and iron absorption. We found that serum erythropoietin (EPO) concentration was dramatically elevated by nearly 5000-fold for the first 2days, and then drop to the basal level on day 6 after PHZ injection. Mirrored with serum EPO concentration, the mRNA expression of erythroferrone (ERFE) was rapidly increased in the bone marrow and spleen 3days after injection of PHZ, and then gradually decreased but was still higher than baseline on day 6. In addition, we also found that the hepcidin mRNA levels were gradually reduced almost up to 8-fold on day 5, and then was ameliorated compared to the untreated control. Mechanistic investigation manifested that the increase of serum EPO essentially determined the induction of ERFE expression particular at the first 3days after PHZ treatment. Lentiviral mediated ERFE knockdown significantly restrained hepcidin suppression under PHZ treatment. Thus, our data unearthed EPO-dependent ERFE expression acts as an erythropoiesis-driven regulator of iron metabolism under PHZ-induced hemolytic anemia.

Topics: Anemia, Hemolytic; Animals; Cytokines; Down-Regulation; Erythropoiesis; Erythropoietin; Hemolysis; Hepcidins; Iron; Male; Mice; Mice, Inbred C57BL; Muscle Proteins; Phenylhydrazines; RNA, Messenger; Up-Regulation

Although the importance of native bone marrow and spleen macrophages in enhancing baseline and stress erythropoiesis has been emphasized over several decades, their kinetic and phenotypic changes during a variety of stress responses have been unclear. Furthermore, whether monocyte-derived recruited macrophages can functionally substitute for inadequate or functionally impaired native macrophages has been controversial and seem to be not only tissue- but also stress-type dependent. To provide further insight into these issues, we made detailed observations at baseline and post-erythroid stress (E-stress) in 2 mouse models with genetically depressed macrophage numbers and compared them to their controls. We documented that, irrespective of the stress-induced (hemolytic or post-erythropoietin [Epo]) treatment, only native CD11b(lo) splenic macrophages expand dramatically post-stress in normal mice without significant changes in the monocyte-derived CD11b(hi) subset. The latter remained a minority and did not change post-stress in 2 genetic models lacking either Spi-C or VCAM-1 with impaired native macrophage proliferative expansion. Although CD11b(lo) macrophages in these mice were one-fifth of normal at their peak response, surprisingly, their erythroid response was not compromised and was similar to controls. Thus, despite the prior emphasis on numerical macrophage reliance to provide functional rescue from E-stress, our data highlight the importance of previously described non-macrophage-dependent pathways activated under certain stress conditions to compensate for low macrophage numbers.

Topics: Animals; Bone Marrow; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Hemolysis; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenylhydrazines; Recombinant Proteins; Spleen; Stress, Physiological; Vascular Cell Adhesion Molecule-1

Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias.

Topics: Acute Disease; Anemia; Anemia, Hemolytic; Animals; Butyrates; Cell Culture Techniques; Cells, Cultured; Chromatin; Chronic Disease; Disease Models, Animal; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Fenofibrate; Glucocorticoids; Humans; Liver; Mice; Phenylhydrazines; Phenylurea Compounds; PPAR alpha; Receptors, Glucocorticoid; Signal Transduction

Samul-tang (Si-Wu-Tang, SMT), a kind of herbal medicines, has been used for the hemato-deficient disease for hundreds of years. In this work, investigate the anti-anemia activity of the H2O extracts from constituent herbal medicines of Samul-tang in an anemia model induced by intravenous infection of phenylhydrazine-HCL (PHZ) at 10 mg/kg for 4 days. After PHZ injection, female Sparague-Dawley rats were administrated extracts from constituent herbal medicines of SMT (300 mg/kg/day, p.o.) daily for 1 week. Results showed that sever hemolysis was induced by PHZ. For Paeonia lactiflora (PL2) H2O extract treated groups, the concentration of hemoglobin, hematocrit and red blood cells number increased much more significantly than PHZ-treated group. Moreover, Angelica gigas (AG), Angelica. acutiloba (AA), Paeonia lactiflora (PL2) and Rehmannia glutinosa (RG) extract administration significantly improved serum erythropoietin concentration. The activity of aminolevulinic acid dehydrates (ALDL) in liver homegenate was increased in Angelica gigas(AA), Paeonia lactiflora (PL2) and Rehmannia glutinosa (RG) treated group.

Topics: Anemia, Hemolytic; Animals; Biomarkers; Disease Models, Animal; Drugs, Chinese Herbal; Erythrocyte Count; Erythrocytes; Erythropoietin; Female; Hematinics; Hematocrit; Hematopoiesis; Hemoglobins; Humans; Liver; Phenylhydrazines; Phytotherapy; Plants, Medicinal; Porphobilinogen Synthase; Rats, Sprague-Dawley; Time Factors

This study examined the effect of fermentation on the ability of antler to act as a stimulator of hematopoietic activity. Hemolytic anemia was induced by phenylhydrazine (PHZ) in female Sprague-Dawley rats. The vehicle or antler extract (nonfermented or fermented) mixed in drinking water was administered from Days 2 to 15 after PHZ injection. On Day 15, red blood cell counts in the fermented antler group (6.33×10⁶/μL) were significantly higher than those in the nonfermented antler group (5.90×10⁶/μL) (P<.05), and rats treated with fermented antler extract tended to have higher hemoglobin compared with rats treated with nonfermented antler extract, but not significantly. In addition, rats treated with fermented antler extract had slightly lower serum erythropoietin levels compared with nonfermented antler extract, which were not statistically different from serum erythropoietin levels of nonanemic rats. We conclude therefore that the hematopoietic activity of antler might be increased by the fermentation process.

Topics: Anemia, Hemolytic; Animals; Antlers; Bacillus subtilis; Deer; Erythrocyte Count; Erythropoietin; Female; Fermentation; Food, Preserved; Hematinics; Hematopoiesis; Hemoglobins; Humans; Male; Materia Medica; Medicine, East Asian Traditional; Osmotic Fragility; Phenylhydrazines; Random Allocation; Rats; Rats, Sprague-Dawley

Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.

Topics: Anemia, Hemolytic; Animals; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Bone Marrow; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Flow Cytometry; Fluorouracil; Immunoprecipitation; Male; Mice; Mice, Knockout; Oxidants; Oxidative Stress; Phenylhydrazines; Phosphorylation; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; rho-Associated Kinases; RNA, Messenger; Signal Transduction; Spleen; Survival Rate; Tumor Suppressor Protein p53

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.

Topics: Anemia; Animals; Cells, Cultured; Cytokines; Erythropoiesis; Erythropoietin; Hematocrit; Kidney; Male; Oxidants; Phenylhydrazines; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trypanosoma brucei brucei; Trypanosomiasis, African

Erythropoietin (EPO) and its receptor (EPOR) are key regulators of red blood cell production in mammals and fish. We aimed to investigate the structural and functional conservation of the EPO-EPOR system in amphibian erythropoiesis, using Xenopus laevis as a model.. X. laevis epo (xlepo) complementary DNA was identified by referring to the Xenopus tropicalis genome database. Biological activity of recombinant xlEPO expressed in COS-1 cells was evaluated using xlEPOR-expressing murine FDC/P2 cells and human EPO-dependent UT-7/EPO cells. Expression of xlepo messenger RNA in adult X. laevis tissues in the normal state and under the condition of phenylhydrazine-induced anemia was evaluated by real-time reverse transcription polymerase chain reaction.. In the encoded protein, the positions of four cysteine residues were conserved; however, xlEPO had only 38% identity with human EPO. N-glycosylation sites were absent. Recombinant xlEPO induced proliferation of cell lines expressing xlEPOR and UT-7/EPO, confirming biological activity and cross-species reactivity. Despite little primary amino acid sequence similarity, the evolutionary highly conserved sequence NFLRGK was identified in the EPOR-binding site 1 region as in the human EPO protein. Strong expression of xlepo messenger RNA was detected in the lung and liver, especially in fractionated hepatocytes. No marked increase in xlepo expression was seen in the lung and liver of phenylhydrazine-induced anemic X. laevis.. We confirmed that xlEPO is the ligand to the previously reported xlEPOR in X. laevis. xlEPO shares structural and functional similarities and differences with mammalian counterparts, and regulation of xlepo expression and its influence on the erythropoietic system appears to be unique.

Topics: Amino Acid Sequence; Anemia, Hemolytic; Animals; Animals, Inbred Strains; Base Sequence; Cell Line; Conserved Sequence; DNA, Complementary; Erythropoiesis; Erythropoietin; Hepatocytes; Humans; Lung; Male; Mice; Molecular Sequence Data; Organ Specificity; Phenylhydrazines; Receptors, Erythropoietin; Recombinant Fusion Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1(-/-) mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)-dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1(-/-) spleens. The ERK1(-/-) phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.

