BMC Cancer 2009, 9:398 http://www.biomedcentral.com/1471-2407/9/398 Topoisomerase II is required to resolve knots and tangles in the genetic material that are produced by physiological processes such as DNA recombination and replication [7- 12]. In the absence of topoisomerase II, cells are unable to segregate daughter chromosomes and die of mitotic fail- ure [13]. In contrast to most drugs that target specific enzymes, etoposide and other topoisomerase II-targeting anticancer agents act in a subtle manner. Rather than blocking the activity of this essential enzyme, etoposide kills cells by increasing the concentration of topoisomerase II-DNA cleavage complexes [7,12,14-16]. This action converts topoisomerase II into a potent cellular toxin that frag- ments the genome. Consequently, etoposide has been deemed a topoisomerase II poison, distinct from drugs that inhibit the overall catalytic activity of an enzyme [7,12,14-18]. It has been known for more than a decade that etoposide stabilizes topoisomerase II-associated DNA breaks, thereby abolishing the ability of the enzyme to ligate cleaved nucleic acid molecules [7,12,16,19-21]. Specifically when etoposide interacts with topoisomerase IIα, it traps the enzyme in a covalently bound form with its DNA substrate [5,22]. The topoisomeriase IIα-DNA complex is stabilized with the etoposide molecule by hydrogen bonds with the nucleic acid bases, and this stabilized complex thus pre- vents re-ligation of DNA by topoisomerase IIα [23,24]. Both double-and single-strand breaks (SSBs) in DNA can be produced by etoposide. The production of free radicals during etoposide metabo- lism has also been observed [25-27]. An orthoquinone metabolite of etoposide can be transformed into a hydro- quinone [21]. When oxidized, hydroquinones give rise to hydroxyl radicals, which may ultimately contribute to etoposide-associated SSBs in DNA [28]. Although, the etoposide mechanism of action is well described in trans- formed cells, is important to know the effects generated in non-transformed whole blood cells as they are also exposed to the antineoplastic drug. The Single Cell Gel Electrophoresis (SCGE) assay, also known as the comet assay, has been proposed as a sensi- tive, reliable and rapid method for detecting DNA SSBs, alkali labile sites (ALSs), and delayed repair sites (DRSs) in eukaryotic cells under extremely alkaline conditions (pH > 13) [29,30]. Meanwhile, the SCGE assay reveals only SSBs and DRSs under less extreme alkali conditions (i.e. pH 12.3). Thus by comparing the SCGE results obtained at pH 12.3 to those obtained pH >13, it is possi- ble to discriminate the accumulation of apurinic and apy- rimidinic sites (AP sites), which produce ALSs, from other forms of DNA damage. In this study, we used the alkaline SCGE assay at pH 12.3 and pH >13 in non-stimulated and PHA-stimulated human blood cells to assess the genotoxicity associated with etoposide-induced oxidative stress in non-trans- formed cells. We performed follow-up assays with exonu- clease III enzyme (Exo III) to detect DNA-AP sites within the genome [31]. The effect of co-treatment with an anti- oxidant, on etoposide genotoxicity was also examined. If etoposide treatment generates the production of reactive oxygen species, principally phenoxyl radicals, in non- stimulated whole blood cells, then exposure to an antioxi- dant should reduce the extent of DNA damage induced. Methods Chemical and reagents Normal agarose, low melting point agarose (LMPA), ethidium bromide, Tris, Na2EDTA, DMSO (dimethyl sul- foxide), Phytohemaglutinin (PHA), Triton X-100, RPMI- 1640 medium, and etoposide were obtained from Sigma Chemical Co. (St. Louis, MO), NaOH and NaCl were obtained from Merck (Mexico) and Baxter (Mexico), respectively. Exo III was obtained from Amersham Life Science (Piscataway, NJ, USA). Ascorbic acid (AA), also known as vitamin C, was obtained from ICN (Mexico). Human blood cells and treatments The protocol was approved by the Ethics committee of Instituto de Investigaciones Biomédicas at Universidad Nacional Autónoma de México. Whole blood samples were obtained by vein punction from normal healthy vol- unteers, who were non-smokers and not taking any med- ications. PHA-stimulated and non-stimulated whole blood cells were treated for 2 or 24 h with different etopo- side concentrations (0, 2.07, 20.7 and 207 μM), in the presence of RPMI-1640 culture medium and maintained at 37°C under 5% CO2 conditions. The etoposide concentrations were determined by assum- ing a body surface area of 1.63 m2 for the volunteers and calculating a dose equivalent to that used clinically for hematological malignancies. The calculated equivalent of the clinical dose was taken as the highest concentration applied in our study. PHA-stimulated whole blood was first incubated for 6 h at 37°C under 5% CO2 conditions in 1 ml of RPMI-1640 culture medium with 71 μl of PHA, and then treated with etoposide as described above. To test the attenuation of etoposide AP-site generation in non-stimulated blood cells, the cultures were treated at the same time with AA (200 μM). Viability The dual cell-stain assay described by Hartman and Speit [32] was employed to determine the viability of the PHA- Page 2 of 9 (page number not for citation purposes)