DNA-AP sites generation by Etoposide in whole blood cells

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.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.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.In the last decade, etoposide (also known as VP-16213) has been one of the most commonly used agents for treating a number of malignancies. Etoposide is a semi-synthetic 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].Topoisomerase II is required to resolve knots and tangles in the genetic material that are produced by physiological