A Naphthoquinone Derivative Can Induce Anemia through Phosphatidylserine Exposure-Mediated Erythrophagocytosis

Abstract

A naphthoquinone derivative, beta-lapachone (beta L; 3,4-dihydro-2,2- dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione), is receiving huge attention for its potent therapeutic effects against various diseases. However, during the preclinical safety evaluation, repeated oral treatment of beta L in rats induced anemia, i.e., a significantly decreased erythrocyte count. In this study, in an effort to elucidate the mechanism underlying the beta L-induced anemia, we investigated the effects of beta L on erythrocytes with freshly isolated human erythrocytes in vitro and rat in vivo. beta L did not induce erythrocyte hemolysis, indicating that direct hemotoxicity was not involved in beta L-associated anemia. Meanwhile, phosphatidylserine (PS) exposure along with spherocytic shape change and microvesicle generation, important factors in the facilitation of erythrophagocytosis, were increased significantly by beta L. The PS exposure on erythrocytes was from beta L-induced reactive oxygen species generation and subsequent depletion of reduced glutathione and protein thiol, which culminated in the modified activities of phospholipid translocases, i.e., inhibition of flippase and activation of scramblase. It is important to note that coincubation of macrophage with beta L-treated erythrocyte in vitro showed increased erythrophagocytosis, demonstrating that the removal of erythrocyte by macrophage can be facilitated by beta L-induced PS exposure. In good accordance with these in vitro results, after oral administration of beta L in rats, increased PS exposure and depletion of glutathione were observed along with enhanced splenic sequestration of erythrocytes. In conclusion, these results suggest that beta L-induced anemia might be mediated through the PS exposure and subsequent erythrophagocytosis, providing novel insight into the drug-induced anemia.