PARTICIPATION OF NAD(P)H OXIDASE ON OXIDATIVE STRESS INDUCED BY CHRONIC ETHANOL CONSUMPTION IN THE RATS CORPUS CAVERNOSUM

Introduction: The vascular damage caused by ethanol involves the generation of reactive oxygen species (ROS) and reduced bioavailability of nitric oxide (NO). The NAD(P)H oxidase plays a key role in generating ROS, including superoxide anion and hydrogen peroxide (H2O2) in vascular smooth muscle cells and endothelial cells. Therefore, the aim of this study is to evaluate the involvement of NAD(P)H oxidase in the effects induced by chronic ethanol consumption in the rat cavernosal smooth muscle (CSM) through its inhibition by apocynin (APO).

Methods: The experimental protocols were approved by the Ethical Committee from University of São Paulo (13.1.471.53.9). Male Wistar rats (200-250g) were divided into 4 groups: Control group (C): received drinking water by oral gavage; Control + APO group (AC): APO (30mg/kg/day, oral gavage); Ethanol group (E): ethanol 20% (v/v) for 6 weeks and drinking water by oral gavage; Ethanol + APO group (AE): ethanol 20% and APO (30mg/kg/day, oral gavage). Reactivity experiments were performed on isolated CSM. Systemic oxidative stress was evaluated by measuring plasma thiobarbituric acid-reacting substances (TBARS) and superoxide anion levels in CSM homogenates evaluated by lucigenin-derived chemiluminescence assay. Nitrate levels were measured in plasma and supernatants from total CSM homogenates. Protein levels of p47phox and Nox1 were assessed by western blot.

Results: Blood ethanol levels in the ethanol-treated rats averaged (E: 1.21 ± 0.21 mg/ml;n=8 AE: 1.39±0.11 mg/ml;n=6). Chronic ethanol consumption reduced the relaxation induced by acetylcholine (C: 41.6±1.5%;n=5 E: 29.7±1.0%;n=7). APO treatment prevented the reduction of acetylcholine-induced relaxation (AC: 42.7%±1.1;n=4 AE: 41.8±0.7%;n=6)(p<0.05, ANOVA). Sodium nitroprusside-induced relaxation was not affected by ethanol consumption (C: 99.9±4.3%;n=5 E: 91.6±2.4%;n=5 AC: 91.5±1.87%;n=5 AE: 100.6±2.5%;n=5). Plasma TBARS levels (nmol/mL) were increased in ethanol-treated rats (34.6±1.5, n=10) compared with control rats (19.7±2.0, n=12). Treatment with APO prevented this response (AC: 18.7 ± 1.9, n=11 AE: 21.2 ± 1.6, n= 12). (p<0.05, ANOVA). Superoxide anion levels (RLU/mg protein) were higher in CSM from ethanol-treated rats (241.9±13.7, n=8) when compared with control (96.4±16.0, n=7). APO prevented the increase in superoxide anion levels induced by ethanol (AC: 75.8 ± 12.1, n=8 AE: 106.7±17.1, n=8)(p<0.05, ANOVA). Chronic ethanol consumption increased p47phox translocation and APO prevented this response. Finally, ethanol consumption reduced plasma and CSM nitrate levels. Treatment with APO prevented this response.

Discussion: Chronic ethanol consumption induced oxidative stress, reduced acetylcholine-induced relaxation and nitrate levels in CSM from ethanol-treated rats, being these response mediated by NAD(P)H oxidase.

Financial Support: CNPq