Topics: Anemia; Animals; Apoptosis; Blotting, Western; Bone Marrow Transplantation; Bone Morphogenetic Protein 4; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Flow Cytometry; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 3; Oxidants; Phenylhydrazines; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spleen

AMP-activated protein kinase (AMPK) is an energy sensor essential for maintaining cellular energy homeostasis. Here, we report that AMPKalpha1 is the predominant isoform of AMPK in murine erythrocytes and mice globally deficient in AMPKalpha1 (AMPKalpha1(-/-)), but not in those lacking AMPKalpha2, and the mice had markedly enlarged spleens with dramatically increased proportions of Ter119-positive erythroid cells. Blood tests revealed significantly decreased erythrocyte and hemoglobin levels with increased reticulocyte counts and elevated plasma erythropoietin concentrations in AMPKalpha1(-/-) mice. The life span of erythrocytes from AMPKalpha1(-/-) mice was less than that in wild-type littermates, and the levels of reactive oxygen species and oxidized proteins were significantly increased in AMPKalpha1(-/-) erythrocytes. In keeping with the elevated oxidative stress, treatment of AMPKalpha1(-/-) mice with the antioxidant, tempol, resulted in decreased reticulocyte counts and improved erythrocyte survival. Furthermore, the expression of Foxo3 and reactive oxygen species scavenging enzymes was significantly decreased in erythroblasts from AMPKalpha1(-/-) mice. Collectively, these results establish an essential role for AMPKalpha1 in regulating oxidative stress and life span in erythrocytes.

Topics: AMP-Activated Protein Kinases; Animals; Blotting, Western; Cell Survival; Enzyme-Linked Immunosorbent Assay; Erythrocyte Count; Erythrocytes; Erythropoietin; Flow Cytometry; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Deletion; Gene Expression; Mice; Mice, Knockout; Oxidants; Oxidative Stress; Phenylhydrazines; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Splenomegaly; Superoxide Dismutase; Survival Analysis; Time Factors

Podocalyxin expression on Ter119(+) erythroblasts is induced following administration of erythropoietin (Epo) or phenylhydrazine treatment, but is notably absent on committed erythroid progenitors during homeostatic red cell turnover. Following high-dose Epo administration in vivo, podocalyxin surface expression is upregulated, in part, via a signal transducers and activators of transcription 5-dependent pathway and this expression has been postulated to play a role in the release of reticulocytes from hematopoietic organs into the periphery under conditions of increased erythropoietic rate. Here we have thoroughly addressed this hypothesis and further examined the expression profile of podocalyxin during Epo-induced erythroblast expansion and stress erythropoiesis.. Following Epo induction, progenitor cells were sorted to characterize podocalyxin expression during stress. In addition, as podocalyxin-deficient mice die perinatally, we used chimeric mice reconstituted with wild-type or podocalyxin-deficient hematopoietic cells to analyze differences in response to high dose Epo administration and chemically induced anemia.. Podocalyxin surface expression is rapidly upregulated in response to stress and marks early erythroid progenitors and erythroblasts. Despite loss of podocalyxin, chimeras exhibit normal basal erythropoiesis and no differences in erythroid progenitor proportions in the spleen and marrow in response to Epo. Further, podocalyxin is dispensable for efficient recovery from models of anemia.. We demonstrate that podocalyxin is a highly specific marker of stress-induced blast-forming unit erythroid and colony-forming unit erythroid progenitors in mouse bone marrow and spleen. In addition, our findings suggest that podocalyxin is not necessary for efficient erythroblast expansion, erythroid differentiation, or reticulocyte release in response to Epo stimulation in vivo.

Topics: Anemia; Animals; Antigens, Differentiation; Bone Marrow; Erythroid Precursor Cells; Erythropoietin; Mice; Mice, Knockout; Oxidants; Phenylhydrazines; Reticulocytes; Sialoglycoproteins; Spleen; STAT5 Transcription Factor; Stress, Physiological; Transplantation Chimera; Up-Regulation

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.

Topics: Anemia; Animals; Bayes Theorem; Bone Marrow Cells; Disease Models, Animal; Erythrocyte Aging; Erythrocyte Count; Erythrocytes; Erythropoietin; Feedback, Physiological; Markov Chains; Mice; Monte Carlo Method; Phenylhydrazines; Reticulocyte Count; Reticulocytes

Erythrocyte production is regulated by balancing precursor cell apoptosis and survival signaling. Previously, we found that BH3-only proapoptotic factor, Nix, opposed erythroblast-survival signaling by erythropoietin-induced Bcl-xl during normal erythrocyte formation. Since erythropoietin treatment of human anemia has limitations, we explored the therapeutic potential of abrogating Nix-mediated erythroblast apoptosis to enhance erythrocyte production. Nix gene ablation blunted the phenylhydrazine-induced fall in blood count, enhanced hematocrit recovery, and reduced erythroblast apoptosis, despite lower endogenous erythropoietin levels. Similar to erythropoietin, Nix ablation increased early splenic erythroblasts and circulating reticulocytes, while maintaining a pool of mature erythroblasts as erythropoietic reserve. Erythrocytes in Nix-deficient mice showed morphological abnormalities, suggesting that apoptosis during erythropoiesis not only controls red blood cell number, but also serves a "triage" function, preferentially eliminating abnormal erythrocytes. These results support the concept of targeting erythroblast apoptosis to maximize erythrocyte production in acute anemia, which may be of value in erythropoietin resistance.

Topics: Anemia; Animals; Apoptosis; Bone Marrow; Cell Differentiation; Cell Proliferation; Cell Survival; Disease Models, Animal; Down-Regulation; Erythroblasts; Erythrocyte Count; Erythrocytes; Erythropoietin; Gene Targeting; Membrane Proteins; Mice; Mice, Knockout; Mitochondrial Proteins; Oxidants; Phenylhydrazines; Recovery of Function

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Topics: Aniline Compounds; Animals; Body Temperature; Bone Marrow Cells; Erythropoietin; Guidelines as Topic; Hyperthermia, Induced; Micronucleus Tests; Mutagenicity Tests; Mutagens; Naphthoquinones; Phenol; Phenylhydrazines; Pyridines; Reserpine; Rodentia; Sensitivity and Specificity; Triazoles

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.

Topics: Anemia, Hemolytic; Animals; Antimicrobial Cationic Peptides; Cation Transport Proteins; Erythropoiesis; Erythropoietin; Ferritins; Gamma Rays; Gene Expression Regulation; GPI-Linked Proteins; Hemochromatosis Protein; Hemolysis; Hepcidins; Iron Compounds; Liver; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Phenylhydrazines; Polycythemia; Receptors, Transferrin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Stress erythropoiesis is usually considered as a compensatory effort to counteract tissue hypoxia. Its homeostatic importance in anemic hypoxia has not been questioned, but researchers, clinicians, and mountain climbers have had second thoughts on polycythemia as to its appropriateness for hypoxic or altitude hypoxia (HA). Therefore, polycythemia, one of the responses to HA seen in nongenetically adapted mammals, could or could not be considered beneficial. The present study was thus performed to obtain further information on the importance of HA polycythemia on acclimation of mice to HA. To this end, the development of polycythemia was prevented by experimental manipulations (administration of 20 mg/kg/d of the hemolytic drug phenylhydrazine or removal of 0.225 mL/d of blood), and the degree of tissue hypoxia was evaluated from plasma erythropoietin (pEPO) concentration, as determined by immunoassay, in adult female mice exposed to air maintained at 506 mbar (380 mmHg) in a simulated HA (SHA) chamber during at least 23.5 h/d for 9 d. Plasma EPO concentration in those treated hypoxic mice whose hematocrit values remained almost unchanged was between 5.55 and 7.89 times higher (depending on the experimental designs) than in control hypoxic mice allowed to develop HA polycythemia. These results, plus the finding of an inverse relationship between the hematocrit value and pEPO concentration in both the polycythemic and normocythemic SHA-exposed mice indicate that HA polycythemia is highly effective in ameliorating tissue hypoxia under SHA conditions, thus giving support to the concept of the important role of the increased hemoglobin mass in nongenetically adapted animals, whereas a left-shifted oxyhemoglobin dissociation curve confers a good degree of adaptation to HA in genetically adapted animals.

