Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

INTRODUCTION: The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. AIM: To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. METHODS: Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10e15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H(2)O(2)) was measured using the Amplex Red assay and intracellular H(2)O(2) using a fluorescent sensor. In addition, ROS superoxide (O(2)•-) was measured using a O(2)•- chemiluminescence enhancer. Both H(2)O(2) and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO(−)) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [(14)C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C(16)-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells’ dysfunction. MAIN OUTCOME MEASURES: Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. RESULTS: Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O(2)•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildena fil protected the endothelial cells from radiation therapy-induced apoptosis. STRENGTHS AND LIMITATIONS: This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O(2)•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. CONCLUSIONS: This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED.