Induction of Micronuclei, Base-pair Substitution Mutation and Excision-repair Deficient by Polluted Water from Asa River in Nigeria

Asa river is a major river designated to supply millions of people of Ilorin, Kwara State, Nigeria potable water for drinking but its managements is of grave concern due to anthropogenic activities. Thus, evaluation of genotoxicity of this river was carried out by subjecting the water samples and fish therein to three bioassays (Micronucleus (MN) assay, Ames test and SOS-chromo test). Physicochemical parameters and heavy metals were analysed at three different stations (Aliara (SI), Unity (SII) and Tuyil (SIII)) of the river. In SII, most of the heavy metals analysed were above the acceptable limits compare to SI and SIII. The peripheral erythrocyte of the fishes (Oreochromis niloticus, Synodontis batensoda, Synodontis eupterus, Clarias gariepinus and Clarias angullaris) at SI and SII stations showed a significant (p<0.05) induction of MN and different nuclear abnormalities (NA). Water samples from the three stations subjected to Ames test (Salmonella typhimurium TA100) and SOS chromotests (Escherichia coli PQ37) at 25%, 50% and 100% concentrations showed statistically significant (p<0.05) induction of DNA damage at all concentrations in the two tester strains, thus indicating base-pair substitution mutation and excision-repairdeficient, respectively, by the water samples. Therefore, drinking of this water and/or consumption of fish from this river should be taken with caution to avoid a carcinogenic risk.

Decrease of Peripheral Monocyte Relative Number and Mean Platelet Volume in Iron Overloaded Mice

Iron overload (IO) because of multiple blood transfusion as a definite therapy for hematological disease with chronic and severe anemia has become a major concern. Deleterious complication contributed by chemically reactive deregulated iron may affect cellular homeostasis systemically lead to tissue and organ damage. When this toxicity occurred in blood cells, alteration of peripheral hematological profile concerning erythrocyte, leucocyte, and platelet most likely to be modified and imperatively need to be evidenced. The experimental IO mice model was established by injecting a low and high dose of iron dextran intraperitoneally. Peripheral erythrocyte, leucocyte and platelet indices measured by hematology analyzer were analyzed. A dynamic tendency of leucocyte absolute cell number and differential cell count of low and high dose iron treatment and a significant decrease of differential monocyte count were found. In addition, high dose iron treatment showed a significantly lower mean platelet volume. In conclusion, this study verified that IO impaired the cellular hematological indices by selectively suppress monocyte number addressing that this mononuclear phagocyte was the most affected immune cell. Furthermore, low mean platelet volume following acquired platelet function defect was evidenced. This research provided an animal experimental model that could be used for further study in finding alternative therapeutic targets on the pathophysiology of iron overload diseases, such as thalassemia.

Splenomegaly with Increased Erythroblast Numbers During Erythropoietic Stress Caused by Ambient Low-Temperature in Mice.

Abstract 4602 Erythropoietic stress such as hypoxia has been well described in mammals. These conditions decrease oxygen supply and then enhance production of erythropoietin (EPO) regulates production of red blood cells. Moreover, spleen becomes the main organ of erythropoiesis due to limited marrow space. Here, we describe a new erythropoietic stress, ambient low-temperature. It has been reported that peripheral blood cell counts are affected by ambient low-temperature in several vertebrates. Cold-acclimated rat and chicken exhibit polycythemia (Dveci et al, J Comp Physiol, 2001; Yahav S, Poult Sci, 1997). The response is considered to increase demand of tissues for oxygen, and then enhance metabolic rate and capacity for heat production to acclimate to ambient low-temperature. However, the physiological mechanisms had not been investigated. First, we examined peripheral erythrocyte levels in C57BL/6 mice putting into 5°C ambient. Hematocrits increased from 48% to a plateau of 53% after fourteen days. Likewise, hemoglobin concentration, initially 15 g/dl, rose to 17 g/dl. Reticulocyte production index significantly increased from 4% to 8% after seven days. These data suggested that mice exposed to low-temperature enhanced production of erythrocytes, so we next examined the anatomy and cell composition of their spleens. On day 5, spleens were about 6 mg/g of body weight, two-fold those on day 0. They gradually decreased to their initial weights on day 14. Flow cytometry showed 38% more Ter119+ splenocytes and four-fold more CD71 high Ter119+ early erythroblasts than normal. These values also gradually declined to their initial numbers by day 14. The results suggested the elevated red blood cell counts were due to an increase in production. To test erythropoietic activity in serum, we used an erythrocyte colony-forming assay. Serum from mice kept at low-temperature showed no ability to stimulate CFU-E colony formation in vitro. However, a combination of the serum with EPO (0.5 U/ml) increased CFU-E numbers 1.5 to 2 times that of a combination of normal serum plus EPO or EPO alone. Whether inducible factors account for these effects is the focus of future investigation. Our findings suggest a low-temperature environment is an erythropoietic stress that may offer insights into the adaptive physiology of red blood cell production in mice. Disclosures: No relevant conflicts of interest to declare.