Topics: Altitude Sickness; Analysis of Variance; Animals; Atmosphere Exposure Chambers; Atmospheric Pressure; Cell Hypoxia; Erythropoiesis; Erythropoietin; Female; Hematocrit; Immunoassay; Mice; Phenylhydrazines; Polycythemia; Secretory Rate

Erythropoietin (Epo) modulates calcium influx through voltage-independent calcium-permeable channel(s). Here, we characterized the expression of transient receptor potential channels (TRPCs) in primary erythroid cells and examined their regulation. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in [Ca](i). Among the classical TRPC channels, expression of three N-terminal splice variants of TRPC2 (clones 14, 17, and alpha) and of TRPC6 were demonstrated in these erythroblasts by both reverse transcriptase-PCR and Western blotting. Confocal microscopy confirmed localization to the plasma membrane. To determine the function of individual TRPC channels in erythropoietin modulation of calcium influx, digital video imaging was used to measure calcium influx through these TRPCs in a Chinese hamster ovary (CHO) cell model. Single CHO-S cells, expressing transfected Epo-R, were identified by detection of green fluorescent protein. Cells that express transfected TRPCs were identified by detection of blue fluorescent protein. [Ca](i) was monitored with Fura Red. Epo stimulation of CHO-S cells transfected with single TRPC2 isoforms (clone 14, 17, or alpha) and Epo-R resulted in a significant increase in [Ca](i). This was not observed in cells transfected with Epo-R and TRPC6. In addition, coexpression of TRPC6 with TRPC2 and Epo-R inhibited the increase in [Ca](i) observed after Epo stimulation. Immunoprecipitation experiments demonstrated that TRPC2 associates with TRPC6, indicating that these TRPCs can form multimeric channels. These data demonstrate that specific TRPCs are expressed in primary erythroid cells and that two of these channels, TRPC2 and TRPC6, can interact to modulate calcium influx stimulated by erythropoietin.

Topics: Alternative Splicing; Animals; Blotting, Western; Brain; Calcium; Calcium Channels; Cell Division; Cell Line; Cell Membrane; Cells, Cultured; CHO Cells; Cricetinae; DNA, Complementary; Dose-Response Relationship, Drug; Erythroblasts; Erythrocytes; Erythropoietin; Green Fluorescent Proteins; Ion Channels; Luminescent Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Video; Phenylhydrazines; Precipitin Tests; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Transfection; TRPC Cation Channels; TRPC6 Cation Channel; TRPM Cation Channels

Nucleated erythroid cells (EC) have been previously reported to possess a potent natural suppressor (NS) activity for B-cell responses. In this study, we demonstrate that murine EC are able to reduce not only lipopolysaccharide (LPS)-driven B-cell proliferation, but also proliferative and cytotoxic T-cell responses generated in a primary allogeneic mixed lymphocyte culture (MLC); and that a soluble low molecular weight factor may be involved in such EC-derived immunoregulation. In addition, the erythroid cell-derived suppressor factor (ESF) was found to be capable of effectively reducing the allergen-driven proliferation of peripheral blood mononuclear cells (PBMC) isolated from allergic patients. From the data presented herein, it appears that ESF is heat-stable (80 degrees C for 20 min) and has molecular weight (MW) lower or close to 0.5 kDa. ESF activity is resistant to both enzyme (trypsin plus chymotrypsin) proteolysis and action of the enzymes such as lipase and phospholipase C. On the other hand, ESF is effectively inactivated by neuraminidase treatment, suggesting the presence in its structure of sialic residue(s). The neuraminidase-sensitive, ESF-like activity is readily detected in the medium conditioned with normal mouse bone marrow (BM) cells. On fractionation of low MW erythroid products on a reversed-phase C16 column in a linear acetonitrile gradient (5-95%), ESF activity is detected in the first peak alone with the shortest time of its retention by the column. The results suggest that (1) by producing ESF, EC may regulate both B- and T-cell-mediated immune processes and (2) based on its physicochemical and biological characteristics, ESF can be distinguished from each of earlier characterised suppressor mediators of bone marrow origin.

Topics: Allergens; Animals; Animals, Newborn; Bone Marrow Cells; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cytotoxicity, Immunologic; Erythroblasts; Erythroid Cells; Erythropoietin; Humans; Immune Tolerance; Immunosuppressive Agents; Leukocytes, Mononuclear; Lipopolysaccharides; Liver; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; Neuraminidase; Peptide Hydrolases; Phenylhydrazines; Phospholipases; Spleen

Deformability and activity of the enzymes: acetylcholinesterase (AChE) and dehydrogenase glucose-6-phosphate (G-6-PD), were assayed for RBC enriched in immature reticulocytes. Reticulocytosis was evoked by administration of two different drugs: recombinant human erythropoietin (rHuEPO) and phenylhydrazine (PHZ) to two groups of Wistar rats. After treatment with the former compound, a group of animals exhibited 17.33% reticulocytes in blood whereas a group of rats treated with the latter drug reached 57.66% of these cells in blood. A marked decrease in RBC deformability was found in both groups of animals. AChE did not significantly change activity neither in PHZ-treated nor in rHuEPO-treated rats, whereas G-6-PD activity was significantly decreased in the PHZ-treated group.

Topics: Acetylcholinesterase; Animals; Erythrocyte Deformability; Erythropoietin; Glucosephosphate Dehydrogenase; Oxidants; Phenylhydrazines; Rats; Rats, Wistar; Recombinant Proteins; Reticulocytes

The hematopoietic, zinc-finger protein FOG-1 is essential for the development of the erythroid and megakaryocytic lineages. FOG-1's function in hematopoiesis is dependent on its ability to interact with the transcription factor GATA-1. FOG-1 has also been observed to interact with the corepressor molecule C-terminal binding protein (CtBP) through a peptide motif shared by all FOG family members. In this study, we confirmed that FOG-1 and CtBP interact by coimmunoprecipitation. We further demonstrate that a FOG-1 mutant unable to interact with CtBP has increased erythropoietic (but not megakaryocytic) rescue (relative to the wild type) of a FOG-1(-/-) cell line. To analyze further the physiological role of the FOG-1-CtBP interaction, we generated knock-in mice that express a FOG-1 variant unable to bind CtBP. Surprisingly, these mice are normal and fertile. Furthermore, erythropoiesis at all stages of development is normal in these mice. Erythrocyte production is similar in mutant and wild-type mice even under conditions of erythropoietic stress stimulated by either exogenously added erythropoietin or phenylhydrazine-induced anemia. Thus, despite conservation of the FOG-CtBP interaction site, the in vivo function of FOG-1 in erythroid development is not affected by its inability to interact with the corepressor CtBP.

Topics: Alcohol Oxidoreductases; Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Carrier Proteins; Cell Line; Conserved Sequence; COS Cells; DNA-Binding Proteins; Erythrocytes; Erythropoiesis; Erythropoietin; Genetic Vectors; Hematocrit; Mice; Mice, Knockout; Mutation; Nuclear Proteins; Phenylhydrazines; Phosphoproteins; Precipitin Tests; Protein Binding; Repressor Proteins; Sequence Homology, Amino Acid; Transcription Factors; Transgenes

The study was designed to examine the impact of anemia on the antitumor efficacy of photodynamic therapy (PDT) in a murine colon-26 adenocarcinoma model syngeneic with BALB/c mice.. Acute hemolytic anemia was induced by a single i.p. injection of phenylhydrazine hydrochloride (150 mg/kg). Anemia induced by i.p. administration of carboplatin (100 mg/kg) was corrected by s.c. treatment with recombinant human erythropoietin (1000 units/kg/day). The effectiveness of PDT (10 mg/kg Photofrin, 150 J/cm2 laser dose) was evaluated by measurements of the footpad edema and tumor volume. All of the RBC-related parameters were measured from the tail vein.. Phenylhydrazine hydrochloride injection resulted in a blunted response of normal tissues to Photofrin-mediated PDT-induced edema formation. Similarly, the antitumor response in mice with hemolytic anemia was nearly completely abrogated. The antitumor effectiveness of PDT was also significantly diminished in a more realistic clinical situation when anemia was induced by administration of carboplatin. Importantly, administration of recombinant human erythropoietin completely restored the sensitivity of the tumor to PDT in carboplatin-treated mice.. These results indicate that anemia can negatively influence the therapeutic effectiveness of PDT. For optimal antitumor response anemia should be corrected before PDT procedure.

