Figure 2: Effect of xylazine, cocaine, 6-MAM, and their combination on RNS production in human umbilical vein endothelial cells (EA.hy926). DAN assay was performed as described previously in the methods section [33, 34]. RNS production after 24 h of exposure to vehicle (negative control), xylazine (XYL, 60 M), cocaine (COC, 160 M), 6-monoacetylmorphine (6-MAM, 160 M), camptothecin (positive control, 50 M), XYL/COC (50 M), XYL/6-MAM (50 M), and XYL/COC/6-MAM (40 M) was determined. Cells exposed to XYL, COC, 6-MAM, and their combinations exhibited no induction of reactive nitrogen species (RNS) in EA.hy926 cells when compared to the negative control; catalytic activity coincides with basal level production of nitrite and nitrate species. Experiments were repeated for at least three times in replicates. 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 to be significant. summary; significantly different from negative control group (), ns = no significant difference from negative control group (). Results percent (%) was calculated by negative control normalization.