Oxidative Stress and Increased Destruction of Red Blood Cells Contribute to the Pathophysiology of Anemia Caused By DMT1 Deficiency
Authors: Zidova Zuzana , Garcia-Santos Daniel , Kapralova Katarina , Koralkova Pavla , Mojzikova Renata , Dolezal Dalibor , Ponka Prem , Divoky Vladimir , Horvathova Monika 
Published: October 2019

Abstract
Inactivating mutations in divalent metal transporter 1 (DMT1) are associated with a severe defect in erythroid iron utilization and cause moderate to severe hypochromic microcytic anemia in human patients and two rodent models. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes the suppression of colony-forming capacity of erythroid progenitors. Using in vitro cultures of fetal liver cells we were able to recapitulate this in vivo defect. We confirmed abnormal pattern of erythroid differentiation and increased apoptosis (2.5-times) of DMT1-mutant erythroblasts when compared to wild-type (wt) fetal liver erythroblats. Determination of 2’,7’-Dichlorofluorescein diacetate-dependent intensity of fluorescence, which is proportional to the concentration of reactive oxygen species (ROS), revealed elevated levels of ROS in DMT1-mutant erythroblats when compared to wt erythroblast. This result suggests that oxidative stress contributes to the apoptosis in DMT1-mutant cells. We also observed that the defective erythroid differentiation of DMT1-mutant erythroblasts is marked by a blunted induction of heme oxygenase-1, an enzyme that co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells (Garcia-Santos et al., Blood, 2014, 123 (14): 2269-77).
In further studies we focused on mature red blood cells (RBC), because it is known that nutritional iron deficiency and certain types of congenital hypochromic anemia are associated with increased levels of ROS and shortened life span of RBC that can be at least partially attributed to a programmed cell death of erythrocytes, so called eryptosis (Lang et al., Int J Biochem Cell Biol, 2012, 44 (8): 1236-43). Using labeling with carboxyfluorescein diacetate succinimidyl ester, we observed an accelerated clearance of DMT1-mutant RBC from circulating blood when compared to wild-type RBC. In vitro, DMT1-mutant RBC exposed to hyperosmotic shock or glucose depletion showed significantly increased levels of phosphatidylserine on the membrane detected by Annexin V binding. Together, these results confirmed eryptosis of DMT1-mutant RBC. As eryptosis is proposed to be triggered via activation of Ca2+ cation channels, we next measured the concentration of cytosolic Ca2+ using Fluo3/AM fluorescent dye and found significantly elevated content of intracellular Ca2+ in DMT1-mutant RBC when compared to wt RBC. In addition, DMT1-mutant RBC had higher levels of ROS than wt RBC despite significantly increased activity of anti-oxidative defense enzymes; glutathione peroxidase (1.6-times), catalase (1.9-times) and methemoglobin reductase (1.9-times). This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBC is not sufficient to eliminate ROS effectively. Furthermore, DMT1-mutant RBC also showed accelerated anaerobic glycolysis as detected by increased activities of hexokinase (2.5-times), pyruvate kinase (2.4-times), glucose-phosphate isomerase (3.2-times). This result together with reduced ATP/ADP (1.6-times) ratio in DMT1-mutant RBC when compared to wt RBC suggests an increased demand for ATP in DMT1-mutant erythrocytes.
In conclusion we propose that increased oxidative stress and accelerated destruction of RBC contribute to the pathophysiology of anemia caused by DMT1-deficiency.
Grant support: Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic, grant No. NT13587, Education for Competitiveness Operational Program, CZ.1.07/2.3.00/20.0164, Internal Grant of Palacky University Olomouc, LF_2014_011 and in part by the Canadian Institutes of Health Research (D.G-S., P.P.).

Disclosures
No relevant conflicts of interest to declare.