A New Animal Model for Anemia Induced by Environmental Low-Temperature: Physiology of Erythrocyte Production and Circulation

To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.

PBI-1402: A Non-Toxic Immunorestorative Small Molecule for the Treatment of Anemia.

Erythropoiesis is regulated by an intricated network of transcription factors and other molecules that mediate differentiation of stem cell progenitors into erythroid lineage. PBI-1402 is a non-toxic, well-defined chemo- and radio-protective orally active small molecule which stimulates erythropoiesis. In vivo studies demonstrate that PBI-1402 has an immunorestorative effect in anemia induced by phenylhydrazine or lethal irradiation. In phenylhydrazine-induced anemia, PBI-1402 treated mice had an increased number of hematopoietic progenitor cells compared to the control mice. This increase was more pronounced in the erythroid lineage (BFU-E and CFU-E). In myeloablated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count and hemoglobin concentration. In conclusion, these results indicate that PBI-1402 plays an important role in the formation of red blood cells. Therefore, PBI-1402 may be useful for the treatment of anemia associated with chemotherapy, radiotherapy, bone marrow transplantation and cancer.

Structure of Erythropoietin in African Clawed Frogs, Xenopus laevis, and Its Role in the Liver Erythropoiesis.

Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.

PBI-1402: A New Candidate for the Treatment of Anemia.

PBI-1402 is an orally active low molecular weight synthetic compound which is currently in a phase Ib/II clinical trial in patients with anemia associated with cancer and/or chemotherapy. PBI-1402 stimulates the in vitro/ex vivo proliferation and maturation of hematopoietic progenitors (erythroid and myeloid populations) with an activity comparable to EPO. Furthermore, an additive effect is observed in combination with EPO. PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Clinical phase I study showed that PBI-1402 is devoid of significant side effects. In addition, the clinical phase I results showed statistical increase (100%, p&lt;0.0001) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment compared to placebo. Preclinical results demonstrated that PBI-1402 yields an increase in erythrocytes in the systemic circulation. In myeloblated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count, hemoglobin, platelet and bone marrow erythroid progenitor cells. PBI-1402 is targeted as an adjunct to cytotoxic drugs, radiotherapy and bone marrow transplantation for the treatment of anemia.

Expression of Erythropoietin Receptor-Like Molecule in Xenopus laevis (xeEPOR) and the Development of Anemia by the Administration of Its Recombinant Soluble Form.

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key ingredient of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. In this study, a cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, named xeEPOR tentatively, was cloned from cDNA library of Xenopus immature erythrocytes. The identities of the deduced entire amino acid sequence to human and murine EPO receptors were 24% and 25%, respectively; whereas transmembrane region and motifs of WSXWS and Box1/2 domains were found in the molecule. The northern analysis revealed that two types of xeEPOR RNAs were expressed in normal peripheral blood cells. In addition, by in situ hybridization and immunostaining with monoclonal antibodies raised against the extracellular domain of xeEPOR (soluble xeEPOR), immature basophilic erythrocytes expressing xeEPOR appeared in peripheral blood of phenylhydrazine-treated adult Xenopus. The fulllength xeEPOR cDNA was introduced into murine FDC/P2 cells and the signaling for the cellular proliferation and differentiation was examined in the presence of serum derived from anemic Xenopus as a stimulator. To further understanding the contribution of the xeEPOR gene expression to primitive and definitive hematopoiesis on Xenopus development, whole mount in situ hybridization was performed. As the binding motif of GATA-1, the hematopoietic specific transcription factor, was located at −24 to −15 base upstream of the translation initiation sequence, the correlated expressions of xeEPOR and Xenopus GATA-1 on developing embryo were evaluated with RT-PCR. The xeEPOR RNA was abundantly expressed at Nieuwkoop stage 30 (blood island formation) and thereafter, and temporally followed the expression of GATA-1, suggesting that the functional expression of xeEPOR was upregulated by GATA-1 in Xenopus as reported in studies on mammalian erythropoiesis. To confirm biological functions of the molecule, soluble xeEPOR was administered into adult Xenopus by intracardiac consecutive injections. The peripheral erythrocyte counts were gradually decreased; meanwhile immature erythrocytes were emerged in the circulation, demonstrating that this molecule plays a significant physiological role in erythropoiesis in Xenopus.