Topics: Acute Disease; Anemia; Anemia, Hemolytic; Animals; Antineoplastic Agents; Carboplatin; Disease Models, Animal; Erythropoietin; Humans; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Phenylhydrazines; Photochemotherapy; Recombinant Proteins; Tumor Cells, Cultured

Gene expression profiles during erythropoietin (Epo)-induced differentiation of erythroid progenitor cells derived from the Friend virus anaemia (FVA) and phenylhydrazine (PHZ) murine models have been examined using differential display polymerase chain reaction (PCR). Ten cDNA fragments upregulated by Epo were isolated. The ribonuclease protection assay confirmed differential expression between Epo-stimulated and Epo-deprived cells for one of these, provisionally named ERIC-1. Sequencing of the full-length cDNA predicted a protein of 558 amino acids, 17 amino acids longer than mTACC3, the third member of a novel family of proteins that contain a coiled-coil domain. The human homologue, cloned using rapid amplification of cDNA ends (RACE)-PCR, encodes a larger protein of 838 amino acids that is identical to hTACC3. In addition to erythroid precursor cells, ERIC-1/TACC3 is expressed at high levels in the testes, at moderate levels in the thymus and peripheral leucocytes, and at lower levels in the spleen and intestinal tissue. Immunohistochemical analysis using an antibody to a GST fusion product of the C-terminus of hERIC-1/TACC3 revealed that it is localized to Sertoli cells in the human testes. Confocal microscopy demonstrated hERIC-1/TACC3 protein concentrated in the perinuclear vesicles of dermal microvascular endothelial cells. Although ERIC-1/TACC3 is expressed in a wide range of tissues, its upregulation by Epo in erythroid progenitors implies that it has a role in terminal erythropoiesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Gene Expression; Humans; Immunohistochemistry; Leukemia, Erythroblastic, Acute; Male; Mice; Microtubule-Associated Proteins; Molecular Sequence Data; Phenylhydrazines; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sertoli Cells; Tumor Cells, Cultured

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Topics: Anemia; Animals; Bone Marrow Cells; Cell Count; DNA-Binding Proteins; Erythroid Precursor Cells; Erythroid-Specific DNA-Binding Factors; Erythropoietin; Female; Flow Cytometry; GATA1 Transcription Factor; Gene Expression; Hematocrit; Hematopoietic Stem Cells; Immunohistochemistry; Male; Megakaryocytes; Mice; Mice, Inbred C57BL; Mutation; Phenylhydrazines; Platelet Count; Spleen; Thrombocytopenia; Transcription Factors

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

Topics: Anemia; Animals; Biological Transport; Cation Transport Proteins; CHO Cells; Cricetinae; Endosomes; Erythrocyte Membrane; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Fluorescent Antibody Technique; Heme; Hemoglobins; Homozygote; Iron; Iron-Binding Proteins; Leukemia, Erythroblastic, Acute; Mice; Mice, Mutant Strains; Microscopy, Confocal; Mutation; Phenylhydrazines; Protein Isoforms; Reticulocyte Count; Reticulocytes; Spleen; Transferrin; Tumor Cells, Cultured

Haptoglobin knockout (Hp-/-) mice are more sensitive to phenylhydrazine-induced hemolysis than Hp+/+ mice.. Hemolysis was induced in Hp-/- and Hp+/+ mice using phenylhydrazine. Relative renal tissue damage and function were then assessed.. Hp-/- mice had higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE). After the administration of phenylhydrazine, levels of 8-hydroxyguanine (but not other products of oxidative DNA damage) were significantly elevated in the renal DNA. There was also increased induction of heme oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of 3H-inulin. This reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced hemolysis. Precipitation of hemoglobin in the kidney was not increased in Hp-/- mice.. Haptoglobin appears to play an important physiological role as an antioxidant, particularly during hemolysis.

Topics: Acute-Phase Reaction; Aldehydes; Animals; Antioxidants; DNA; Erythropoietin; Gene Expression Regulation, Enzymologic; Guanine; Haptoglobins; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemoglobins; Hemolysis; Inulin; Kidney; Kidney Function Tests; Lipid Peroxidation; Liver; Malondialdehyde; Membrane Proteins; Mice; Mice, Knockout; Oxidative Stress; Phenylhydrazines; Tritium

The dynamics of gene expression during terminal erythroid differentiation have been examined in three murine models; the erythroleukaemia cell line HCD-57 and splenic erythroblasts isolated from mice treated with either the anaemia-inducing strain of Friend virus (FVA cells) or the haemolytic agent phenylhydrazine (PHZ cells). In response to erythropoietin (EPO) and haemin, HCD-57 cells proliferated and synthesized haemoglobin, but failed to complete terminal differentiation as indicated by lack of change in both gene expression and morphological appearance. In contrast, EPO-induced terminal differentiation in FVA and PHZ cells in vitro was accompanied by increases in haemoglobin positivity, morphological maturation and a shared pattern of gene expression. EPO receptor (EPO-R) mRNA levels peaked before globin gene expression which was maximal at 24 h. Peak GATA-1 and EKLF mRNA levels also preceded the globin gene peak, but the highest NF-E2 levels coincided with maximal globin levels, suggesting a role for NF-E2 in the maintenance, rather than the initiation of globin gene expression. Peak expression of delta-aminolaevulinic acid synthase (ALAS) coincided with peak globin expression. FVA and PHZ cells represent more effective models than the HCD-57 cell line for the investigation of erythroid gene expression during EPO-regulated terminal erythropoiesis.

Topics: Animals; Cell Size; Erythroblasts; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Gene Expression; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Phenylhydrazines; Tumor Cells, Cultured

Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis. COX-2 is inducible and is the major isoform of inflammatory cells. COX-2-deficient mice were shown to have normal basal hematopoiesis and hematology. We hypothesized that COX-2 induction plays a role in the recovery phase of 5-fluorouracil (5-FU) induced bone marrow injury, because significant macrophage-driven phagocytic removal of necrotic debris and stromal cell reorganization of repopulating marrow occur after 5-FU induction of bone marrow necrosis. Hematologic recovery was markedly delayed with moderately severe leukopenia, thrombocytopenia and reticulocytopenia compared to heterozygotes on day 8 or 12 in Cox-2-/- mice. Mild anemia was present in 5-FU-treated Cox-2-/- and Cox-2+/- mice on days 8 and 12, which was more severe in Cox-2-/- mice. Cox-2-/- mice had markedly decreased bone marrow cell counts per femur and reduced numbers of erythroid and myeloid colony-forming cells compared to heterozygote mice on days 8 and 12 post 5-FU. Histologic examination of 5-FU-treated Cox-2-/- mice revealed a failure to repopulate the intact marrow stroma with hematopoietic cells. Accelerated erythropoiesis following phenylhydrazine-induced hemolytic anemia, however, was comparable between Cox-2-/- and Cox+/- mice, as were induced levels of renal erythropoietin mRNA. COX-2 induction is likely a central event in the accelerated hematopoiesis following myelotoxic injury, because recovery from 5-FU-induced myeloablation is markedly impaired in Cox-2-/- mice but is normal after phenylhydrazine induction of anemia.

Topics: Animals; Bone Marrow; Bone Marrow Examination; Cell Count; Cyclooxygenase 2; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fluorouracil; Isoenzymes; Kidney; Leukopoiesis; Liver; Mice; Mice, Knockout; Phenylhydrazines; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Time Factors

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated by interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.

Topics: Anemia, Hemolytic; Animals; Erythropoietin; Hematocrit; Hematopoiesis; Hematopoietic Stem Cells; Kidney; Male; Mice; Phenylhydrazines; Spleen

The effects of cisplatin on erythropoietin (EPO) production were investigated in comparison with the effects of phenylhydrazine in rats. Cisplatin (4.5 mg/kg i.p. bolus) decreased red blood cell count (RBC), hematocrit (Hct) and hemoglobin concentration (Hgb) for 14 days after dosing. These decreases were accompanied with increases of blood urea nitrogen (BUN) and serum creatinine (s-CRE). Both serum EPO concentration and kidney EPO mRNA content were significantly decreased in cisplatin-treated rats. On the other hand, phenylhydrazine (10 mg/kg p.o. once a day for 8 days) decreased RBC, Hct and Hgb, and increased serum EPO concentration and kidney EPO mRNA content. Phenylhydrazine had little effect on BUN or s-CRE. These results suggest that a suppression of EPO production is involved in the pathophysiology of cisplatin-induced renal anemia and that measurement of serum EPO concentration and kidney EPO mRNA content is available for distinguishing renal anemia from hemolytic anemia.

