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Vascular endothelial dysfunction is recognized as a contributor to a wide array of cardiovascular disease states, but the initiating events involved are incompletely defined. Elevated plasma levels of free 3-nitro-L-tyrosine (3NT, biomarker of peroxynitrite formation) have been measured in settings of endothelial dysfunction, but its pathologic significance is unknown. We tested the hypothesis that clinically demonstrated concentrations of 3NT can induce vascular and endothelial dysfunction in vitro. Further studies evaluated involvement of DNA fragmentation and/or apoptosis as a potential mechanism. Preincubation of rat thoracic aorta segments with 3NT (100, 250 μM) resulted in selective, concentration-dependent impairment of acetylcholine (ACH) maximal response, with no change in KCL, phenylephrine, nitroprusside, or ACH EC50 effects (ACH Emax, 53 ± 2, 42 ± 5, 31 ± 2%; Control, 100 μM, 250 μM 3NT). Vascular segments treated with 3NT also demonstrated concentration-dependent DNA damage, assessed using DNA nick-end labeling techniques (TUNEL staining), compared with control (TUNEL-positive nuclei/linear mm: 5.4 ± 1.2, 13.7 ± 1.2, 16.9 ± 3.2; Control, 100 μM, 250 μM 3NT), which was confined to the endothelial layer. Equimolar tyrosine had no significant effects. Frequency of positively stained nuclei was statistically correlated to extent of endothelial dysfunction (p < 0.01). Free 3NT is apparently more than a benign biomarker in vivo, and may contribute to vascular endothelial dysfunction through promotion of DNA damage and/or apoptosis.