Toxicology 300 (2012) 57–66 Contents lists available at SciVerse ScienceDirect Toxicology jou rn al hom epage: www.elsevier.com/locate/toxicol Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle Sara Finaurini a,∗ , Nicoletta Basilico a,2 , Yolanda Corbett a,1 , Sarah D’Alessandro a,1 , Silvia Parapini a,1 , Piero Olliaro b , Richard K. Haynes c , Donatella Taramelli a,∗,1 a Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy b UNICEF/UNDP/World Bank/WHO Special Programme on Research and Training in Tropical Diseases, World Health Organization, 20, Avenue Appia, CH 1211 Geneva 27, Switzerland c Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China a r t i c l e i n f o Article history: Received 27 March 2012 Received in revised form 26 May 2012 Accepted 26 May 2012 Available online 5 June 2012 Keywords: Dihydroartemisinin K562 erythroid cell differentiation Malaria in pregnancy Erythro-toxicity Cytotoxicity a b s t r a c t Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro- erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure–activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deox- yartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy- artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In partic- ular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the - globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24 h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Artemether, artesunate and their common active metabolite dihydroartemisinin (DHA) are derived by semi-synthesis from artemisinin, the sesquiterpene peroxide extracted from the leaves Abbreviations: DHA, dihydroartemisinin; ATS, artesunate; ARM, artemisinin; AMS, artemisone; C10-ARM, 10-deoxoartemisinin; Deoxy-ARM, deoxyartemisinin; CQ, chloroquine; RT- PCR, reversed transcribed polymerase chain reaction; DMSO, dimethyl sulfoxide; Hm, haemin; PpIX, protoporphyrin IX; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MW, molecular weight; GpA, glycophorin A receptor. ∗ Corresponding authors. Tel.: +39 02 50315071; fax: +39 02050315068. E-mail addresses: sarafinaurini@gmail.com (S. Finaurini), donatella.taramelli@unimi.it (D. Taramelli). 1 Present address: Dipartimento di Scienze Farmacologiche e Biomolecolari, degli Studi di Milano, Via Balzaretti 10, 20133 Milano, Italy. 2 Present address: Dipartimento di Scienze Biomediche, Chirurgiche e Odon- toiatriche, degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy. of Artemisia annua (White, 2008). The common feature and hall- mark of the activity of these compounds is a 1,2,4-trioxane ring incorporating a peroxide bridge (Efferth, 2007). These drugs consti- tute today the cornerstone of malarial control (Krishna et al., 2008; WHO, 2009). The vast majority of antimalarial drugs are used for uncomplicated malaria, for which the World Health Organization (WHO) recommends a three-day regimen with an artemisinin- based combination therapy (ACT), though with a restriction on the first trimester of pregnancy (WHO, 2006b, 2009). Artemisinins are remarkably well tolerated, but some safety issues exist (Efferth and Kaina, 2010). Among these, embryotoxicity and haematological toxicity such as anaemia may have a common mechanism (Clark, 2009; Clark et al., 2010, 2011; WHO, 2006a, 2007). Experimental models indicate that all current artemisinin derivatives and synthetic peroxides cause a depletion of embryonic erythroblasts during a specific time window in the first trimester of pregnancy (Clark et al., 2004, 2008a,b, 2010; Longo et al., 2006a,b, 2008, 2010; White and Clark, 2008). Our previous studies 0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2012.05.024