Arsenite and its primary metabolite monomethylarsonous acid impair early human red blood cell development

Abstract Abstract: Chronic exposure to heavy metals, including arsenic, is linked to hematological disorders such as anemia. Both in vivo and in vitro models have demonstrated the trivalent inorganic form of arsenic (arsenite, As3+) causes hematotoxicity by impairing red blood cell (RBC) differentiation and maturation. However, little is known regarding As3+-derived metabolites and how they contribute to impairment of early RBC development. Preliminary in vitro studies indicate that the major trivalent As3+ metabolite, monomethylarsonous acid (MMA3+), is more toxic than its parent inorganic arsenical. Therefore, the aim of this study was to investigate the differential hematotoxicity of As3+ and MMA3+ in primary human bone marrow CD34+ hematopoietic progenitor cells (HPCs) from male and female donors (i.e., obtained commercially from STEMCELL Technologies). To evaluate As3+- and MMA3+-mediated effects on early RBC growth and differentiation, CD34+ HPCs were cultured in two parallel systems: 1) HemaTox Erythroid™ medium, selective for erythroid development, and 2) H4330 MethoCult™ medium, which supports growth of both erythroid and myeloid progenitors. HPCs in both systems were exposed to low concentrations of As3+ or MMA3+. After 3 and 7 days, viability and growth of early RBCs was assessed using acridine orange/propidium iodide staining, and erythroid differentiation was assessed by flow cytometry. As3+ and MMA3+ reduced the viability and differentiation of early human RBCs in a dose-dependent manner. Flow cytometry revealed impaired erythroid differentiation following MMA3+ treatment, with early RBCs not progressing to later stages of maturation. Broader hematopoietic impacts were further evaluated by colony formation in H4330 MethoCult™ after 14 days. Colony-forming assays, likewise, showed significant reductions in erythroid colonies (BFU-E and CFU-E) after exposure to both arsenicals. Differential hematotoxic effects were observed between male and female donors at the 7-day timepoint. Collectively, these findings indicate that early stages of human RBC development are highly susceptible to hematotoxicity by As3+ and MMA3+, with MMA3+ generally eliciting greater toxicity compared to As3+. Ongoing mechanistic studies are focused on elucidating the mechanisms by which arsenic biotransformation disrupts erythropoiesis and underlies hematological disorders, such as anemia.Funding: This work was supported by National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health 1R16 GM146669; NIGMS Institutional Development Award (IDeA) P20 GM103451; UNM Center for Metals in Biology and Medicine through NIGMS Grant Number P20 GM130422; and the NIEHS UNM METALS Superfund Research Program Grant Number P42 ES025589.