contact of nanotubes with erythrocytes that was possible in case of intravenous administration only, would not have taken place. Significant anemia in response to intratarcheal administration of AF-SWCNTs could be a result of the relatively easy diffusion of nanotubes through thin alveolar membranes as reported earlier also [16]. Diffusion of CNTs from peritoneal cavity and stomach into bloodstream may be slow and could be the reason for the lack of effect of AF-SWCNTs in these cases. In another recent study, SWCNTs were not found to induce significant toxic effects in nude mice [17]. The processes of preparation of stable suspensions of SWCNTs in this study were however very different from the one used in our study and these differences could be responsible of the results in these two studies. Acute anemia is known to stimulate the release of erythrocytes from spleen and bone marrow in order to compensate for the loss of erythrocytes [18], [19]. Recovery of erythrocytes count in blood may represent a homeostatic response to fall in blood erythrocyte count. This propositions is supported by our observation that a significant increase in blood reticulocyte count was observed in blood with few hours of i.v. infusion of AF-SWCNTs (results not shown). A second dose of AF-SWCNTs given intravenously extended the duration of anemia. The fact that the anemia induced by intravenous administration of AF-SWCNTs was transient in nature suggests that the nanotubes may have a short half life in circulation. In a recent study, radio-labelled polydispersed SWCNTs were shown to accumulate in mouse liver [20]. It is therefore possible that the nanotubes may be removed from circulation by organs like liver and kidney. There could be several possible mechanisms of induction of anemia in response to AF-SWCNTs. A direct cytotoxic effect of nanotubes on erythrocytes is suggested by our in vitro experiments. It was important to understand whether the cytotoxic effect of nanotubes was exerted uniformly on all erythrocytes in circulation or it was related to the age of erythrocytes in circulation. This question could be addressed by help of a technique involving two steps of in vivo biotinylation of circulating erythrocytes that has recently been developed in our laboratory. By using this technique it became clear that the oldest subpopulation of erythrocytes (age . 40 days) was specifically sensitive to nanotubes. Blood count of References 1. Lam CW, James JT, McCluskey R, Hunter RL (2004) Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicol Sci 77: 126–134. 2. Lam CW, James JT, McCluskey R, Arepalli S, Hunter RL (2006) A review of carbon nanotube toxicity and assessment of potential occupational and environmental health risks. Crit Rev Toxicol 36: 189–217. 3. 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Biochem and Biophy Res Commun 362: 651–657. 9. Khandelwal S, Saxena RK (2006) Assessment of survival of aging erythrocyte in circulation and attendant changes in size and CD147 expression by a novel two step biotinylation method. Exp Gerontol 41: 855–861. 10. Khandelwal S, van Rooijen N, Saxena RK (2007) Reduced expression of CD47 during murine red blood cell (RBC) senescence and its clearance from the circulation. Transfusion 47: 1725–732. Effect of Carbon Nanotubes on Erythrocytes young erythrocytes (,10 days) actually increased after adminis- tration of AF-SWCNTs. This increase could be a consequence of release of fresh erythrocytes into circulation from spleen and bonemarrow. Interestingly the erythrocytes population of inter- mediate age group (10–40days) didn’t change in response to AF- SWCNTs. These results suggest that the older erythrocytes in circulation that have become weakened by oxidative damage may be easier targets for the nanotubes. It should however be stressed that even though greater susceptibility of older erythrocytes to AF- SWCNTs is indicated by the in vivo experiments, a more generalized erythrolysis observed in vitro (Figure 1) suggests that age related differences in susceptibility observed in vivo may only be relative and not absolute. In conclusion, we have shown an acute cytotoxic effect of polydispersed SWCNTs in vitro as well as in vivo. Rapid induction of anemia within a day is followed by a recovery of blood count of erythrocytes. Effect of AF-SWCNTs on the erythropoietic activity in spleen and bone marrow will further clarify if the fall in blood count of erythrocytes is accompanied with changes in erythropoi- etic activity. These studies using fluorescence tagged AF-SWCNTs are currently under way in our laboratory. Supporting Information Video S1 Time lapse recording demonstrating the lysis of erythrocytes incubated with AF-SWCNTs. 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(2007) PET Imaging of Soluble Yttrium-86-Labeled Carbon Nanotubes in Mice. Plos one 2(9): e907. PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22032