Topics: Anemia; Animals; Antineoplastic Agents; Blood Urea Nitrogen; Cisplatin; Creatinine; Erythrocyte Count; Erythropoietin; Hematocrit; Hemoglobins; Immunoenzyme Techniques; Kidney; Kidney Diseases; Male; Phenylhydrazines; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic

Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells--an erythropoietin-responsive model progenitor cell line--to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17- to 18-fold and 14- to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11- to 48-fold and 20- to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Cell Differentiation; Cell Division; Cells, Cultured; Cytokines; Erythropoietin; Fibroblasts; Hematopoietic Stem Cells; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Models, Biological; Phenylhydrazines; Platelet-Derived Growth Factor; Recombinant Proteins; Spleen; Time Factors; Tumor Cells, Cultured

In a study to employ progenitor-derived clonogenic assays of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid colony-forming units (CFU-E) for the assessment of chemically induced hematotoxicity in rats, we sought the appropriate culture medium for each assay. Then, the effect of cyclophosphamide or phenylhydrazine on bone marrow cells was examined in vivo and in vitro. Oral treatment of rats with 25 mg/kg of cyclophosphamide significantly decreased the number of CFU-GM, which was an earlier and more sensitive index than were other hematological parameters tested. Direct exposure of the culture to cyclophosphamide had little effect on CFU-GM production, but addition of sera from rats pretreated with cyclophosphamide strongly depressed their formation, which suggested that the active metabolites of cyclophosphamide produced in the body may play an important role in toxicity. Subcutaneous injection of rats with 40 mg/kg of phenylhydrazine produced a marked increase in CFU-E 6-24 hr after injection. Direct exposure of the culture to phenylhydrazine had severe cytotoxic effects, but addition of sera from rats receiving phenylhydrazine resulted in increased numbers of CFU-E, probably indicative of increases in endogenous erythropoietin in donor rats. These results demonstrate that the proper use of progenitor assays in the in vivo and in vitro studies may be a valuable tool for approaching the mechanism underlying hematotoxicity.

Topics: Animals; Cells, Cultured; Colony-Forming Units Assay; Culture Media, Conditioned; Cyclophosphamide; Erythropoietin; Female; Hematopoietic Stem Cells; Phenylhydrazines; Rats; Rats, Inbred F344

Zinc metabolism and metallothionein induction in rat bone marrow were investigated during induced erythropoiesis. Redistribution of body zinc was measured with 65Zn after acute blood loss in rats fed zinc-restricted or zinc-adequate diets. Uptake of 65Zn by bone marrow was related to time after blood loss, metallothionein induction, and dietary zinc status. Increased 65Zn uptake by marrow of zinc-restricted rats suggests a minimal amount of zinc is necessary to support expansion of the erythrocytic compartment. Zinc induction of marrow metallothionein also occurred in rats in which anemia was produced using phenylhydrazine. Anemic rats which were administered zinc had higher concentrations of marrow metallothionein compared with control rats. Induction of marrow metallothionein by zinc in nonanemic rats required prior treatment with erythropoietin. Percoll fractionation showed marrow metallothionein was most abundant in erythroblasts. These experiments suggest metallothionein synthesis occurs in erythropoietin-sensitive precursor cells in the marrow in response to increased zinc accessibility.

Topics: Anemia, Hemolytic; Animals; Biological Transport; Bone Marrow; Erythropoiesis; Erythropoietin; Hematocrit; Hematopoietic Stem Cells; Humans; Liver; Male; Metallothionein; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Spleen; Zinc

The role of the submandibular salivary gland in erythropoiesis in the male mouse (MRC TO strain) was evaluated by subjecting mice without submandibular salivary glands (SX) and control (C) sham-operated mice to a variety of stimuli intended to stress the erythropoietic system. In SX mice, after removal of the submandibular glands at age 4 weeks and observation for 8 weeks, mean hematocrit was the same as in C mice, but mean body weight was less. Bilateral removal of the submandibular glands at age 6 weeks neither affected the rate of fall and subsequent recovery of hematocrit which followed treatment with phenylhydrazine (80 mg/kg intraperitoneally [i.p.] 9 days after operation) nor altered the rate of increase in hematocrit or change in body weight which occurred during hypobaric hypoxia (0.5 atm, > 23 hours/day) for 23 days. Mean (SEM) estimates of serum immunoreactive erythropoietin after 17 hours' continuous hypobaric (0.5 atm) exposure were not significantly different between SX [186 (30) mU/mL, n = 7] and C mice [232 (17) mU/mL, n = 7]. In mice given bilateral nephrectomies at age 6 weeks--2 weeks after SX or C surgery--and then both treated with phenylhydrazine (60 mg/kg i.p.) and exposed for 17 hours to hypobaric (0.5 atm) hypoxia, mean estimates of serum immunoreactive erythropoietin were 22.6 (10.6) mU/mL and 22.3 (5.4) mU/mL in SX (n = 5) and C (n = 5) mice. Results of the study do not support the premise that the submandibular salivary glands either contribute to the erythropoietic response or are a source of extrarenal erythropoietin.

Topics: Animals; Atmospheric Pressure; Body Weight; Erythropoiesis; Erythropoietin; Hematocrit; Hypoxia; Male; Mice; Phenylhydrazines; Submandibular Gland

Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross-linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.

Topics: Animals; B-Lymphocytes; Cell Line; Cross-Linking Reagents; DNA; Erythropoietin; Gene Expression; Immunoblotting; Interleukin-3; Kinetics; Mice; Phenylhydrazines; Phosphorylation; Phosphotyrosine; Receptors, Erythropoietin; RNA, Messenger; Signal Transduction; Transfection; Tyrosine

The proto-oncogene c-myc has been identified as an early response gene for erythropoietin (Epo) in transformed murine erythroleukemia cells. Epo activation of c-myc in these cells requires protein kinase C. We now show the fidelity of this signaling pathway in normal erythroid cells isolated from the spleens of phenylhydrazine-treated mice. Mouse spleen cells rich in erythroid progenitors were washed free of endogenous Epo and then incubated in the absence of Epo. Subsequent addition of Epo for 1 hour led to a dramatic elevation of c-myc transcript. Addition of the protein synthesis inhibitor cycloheximide did not prevent the c-myc response, thus identifying c-myc as an Epo early response gene in normal cells. We used this c-myc response as a reporter for signals initiated by the Epo receptor. Using a series of inhibitors with known specificities and established rank-orders of potency for different kinases, we determined that the c-myc response to Epo was blocked with the following rank order: staurosporine much greater than H7 greater than sangivamycin greater than H8. This sequence is identical to that obtained using transformed cells and is diagnostic of a protein kinase C-dependent signal. Because direct activation of protein kinase by phorbol esters does not induce terminal differentiation of normal cells, the pathway to c-myc established by these studies must represent one part of a signal transduction mechanism.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Erythroid Precursor Cells; Erythropoietin; Gene Expression; Genes, myc; Isoquinolines; Mice; Mice, Inbred C57BL; Phenylhydrazines; Piperazines; Protein Kinase C; Pyrimidine Nucleosides; Signal Transduction; Spleen; Staurosporine; Transcription, Genetic

The metabolic fate of erythropoietin (EPO) remains unknown. Urinary excretion does not appear to play a major role and liver catabolism has been shown to occur only after terminal sugars on the hormone have been removed. However, it has been proposed that EPO is eliminated by consumption in the bone marrow. In order to examine the extent of such consumption we measured the half-life of radioidinated recombinant EPO injected intravenously (IV) to rats with bone marrows suppressed by cyclophosphamide or hypertransfusion and marrows stimulated by phenylhydrazine or bleeding. The mean half-life or erythropoietin in normal rats was 179 +/- 16 min, with similar half-lives found in the other rats regardless of decreased or increased bone marrow activity. The results indicate that it is unlikely that erythroid activity determines EPO life span and catabolism.

Topics: Animals; Bone Marrow; Cyclophosphamide; Erythropoietin; Half-Life; Hemorrhage; Hyperplasia; Injections, Intravenous; Male; Phenylhydrazines; Rats; Rats, Inbred Strains; Recombinant Proteins

The present study was undertaken to determine plasma EPO levels in response to acute exposure to hypobaria in transfused-polycythemic mice previously exposed to different forms of erythropoietic stimulation. The erythrocyte production rate (EPR) was stimulated by one of the following conditions: (1) discontinuous exposure to 456 mb during 4 weeks, (2) one weekly injection of phenylhydrazine (PHZ) during 4 weeks for induction of a compensated hemolytic state, and 3) three weekly injections of rHuEPO at a dose level of 1500 U/Kg body weight for 4 weeks. Treated and control mice received different volumes of packed red cells in order to obtain hematocrit values around 65% in all groups. Mice from each group were exposed to 456 mb during 14 h, 4 days after transfusion. Mice were bled immediately through cardiac puncture after removal from the hypobaric chamber. Plasma EPO titer was estimated by radioimmunoassay. Two observations were worth noting: (1) plasma iEPO level was very low in mice with transfusion polycythemia exposed to hypobaria, the value of 12 +/- 1.3 mU/ml being significantly different from that of 180 +/- 18 mU/ml found in normocythemic mice similarly exposed, and (2) plasma iEPO levels during EPRs were previously stimulated by different conditions (hypobaria, PHZ and rHuEPO) were at least as high as those found in normocythemic mices similarly exposed. All these values were significantly higher than the value corresponding to polycythemic mice whose EPRs were not stimulated before exposure to hypobaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atmosphere Exposure Chambers; Atmospheric Pressure; Blood Transfusion; Erythropoiesis; Erythropoietin; Female; Hematocrit; Mice; Phenylhydrazines; Polycythemia; Recombinant Proteins; Secretory Rate

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.

