0 μM 2.07 μM 20.7 μM 207 μM 0 μM 2.07 μM 20.7 μM 207 μM 100 ± 5.5 95.5 ± 8.5 99.2 ± 2.2 91.1 ± 8.2 100 ± 2.3 100 ± 3.7 100 ± 8.0 93.8 ± 8.2 Viability % ± SD. Each cell represents the average of three independent experiments. BMC Cancer 2009, 9:398 http://www.biomedcentral.com/1471-2407/9/398 Table 1: Viability of non-stimulated and PHA-stimulated blood cells treated with etoposide as determined by dual-cell stain method. Etoposide 2 h treatment non-stimulated blood cells 2 h treatment PHA-stimulated blood cells Etoposide 24 h treatment non-stimulated blood cells 24 h treatment PHA-stimulated blood cells 100 ± 5.6 88.6 ± 3.9 83.2 ± 8.2 75.6 ± 7.6 100 ± 3.9 96.7 ± 3.4 96.7 ± 3.4 81.4 ± 9.9 Alkali labile site (ALS) index The ALS index was determined as the difference between the DNA damage detected at pH 12.3 and the damage esti- mated at pH>13 by the SCGE assay. All data were normal- ized with respect to the controls. As shown in Figure 3, non-stimulated whole blood cells had higher ALS index values than PHA-stimulated cells treated with etoposide. Control cells differed from etoposide-treated cells in all conditions except in PHA-stimulated cells treated for 24 h. The ANOVA analysis showed that there was a significant effect of etoposide concentration (0.0, 2.07, 20.7, 207.0) on the formation of ALS in all treatments: 2 h non-stimu- lated (F3 = 442.97, P < 0.0000), 24 h non-stimulated (F3 = 246.66, P < 0.0000) 2 h stimulated (F3 = 50.35,> P < 0000) 24 h stimulated (F3 = 9.52, P < 0.01).; these find- ings were consistent with the SCGE data (Figure 3). DNA AP-sites detection To test the hypothesis that ALSs generated by etoposide- oxidative stress could develop into DNA-AP sites, we used the enzyme Exo III, which recognizes this kind of DNA lesions. This analysis was performed in non-stimulated whole blood cells treated for 2 h with etoposide, a condi- tion which produces a high rate of ALS induction, and the results were compared with the data from the PHA-stimu- lated whole blood cells exposed to etoposide for 24 h. As shown in figure 4, we observed a relationship between percentage of AP-sites and etoposide concentration in non-stimulated whole blood cells relative to PHA-stimu- lated blood cells subjected to the longer treatment. Because the presence of ALSs in non-stimulated whole blood cells was inferred indirectly by comparing the SCGE assays under the two pH conditions, it was important to test whether these putative ALSs involved AP-sites. There- fore we examined whether there was an association between the ALS index data and the DNA AP-sites data, as revealed by the use of Exo III. Indeed, we found a signifi- cant positive correlation (r = 0.90; p < 0.01) between ALS index value and the percent of DNA-AP sites detected by Exo-III (Figure 5). If etoposide treatment induces ROS, principally phenoxyl radicals in non-stimulated whole blood cells, then an antioxidant exposure should reduce the DNA damage induced by etoposide. As shown in figure 6, we found that a 2-h treatment course with the antioxidant AA (200 μM) concurrent with the 2-h etoposide treatment reduced the DNA damage induced by etoposide in non-stimulated whole blood cells. This finding indicates that AA provided some level of protection for the non-stimulated blood cell DNA in the oxidative micro-environment generated by the etoposide. Discussion Etoposide affects chromatin function by directly and physically interfering with topoisomerase IIα enzyme activity. Topoisomerase IIα is considered to be an impor- tant player in the maintenance of the DNA double helix, due to its capacity to regulate conformational changes in DNA, in normal processes such as replication, transcrip- tion or condensation and segregation of chromosomes [4- 6]. Topoisomerase IIα activity fluctuates with the cell cycle; its levels elevate as the cells progress through the cycle toward mitosis [36-39]. Thus as structural maintenance of DNA is most challenged during DNA replication, it is expected that cycling cells would be the most susceptible to damage in the presence of etoposide. Because non- stimulated cells are not cycling, they have relatively low Topoisomerase II activity compared to PHA-stimulated whole blood cells, and thus would be expected to be rela- tively insensitive to DNA-damaging effects of etoposide. The present findings of etoposide-induced DNA damage in non-stimulated cells differ from the findings of Olive and Banath [40] which indicated an absence of DNA dam- Page 4 of 9 (page number not for citation purposes)