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Induction of micronucleated erythrocytes by recombinant human erythropoietin
1Research Institute of Life Science, Snow Brand Milk Products Co. Ltd, Tochigi, Japan.
Abstract:
Induction of micronucleated erythrocytes by a recombinant human erythropoietin (rhEPO) was examined using both in vitro and in vivo test systems. A small, significant and dose-related increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of mice administered i.p. with 12,500-50,000 IU/kg rhEPO was induced at 48 h sampling time. A clear positive dose--response relationship and significant increase in the frequency of micronucleated reticulocytes (MNRET) in peripheral blood of mice administered i.p. with 400-50,000 IU/kg rhEPO was noted at 48, 72 and 96 h sampling times. Conversely, in bacterial reverse mutation tests, no noticeable increase of auxotrophic revertants was observed in Salmonella typhimurium, TA100, TA98, TA1535, TA1537, or Escherichia coli, WP2 uvrA-, by treatment with 188-6000 IU/plate of rhEPO, with or without S9 mix. Furthermore, rhEPO at 750-6000 IU/ml did not induce chromosomal aberrations in vitro in CHL cells or human peripheral blood lymphocytes in a direct method nor in a metabolic activation method. Moreover, chromosomal aberrations were not detected in bone marrow cells of CD-1 male mice, even at high rhEPO concentrations (100,000 IU/kg) in vivo. Consequently, it was concluded that errors in the process of enucleation or differentiation of the erythrocytes should be equally considered as possible mechanisms for the increased frequencies of MNPCE and MNRET alongside induction of DNA damage or errors in the process of DNA repair.
Insights
Recombinant human erythropoietin (rhEPO) induced micronucleated erythrocytes in mice, suggesting potential enucleation or differentiation errors. Bacterial and chromosomal aberration tests showed no genotoxicity, indicating a complex mechanism for rhEPO
Area of Science:
- Toxicology
- Hematology
- Genetics
Background:
- Recombinant human erythropoietin (rhEPO) is a therapeutic protein used to treat anemia.
- Understanding the genotoxic potential of rhEPO is crucial for patient safety.
Purpose of the Study:
- To evaluate the genotoxicity of rhEPO using in vitro and in vivo assays.
- To investigate the mechanisms underlying micronucleus formation induced by rhEPO.
Main Methods:
- In vivo micronucleus tests in mouse bone marrow and peripheral blood.
- In vitro bacterial reverse mutation assays (Ames test) with Salmonella typhimurium and Escherichia coli.
- In vitro chromosomal aberration tests in mammalian cells (CHL cells, human lymphocytes).
Main Results:
- A dose-related increase in micronucleated polychromatic erythrocytes (MNPCE) and micronucleated reticulocytes (MNRET) was observed in vivo.
- No genotoxic effects were detected in bacterial mutation assays or in vitro chromosomal aberration tests.
- Chromosomal aberrations were also absent in in vivo mouse bone marrow cells, even at high rhEPO doses.
Conclusions:
- rhEPO can induce micronucleated erythrocytes in vivo, suggesting potential non-DNA-damaging mechanisms.
- Possible mechanisms include errors in erythrocyte enucleation or differentiation, or DNA repair processes.
- rhEPO does not appear to be directly genotoxic based on bacterial and chromosomal aberration assays.