Topics: Anemia; Animals; Autoradiography; Bloodletting; Bone Marrow; Cross-Linking Reagents; Erythropoietin; Female; Hematopoietic Stem Cells; Iodine Radioisotopes; Kinetics; Phenylhydrazines; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Erythropoietin

The polycythemia-inducing strain of the Friend spleen focus-forming virus (SFFVP) induces an acute erythroleukemia in mice. Erythroid cells from these mice differ from normal erythroid cells in that they can proliferate and differentiate in the apparent absence of the erythroid hormone erythropoietin (Epo). Although it was recently shown that the unique envelope protein encoded by SFFV is responsible for altering the hormonal requirements of erythroid cells for growth and differentiation, the mechanisms by which this occurs is not known. Since the SFFV envelope protein appears to interact with a target present only in erythroid cells and since Epo is specific for these cells, it is possible that the virus is exerting its effect through this hormone. In an effort to ascertain if this is the case, we examined cells from SFFVP-infected mice to determine (a) if they produce Epo or other erythroid growth factors that stimulate erythroid cells to grow in an autocrine-like manner and (b) if they express elevated numbers of Epo receptors that may result in a reduced requirement for the level of Epo needed for growth and differentiation. Our results indicate that SFFVP-infected cells do not secrete Epo or any other erythroid growth factors that could account for the reduced hormonal requirements of these cells. Also, our studies using iodinated Epo in cell binding assays and cross-linking studies indicate that SFFVP-infected cells are not significantly different from normal erythroid cells in the number, affinity, or size of their Epo receptors.

Topics: Animals; Erythropoiesis; Erythropoietin; Leukemia Virus, Murine; Mice; Phenylhydrazines; Polycythemia; Receptors, Cell Surface; Receptors, Erythropoietin; RNA, Messenger; Spleen; Spleen Focus-Forming Viruses; Viral Envelope Proteins

A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep). A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions. Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters. After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with 125I-labeled human recombinant Ep (125I-rEp), and nitrocellulose bound radioactivity was determined thereafter. Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found. Specific binding was saturable, reaching a plateau at 2.5 nM. Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells. No appreciable binding of 125I-rEp to control membranes at low Ep-receptor content was observed. Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of 125I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion. The technique described herein may be of use in the study of the Ep receptor and as an assay for its purification. Moreover, it may also be of general application in the study of receptor-ligand interactions.

Topics: Anemia; Animals; Biomarkers; Collodion; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Humans; Iodine Radioisotopes; Male; Mice; Microchemistry; Phenylhydrazines; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Spleen

A mathematical model of erythropoietic cell production and its regulation process has been proposed in a preceding paper. It is primarily based on the assumption that the number of cell divisions taking place in the CFU-E and erythropoietic precursor stages is regulated depending on the oxygen supply of the tissue. Quantitative dose-response relationships for in vivo erythropoiesis are suggested. Here, we demonstrate that this model adequately reproduces data obtained in situations of stimulated erythropoiesis in mice and rats. In detail, this implies a quantitative description of the following processes: (1) Changes in tissue oxygen tension (Pto2) following removal of red cells (bleeding, haemolytic anaemia) or increase in plasma volume (dilution anaemia) or decrease in atmospheric oxygen pressure (hypoxia). (2) Pto2 dependent erythropoietin (EPO) production. (3) Dose-response of EPO on erythropoietic amplification (up to two to four additional mitoses). (4) The changes of the marrow transit time. Model simulations are compared with experimental data for changes of erythropoiesis during hypoxia, EPO-injection, and different forms of anaemia. A satisfactory agreement suggests that the model adequately describes and correlates different direct and indirect ways to stimulate erythropoiesis. It quantifies the role and relative contribution of the haematocrit, haemoglobin concentration, atmospheric oxygen pressure, tissue oxygen pressure, and plasma volume as triggers in erythropoietic stimulation under various conditions. Furthermore, the model may allow to optimize the scheme of EPO-administration and to find the maximum increase of erythropoiesis for a given amount of erythropoietin.

Topics: Anemia; Animals; Blood Volume; Erythropoiesis; Erythropoietin; Hematocrit; Hematopoietic Stem Cells; Hemorrhage; Hypoxia; Mice; Phenylhydrazines; Rats; Reticulocytes

The simple, specific and sensitive erythropoietin bioassay in serum and plasma from phenylhydrazine treated mice is described, based on H3-thymidine incorporation into divided hemopoietic cells. Spleen cells taken from mice on the third day following the second of 2 daily injections of phenylhydrazine were cultured in 24 hours in the presence of test material. Following incubation for 2 hours with H3-thymidine solution radioactivity was measured.

Topics: Anemia; Animals; Biological Assay; Cells, Cultured; Erythropoietin; Female; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Phenylhydrazines; Spleen

Erythropoiesis is regulated by the glycoprotein hormone erythropoietin (Epo) and by several other factors including interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor. The possibility that IL-3 and GM-CSF may act by modulating Epo receptor expression was investigated using erythroblasts purified from the spleens of phenylhydrazine-treated mice. AT 37 degrees C, in the presence of sodium azide to inhibit receptor internalization. 125I-labeled human recombinant Epo bound to a single class of high-affinity receptors on splenic erythroblasts (450 sites/cell, Kd = 700 pM). Autoradiographic studies indicated that 94% of specifically bound Epo was associated with erythroblasts, decreased Epo binding being observed with increasing erythroid cell maturation. Whereas recombinant mouse IL-3 and GM-CSF did not compete with 125I-Epo for binding to the Epo receptor, preincubation of cells with IL-3 resulted in a concentration-dependent loss of 125I-Epo binding without altering the affinity of residual receptors for Epo. Complete loss of Epo receptors was effected within 2 h at IL-3 concentrations above 2500 U/ml. Preincubation with recombinant mouse GM-CSF had no effect on binding, even at 100,000 U/ml. In comparison, preincubation of cells with Epo (50 U/ml) caused complete loss of 125I-Epo binding within 30-60 min, an effect not explained by receptor saturation with unlabeled Epo. Thus, in addition to trans-down-modulating growth factor receptors of the granulocyte-macrophage series, IL-3 also trans-down-modulates a growth factor receptor of the erythroid lineage.

Topics: Animals; Binding, Competitive; Colony-Stimulating Factors; Erythroblasts; Erythropoietin; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Interleukin-3; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Phenylhydrazines; Receptors, Cell Surface; Receptors, Erythropoietin; Spleen

Rauscher murine erythroleukemia cells grow continuously in vitro and undergo terminal differentiation in response to the physiological inducer erythropoietin. In the course of this developmental process they express many erythroid-specific markers. In order to investigate the expression of cell surface determinants during Rauscher cell differentiation we generated monoclonal antibodies to uninduced cells. Using an anti-Rauscher cell monoclonal antibody, we have identified a cell surface determinant, designated ERY-1, that is present on normal murine erythroid cells. This determinant is apparently absent from the early progenitor BFU-E, but is present on the more mature progenitors CFU-E and CFC-E. It disappears during erythroid maturation and is absent from the mature erythrocyte. This pattern of ERY-1 expression is exhibited with remarkable fidelity during the erythropoietin-induced differentiation of Rauscher cells. Such differentiation-specific expression of the ERY-1 determinant suggests that it may play a functional role in erythropoiesis.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Surface; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Erythrocytes; Erythropoiesis; Erythropoietin; Flow Cytometry; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Mice; Phenylhydrazines; Spleen; Time Factors

The reproducibility and utility of an in vitro bioassay for erythropoietin were evaluated, and modifications, including the use of frozen target cells, were made. The assay, based on 3H-thymidine incorporation into phenylhydrazine-treated mouse spleen cells, showed a dose-response curve from 0.2 to 20 mU of sheep plasma or human urinary erythropoietin, with a 50-100-fold increase in 3H-thymidine incorporation. The dose-response curve, using purified, recombinant erythropoietin was identical to that from material obtained from other sources. The use of frozen cells simplified the methodology, reduced the time for assay preparation, and lowered costs. The freezing process resulted in 15% attrition of the cells, but after one year of storage, this level was not increased nor was the response to erythropoietin diminished. The nonspecificity of the assay was reduced by supplementing the cells with critical nutrients, depleting fetal bovine serum of endogenous erythropoietin, and reducing solute variability by ultrafiltration and reconstitution of samples with culture medium. Heat inactivation diminished inhibitors in sera samples, but affinity chromatography using wheat-germ lectin-Sepharose was most effective for extracting erythropoietin and reducing nonspecific effects. The assay is sensitive, can be used to measure multiple samples rapidly, and should prove useful for complementing immunoassay techniques.

