Figure 3: Effect of xylazine, cocaine, 6-monoacetylmorphine, and their combination on DNA fragmentation content in human umbilical vein endothelial cells (EA.hy926). DNA fragmentation content in EA.hy926 cells after 24 h of exposure to vehicle (negative control group, NC), camptothecin (positive control group, PC, 50 M), xylazine (XYL, 60 M), cocaine (COC, 160 M), 6-monoacetylmorphine (6-MAM, 160 M), XYL/COC (50 M), XYL/6-MAM (50 M), and XYL/COC/6-MAM (40 M) was evaluated in each phase independently and compared to negative control group. DNA fragmentation content assay was performed to measure cells containing less than 1DNA equivalent (Sub-G1) after degradation of DNA triggered by endonucleases or drug interaction [35]. Cells were harvested after drug treatment, fixed with 70% ethanol, incubated 24 hours at 4°C, stained with 1 g/ml DAPI (5 min incubation), and image-analyzed with NucleoCounter NC-3000 instrument. Results obtained in phase G0/G1 (a) show significant difference () in cells exposed to XYL, COC, and 6-MAM. Cells treated with XYL in combination with COC or 6-MAM presented significant difference (, respectively), but lower than previously described. Phase G2/M (b) presented no significant difference (, ns) in cells treated with XYL. Cells treated with these three-drug combination presented significant difference () when compared with the negative control group. The experiment was repeated for at least three times. All values are expressed as mean SD of 6–9 replicates, from 2 to 3 experiments. Statistical analysis performed was one-way ANOVA, with Tukey post hoc test, where was considered significant. summary; significantly different when compared to negative control group, , , ns = no significant difference from negative control group (). Results percent (%) was calculated by negative control normalization.