BMC Cancer 2009, 9:398 http://www.biomedcentral.com/1471-2407/9/398 ulated cells, we used the SCGE assay in combination with Exo III, an enzyme that recognizes AP sites in DNA. The results corroborated the presence of DNA-AP sites in the genomes of the non-stimulated cells treated with etopo- side. Moreover, when non-stimulated cells were exposed to etoposide in the presence of the antioxidant AA, less DNA damage was observed. Thus the ability of the anti- oxidant to protect the DNA suggests that phenoxyl radi- cals are the major radicals involved in the oxidative DNA- damage induced by etoposide, especially in non-stimu- lated cells [[27,43], and [44]]. Interestingly, our results suggest that the molecular events by which DNA breaks are generated in PHA-stimulated and non-stimulated cells are quite different. The damage observed in the PHA-stimulated whole blood cells could be explained by the classical events ascribed to topoi- somerase II poisons [12]. These have two principal components: DNA strand breaks due to the inhibition of topoisomerase II by etoposide and etoposide-quinone free radical effects [25,45]. Tor- nov and colleagues [46] observed DNA damage in leuko- cytes evidenced by SCGE assay at pH 13, suggesting that etoposide might cause oxidative damage in leukocytes by a mechanism involving inhibition of the enzyme topoi- somerase IIβ. Although the exact function of this enzyme has not been resolved, it is known that its concentration is generally independent of cell cycle and cell growth [[12,37,39], and [46]]. In addition, topoisomerase IIβ is expressed at a higher level than topoisomerase IIα in human peripheral blood cells [47]. If the AP site damage observed in the present study was dependent upon an interaction with this enzyme, we should have also observed this kind of damage in PHA-stimulated whole blood cells. However our findings were not consistent with this prediction. It is our view that the induction of damage at DNA-AP sites observed in non-stimulated whole blood cells herein was due to interaction of the drug metabolites with cellu- lar targets beyond topoisomerase IIβ (which was not present at high levels), such as DNA and/or proteins. Interaction of the drug metabolites with these alternative targets can cause DNA damage by the generation of free radicals [28]. Another possible explanation could be that DNA repair status in non-stimulated whole blood cells are less active than stimulated whole blood cells to remove this kind of DNA damage [48]. Moreover, the reduced DNA damage in the presence of the antioxidant AA sug- gests that etoposide-hydroquinone phenoxyl radical is the responsible mediator of these effects [27,49]. Conclusion In summary, our data show that etoposide can produce differential forms of DNA damage in PHA-stimulated and non-stimulated blood cells. These results could have important implications for elucidating the mechanisms associated with the development of secondary malignan- cies (principally acute mielocitic leukemia) that are asso- ciated with the use of etoposide as an antineoplastic drug [50]. Competing interests The authors declare that they have no competing interests. Authors' contributions ER was the project leader and directed the study; PM par- ticipated in data acquisition and generated experimental data. ET performs the statistical analysis of the data. MV was the main investigator of the study, performed the experimental design and the manuscript draft. She is the corresponding author. All authors reviewed and approved the final manuscript. Acknowledgements The authors thank Maria del Carmen López for her technical support. A professional scientific editor at Write Science Right was consulted during the preparation of this manuscript. References 1. van Maanen JMS, Retel J, de Vries J, Pinedo HM: Mechanisms of action of antitumor drug etoposide: a review. J Natl CancerInst 1998, 80:1526-1533. 2. Hainsworth JD, Greco FA: Etoposide: Twenty years later. Annals Oncol 1995, 6:325-341. 3. Hande KR: Clinical Oncology Update, Etoposide: Four dec- ades of development of a topoisomerase II inhibitor. Eur J Cancer 1998, 34(10):1514-1521. 4. Markovits J, Pommier Y, Kerrigan D, Covey JM, Tilchen EJ, Kohn KW: Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and in the cell cycle in NIH-3T3 fibroblasts and L1210 leukemia cells. Cancer Res 1987, 47:2050-2055. 5. McPherson JP, Goldenberg GJ: Induction of apoptosis by dereg- ulated expression of DNA topoisomerase IIL1. Can Res 1998, 58:4519-4524. 6. Boos G, Stopper H: Genotoxicity of several clinically used topoisomerase II inhibitors. Toxicol Lett 2000, 116:7-16. 7. Burden DA, Osheroff N: Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim- Biophys Acta 1998, 1400:139-154. 8. Wang JC: DNA topoisomerases. Ann Rev Biochem 1996, 65:635-692. 9. Wang JC: Moving one DNA double helix through another by a type II DNA topoisomerase: The story of a simple molec- ular machine. Quart Rev Biophys 1998, 31:107-144. 10. Wang JC: Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 2002, 3:430-440. 11. Nitiss JL: Investigating the biological functions of DNA topoi- somerases in eukaryotic cells. Biochim Biophys Acta 1998, 1400(1-3):63-81. 12. Fortune JM, Osheroff N: Topoisomerase II as a target for anti- cancer drugs: When enzymes stop being nice. Prog Nucleic Acid Res Mol Biol 2000, 64:221-253. 13. Bromber KD, Burgin AB, Osheroff N: A two drug model for etoposide action against human topoisomerase II. J Biol Chem 2002, 277:31201-31206. Page 8 of 9 (page number not for citation purposes)