Topics: Animals; Biological Assay; Cells, Cultured; Dose-Response Relationship, Drug; Erythropoietin; Freezing; Hot Temperature; Humans; In Vitro Techniques; Mice; Phenylhydrazines; Sheep; Spleen; Thymidine

The hemin regulation of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid (ALA) biosynthesis in mammalian system was studied in different conditions: phenylhydrazine induced anemia, polycythemia by erythropoietin to anemic rats, treatment with cobalt chloride, a porphyrogenic drug. The activity of L- alanine:4,5-dioxovalerate transaminase in liver and kidney is stimulated in phenylhydrazine, whereas, erythropoietin injection to anemic rats prevents such stimulation. Further treatment with cobalt chloride to erythropoietin treated anemic rats stimulates the enzyme activity. Actinomycin D, however, inhibits the stimulation of L-alanine:4,5-dioxovalerate transaminase by phenylhydrazine suggesting that induction is at the level of transcription. Induced level of this enzyme in anemic condition was estimated quantitatively by radial immunodiffusion using antibody raised against L-alanine:4,5-dioxovalerate transaminase. Moreover, our studies reveal that stimulation of L-alanine:4,5-dioxovalerate transaminase in anemic condition is dependent on depletion of heme level. The regulatory role of intracellular heme pool on the induction of this enzyme suggests its physiological importance in heme biosynthesis.

Topics: Anemia; Animals; Cobalt; Dactinomycin; Erythropoietin; Hematocrit; Heme; Immune Sera; Immunodiffusion; Kidney; Kinetics; Liver; Male; Phenylhydrazines; Rats; Rats, Inbred Strains; Transaminases

A compensated hemolytic state was induced in rats by injection of phenylhydrazine (PHZ) over a 6-week period. The liver and kidney were perfused to determine the levels and time of appearance of a hepatic erythropoietic factor (HEF) which induces the production of hepatic erythropoietin (Ep) and its antagonist, a renal inhibitory factor (RIF). Erythropoietin assays on the perfusates have been previously reported. The amount of HEF in perfusates recovered from the livers of the PHZ-treated rats was significantly higher during the 4th to 5th weeks of treatment, coinciding with the time of increased liver production of Ep. During the 6th week of PHZ treatment, the titer of RIF in perfusates recovered from the kidney was markedly increased and the HEF titer was decreased to near the control level, suggesting inhibitory action of RIF on HEF synthesis and/or effect. These findings indicate that an HEF/RIF control mechanism regulates hepatic Ep production in this compensated hemolytic condition.

Topics: Animals; Blood Proteins; Erythropoietin; Female; Hemolysis; Hepatocyte Growth Factor; Hypoxia; Kidney; Liver; Male; Mice; Perfusion; Phenylhydrazines; Rats; Time Factors

Serum obtained from (C3H X C57BL) F1 hybrid mice injected with parental (C57BL) lymph node cells after 600 rad irradiation contained a high level of an erythropoietic factor, which enhanced erythroid colony formation by fetal C57BL liver cells in methylcellulose cultures. The serum level of this erythroid colony-stimulating activity increased as the dose of lymph node cells injected increased and as suppression of endogenous spleen colonies (indicative of GVH severity) became more marked. Erythropoietin was also elevated in the sera of these mice. When these animals were transfused with washed packed red cells to induce plethora after sublethal irradiation and injection of lymph node cells, the erythropoietin level decreased, but erythroid colony-stimulating activity remained elevated. On the other hand, in anemic mice treated with phenylhydrazine, this erythropoietic factor was not detected, although the serum level of erythropoietin was elevated. These results suggest that a graft-versus-host (GVH) reaction may induce the production of an erythropoietic factor other than erythropoietin and that anemia itself may not be a stimulus for its in vivo production.

Topics: Anemia; Animals; Blood Transfusion; Cells, Cultured; Colony-Forming Units Assay; Colony-Stimulating Factors; Erythrocytes; Erythropoietin; Graft vs Host Disease; Hematopoietic Stem Cells; Liver; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Phenylhydrazines; Spleen

Effect of Ep on [14C]acetate incorporation into different lipid fractions of RBC membranes in starved and phenylhydrazine-treated rats was studied. The incorporation was increased into both neutral and phospholipid fractions on Ep treatment to starved or phenylhydrazine-treated rats. A slight decrease in the ratio of neutral lipid to phospholipid was observed under the influence of Ep in starved rats (23%) or in phenylhydrazine-treated rats (36%). Incorporation of radioactivities into different phospholipid fractions of RBC membrane increased on Ep treatment to starved rats, whereas, the relative percentages of these phospholipids (except LPC) remained more or less unchanged under similar conditions. Phenylhydrazine treatment increased the relative percentage of PC and concomitantly decreased the percentage of Sph. Percentage composition of both these two phospholipids showed a tendency to return to their normal levels on administration of Ep to phenylhydrazine-treated rats. Ep decreased the sigma saturated/sigma unsaturated ratio of fatty acids in PE, PS, and PC of RBC membrane in starved rats. On the other hand, no significant change was observed in this ratio of fatty acids in the phospholipids except Sph of RBC membrane in the presence of phenylhydrazine and Ep. In Sph, the ratio went down under similar conditions.

Topics: Acetates; Animals; Chemical Phenomena; Chemistry; Cholesterol; Erythrocyte Membrane; Erythropoietin; Fatty Acids; Fatty Acids, Unsaturated; Membrane Lipids; Phenylhydrazines; Phospholipids; Rats; Starvation

We have compared the response of mice heterozygous for the Slj allele-which have a stromal defect resulting in mild macrocytic anemia-and their normal +/+ littermates to manipulations of the erythron to further characterize the effects of the Slj allele on hemopoiesis. CFU-S kinetics in Slj/+ mice following plethorization or induction of anemia did not differ from similarly treated +/+ littermates. Under all circumstances, however, Slj/+ mice had a smaller splenic CFU-S population than similarly treated +/+ mice. Slj/+ and +/+ mice responded similarly to induction of hemolytic anemia with a large rise in 59Fe incorporation in spleen and blood. Hypertransfused Slj/+ mice were shown to have a diminished response to exogenous erythropoietin in spleen and blood as compared with their +/+ littermates. We conclude that the regulation of CFU-S kinetics in mutant Slj/+ mice is not anomalous as has been reported to be the case in Sl/Sld mice. The defective stromal tissue of Slj/+ mice, however, does not support a normal-sized CFU-S population, which leads to decreased influx of progenitor cells into the erythropoietin-responsive cell compartment, causing the diminished response of plethorized Slj/+ mice to exogenous erythropoietin.

Topics: Anemia, Hemolytic; Anemia, Macrocytic; Animals; Blood Transfusion; Bloodletting; Bone Marrow; Erythropoietin; Female; Hematopoiesis; Hematopoietic Stem Cells; Male; Mice; Mice, Mutant Strains; Phenylhydrazines; Polycythemia; Spleen

The effects of pure and crude human urinary erythropoietin, crude sheep plasma erythropoietin and other growth factors on the incorporation of labeled thymidine were studied using spleen cells from mice previously treated with phenylhydrazine hydrochloride. Erythropoietin at 400 mU/ml caused a 40-80 fold increase in the incorporation of the labeled nucleoside. The slope of the dose-response curve found for pure erythropoietin was not significantly different from that found for a crude urinary erythropoietin preparation or for crude sheep plasma erythropoietin. Colony-stimulating factor, interleukin 2, interleukin 3 and the lectin, concanavalin A were also stimulatory but at concentrations from one hundred to one million times higher than that found for erythropoietin.

