BMC Cancer Research article DNA-AP sites generation by Etoposide in whole blood cells Emilio Rojas1, Patricia Mussali1, Efrain Tovar2 and Mahara Valverde*1 Address: 1Departamento de Medicina Genómica y Toxicología Ambiental Instituto de Investigaciones Biomédicas. Universidad Nacional Autónoma de México D.F. C.P. 04510, México and 2Centro de Educación Ambiental e Investigación, Sierra de Huautla (CEAMISH), UAEM. Av. Universidad No. 1001, Col. Chamilpa, Cuernavaca, Morelos, CP 62210, México Email: Emilio Rojas - emilior@servidor.unam.mx; Patricia Mussali - pmussali@yahoo.com.mx; Efrain Tovar - etovar@yahoo.mx; Mahara Valverde* - mahara@biomedicas.unam.mx * Corresponding author Published: 16 November 2009 BMC Cancer 2009, 9:398 doi:10.1186/1471-2407-9-398 This article is available from: http://www.biomedcentral.com/1471-2407/9/398 Received: 28 May 2008 Accepted: 16 November 2009 © 2009 Rojas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central Open Access Abstract Background: Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies. Methods: To determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured. Results: Alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide. Conclusion: Our results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy. Background In the last decade, etoposide (also known as VP-16213) has been one of the most commonly used agents for treat- ing a number of malignancies. Etoposide is a semi-syn- thetic derivative of epipodophyllotoxin derived from the plant Podophyllum peltatum [1-3]. Its primary intracellular target, topoisomerase II, alters DNA topology by passing an intact double helix through a transient double stranded break that it generates in a separate nucleic acid segment [4-6]. Page 1 of 9 (page number not for citation purposes)