Topics: Animals; Colony-Stimulating Factors; Concanavalin A; DNA; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Female; Humans; Interleukin-2; Mice; Mice, Inbred Strains; Phenylhydrazines; Spleen

Spleens taken from B6C3F1 mice on the third day following the second of 2 daily injections of phenylhydrazine HCl (60 mg/kg) contain approximately 10 times the number of cells (i.e., 10(9) cells) found in spleens from normal mice. More than 90% of these spleen cells are recognizable erythroid and form the basis of a new in vitro microassay for erythropoietin (Ep) which uses 3H-thymidine incorporation as an endpoint. This assay takes 24 h and can be carried out in the presence of either 20% FCS or a well-defined serum substitute. Under the conditions used, Ep levels from 30 mU/well down to as little as 0.2 mU/well can be accurately measured with a corresponding variation in counts of 50-100-fold. One spleen is sufficient for 1000 triplicate Ep determinations, and the use of this microassay procedure requires only very small (1-60) microliters) samples for evaluation. Preliminary studies with human plasma suggest that this assay may be more specific for Ep than established in vitro 59Fe bioassay methods.

Topics: Animals; Biological Assay; Cells, Cultured; Colony-Forming Units Assay; Erythropoietin; Fetal Blood; Humans; Immune Sera; Kinetics; Mice; Mice, Inbred Strains; Phenylhydrazines; Rabbits; Spleen; Thymidine

Synchronized erythroid precursors obtained from the bone marrow of rabbits and plated in methyl-cellulose were used as a bioassay for the measurement of erythropoietin (Ep). Rabbits were given five daily injections of phenylhydrazine followed by a single dose of actinomycin-D. This treatment resulted in a rapid repopulation of bone marrow by synchronized erythroid precursors which can be stored at -180 degrees C for long periods. Grown in vitro for 2 days in the presence of added Ep these cells divided to form colonies (CFUE). The erythroid nature of these colonies was confirmed by 59Fe incorporation into heme. Preliminary studies indicate that this system is suitable for the measurement of Ep in human sera. It is simple, inexpensive, reproducible, and permits measurements at the physiologic range of Ep concentrations.

Topics: Animals; Bone Marrow Cells; Cell Aggregation; Cell Division; Colony-Forming Units Assay; Dactinomycin; Dose-Response Relationship, Drug; Erythrocytes; Erythropoiesis; Erythropoietin; Hemolysis; Phenylhydrazines; Rabbits

Erythropoietin production in response to hypoxic-hypoxia is markedly reduced in the newborn when compared to the adult rat. This response improves steadily with age and reaches adult values at about 4 wk. When animals of the same age are stimulated with anemic-hypoxia, considerably higher levels of erythropoietin are found. The erythropoietin level is proportional to the degree of anemia and independent of the age of the animal. Extraction of erythropoietin from tissue homogenates revealed a parallelism between the plasma and kidney erythropoietin content, while no erythropoietin could be extracted from liver tissue at any age. The lack of response to hypoxia in the newborn appears to be related to the high hemoglobin oxygen affinity during the neonatal period, which facilitates oxygen loading. Newborn rats have a very low intraerythrocytic concentration of 2-3 DPG and a marked shift to the left in the oxygen hemoglobin dissociation curve that slowly increases to adult values at 4 wk of age. The response to anemia on the other hand, appears to be normal and not affected by age or by hemoglobin oxygen affinity. These studies suggest that the newborn rat, when properly stimulated, is able to produce normal amounts of erythropoietin, most likely renal in origin.

Topics: Anemia; Animals; Animals, Newborn; Diphosphoglyceric Acids; Erythrocytes; Erythropoietin; Hypoxia; Kidney; Liver; Phenylhydrazines; Rats

Employing the plasma clot culture system, the effects of anemia or plethora on 10-day-old neonatal rat bone marrow and splenic erythrocytic colony-forming units (CFU-E) were examined. Anemia induced by phenylhydrazine resulted in a decreased CFU-E population in the bone marrow and dramatically increased CFU-E numbers in the spleen. Transfusion-induced plethora increased CFU-E numbers in both the marrow and the spleen. These in vitro findings lend support to studies performed in vivo and further suggest that regulation of erythropoiesis in the neonatal rat differs from that observed in the adult.

Topics: Anemia; Animals; Animals, Newborn; Bone Marrow Cells; Cells, Cultured; Colony-Forming Units Assay; Erythrocytes; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Phenylhydrazines; Rats; Reticulocytes; Spleen

Topics: Animals; Cell Separation; Erythrocytes; Erythropoietin; Female; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Phenylhydrazines; Spleen; Time Factors

Topics: Adrenocorticotropic Hormone; Animals; Epoetin Alfa; Erythropoiesis; Erythropoietin; gamma-Globulins; Humans; Hydrocortisone; Immune Sera; Iron Isotopes; Kidney; Liver; Luteinizing Hormone; Mice; Neutralization Tests; Phenylhydrazines; Rabbits; Renin; Research; Sheep; Spleen; Thyroxine

Topics: Adaptation, Physiological; Altitude; Anemia; Biochemical Phenomena; Biochemistry; Epoetin Alfa; Erythropoietin; Hypoxia; Iron Isotopes; Kidney; Phenylhydrazines; Rats; Research; Serum Albumin; Serum Albumin, Radio-Iodinated

Topics: Animals; Blood Cell Count; Blood Platelets; Eosinophils; Epoetin Alfa; Erythrocyte Count; Erythropoiesis; Erythropoietin; Leukocyte Count; Lymphocytes; Mice; Monocytes; Phenylhydrazines; Polycythemia; Rats; Research

Topics: Anemia; Animals; Epoetin Alfa; Erythrocyte Count; Erythropoietin; Hemoglobinometry; Hemolysis; Mice; Phenylhydrazines; Physiology; Polycythemia; Rabbits; Research; Toxicology

Topics: Animals, Newborn; Autoradiography; Bone and Bones; Epoetin Alfa; Erythropoietin; Iron; Iron Isotopes; Pharmacology; Phenylhydrazines; Rats; Research; Tibia; Urine

Topics: Anemia; Animals; Bone Marrow Transplantation; Epoetin Alfa; Erythropoietin; Phenylhydrazines; Plasma; Rabbits; Radiation Injuries; Radiation Injuries, Experimental; Rats; Research

Topics: Epoetin Alfa; Erythropoietin; Phenylhydrazines; Protein Deficiency; Rats; Research; Reticulocytes

Topics: Anemia; Anemia, Hemolytic; Animals; Biomedical Research; Bone and Bones; Bone Marrow; Epoetin Alfa; Erythropoiesis; Erythropoietin; Hematocrit; Hemoglobinometry; Hemorrhage; Humans; Iron Isotopes; Liver; Male; Pharmacology; Phenylhydrazines; Physiology, Comparative; Rabbits; Rats; Research; Spleen

Topics: Angiotensins; Epoetin Alfa; Erythropoiesis; Erythropoietin; Juxtaglomerular Apparatus; Kidney; Kidney Glomerulus; Nephrectomy; Pharmacology; Phenylhydrazines; Physiology; Rats; Research

Topics: Adrenocorticotropic Hormone; Animals; Antigen-Antibody Reactions; Ceruloplasmin; Cobalt; Epoetin Alfa; Erythropoiesis; Erythropoietin; Freund's Adjuvant; gamma-Globulins; Hematocrit; Hydrocortisone; Immune Sera; Immunization; Iron; Iron Isotopes; Mice; Mucoproteins; Neuraminic Acids; Neutralization Tests; Phenylhydrazines; Polycythemia; Rabbits; Research; Thyroxine; Transferrin; Urine

Topics: Anemia; Anemia, Hemolytic; Animals; Biomedical Research; Bone Marrow; Bone Marrow Examination; Epoetin Alfa; Erythropoiesis; Erythropoietin; Humans; Iron Isotopes; Male; Phenylhydrazines; Rabbits; Radiography; Radioisotopes; Radionuclide Imaging; Rats; Research; Skeleton

Topics: Cell Division; Colchicine; Dietary Proteins; Epoetin Alfa; Erythroblasts; Erythropoietin; Mitosis; Phenylhydrazines; Protein Deficiency; Reticulocytes

Topics: Animals; Epoetin Alfa; Erythropoietin; Hemorrhage; Phenylhydrazines; Sheep

Topics: Anemia; Blood Chemical Analysis; Bone Marrow; Dietary Proteins; Epoetin Alfa; Erythrocytes; Erythropoietin; Hemoglobins; Hemolysis; Iron; Iron Isotopes; Liver; Phenylhydrazines; Protein Deficiency; Rats; Research; Reticulocytes

Topics: Anemia; Animals; Biological Assay; Epoetin Alfa; Erythropoietin; Guinea Pigs; Hematocrit; Hypoxia; Iron Isotopes; Pharmacology; Phenylhydrazines; Polycythemia; Rabbits; Research