ABC | Volume 112, Nº4, April 2019

Original Article Vassallo et al Mercury increases ACE activity and oxidative stress Arq Bras Cardiol. 2019; 112(4):374-380 Additionally, at the vascular level, the vasoconstrictor response to phenylephrine was increased in caudal, mesenteric, coronary arteries and in the rat aorta, effects commonly related to reduced bioavailability of nitric oxide (NO) and increased oxidative stress. 4,18,19 Interacting with NO, superoxide anion (O 2 .- ) forms peroxynitrite, decreasing NO availability for smooth muscle relaxation. 20-22 We reported that mercury administration increases local angiotensin converting enzyme (ACE) activity, 18 releasing more angiotensin II that enhances the production of free radicals. 23 These results show that mercury pressor effects might depend on angiotensin II generation and are involved in oxidative stress generation. Previous studies showed that mercury could increase local ACE activity and oxidative stress with subsequent oxidative damage in several organs and systems, 5,11,24-27 but the in vivo effects of mercury chronic exposure on cardiovascular activity are not yet completely understood. Moreover, investigations on mercury effects have been mainly focused on the cardiovascular systems of normotensive animals. However, little information exists about the chronic effects of low doses of inorganic mercury regarding ACE activity in organs and tissues of normotensive and hypertensive animals. To investigate such effects, increased mercury levels were induced to produce blood level concentrations similar to those of exposed individuals. Therefore, we aimed to determine whether chronic exposure to inorganic mercury increases the activity of ACE and the relationship of such exposure with oxidative stress on heart, aorta, lung, brain and kidney in hypertensive compared to normotensive animals. Methods Animals Three-month-old male normotensive Wistar rats and SHRs (spontaneously hypertensive rats) were obtained from the Federal University of Espirito Santo breeding laboratories. During treatment, rats were housed at a constant room temperature, humidity, and 12:12-h light-dark cycle. Rats had free access to tap water and were fed with standard chow ad libitum . All experiments were conducted in compliance with the guidelines for biomedical research as stated by Conselho Nacional de Controle de Experimentação Animal-CONCEA, and in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocols were approved by the Ethics Committee of Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, Brazil (CEUA‑EMESCAM 003/2007). Wistar rats and SHRs were divided into four groups: control Wistar rats (n = 6) and SHRs (n = 9) treated with vehicle (saline solution, im ), and Wistar rats (n = 8) and SHRs (n = 9) treated with mercury chloride (HgCl 2 ) for 30 days (1 st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, im to cover daily loss). We used the model described by Wiggers et al. 4 to reach blood level concentrations (7,97 ng/ml) similar to those of exposed individuals. Blood pressure measurements Indirect systolic blood pressure was measured at both the beginning and the end of the treatment using tail‑cuff plethysmography (IITC Life Science Inc.). For this measurement, conscious rats were restrained for 5–10 min in a warm and quiet room and were conditioned to numerous cuff inflation‑deflation cycles by a trained operator. Subsequently, systolic blood pressure was measured, and the mean of three measurements was recorded. Hemodynamic parameter measurements At the end of treatment, control and HgCl 2 -treated rats (n = 26) were anaesthetized with urethane (1.2 g/kg, Sigma, St Louis, MO, USA), and the carotid artery and jugular vein were cannulated. A polyethylene catheter (PE50/ Clay-Adams ) filled with heparinized saline (50 U/mL) was introduced into the carotid artery to measure systolic blood pressure (SBP) and diastolic blood pressure (DBP). The carotid artery catheter was introduced into the left ventricle, and the jugular vein cannula was advanced into the right ventricular chamber to measure the left and right ventricular systolic pressures (LVSP and RVSP) and their positive and negative time derivatives (+dP/dt and - dP/dt, respectively) along with the left and right ventricular end-diastolic pressures (LVEDP and RVEDP). Recordings were performed over 30 min with a pressure transducer (TSD 104A-Biopac) and with an interface and software for computer data collection (MP100A, Biopac System, Inc., Santa Barbara, CA, USA). Heart rate (HR) was determined in the interbeat intervals. Measurement of malondialdehyde (MDA) production. Levels of MDA in plasma, heart, aorta, brain, kidney and lung were measured using a modified thiobarbituric acid (TBA) assay. 28 Plasma and tissue samples were mixed with 20% trichloroacetic acid in 0.6MHCl (1:1, v/v), and tubes were kept on ice for 20 min to precipitate plasma components to avoid possible interferences. Samples were centrifuged at 1500 x g for 15 minutes before adding TBA (120 mM in Tris 260 mM, pH 7) to the supernatant in a proportion of 1:5 (v/v); then, the mixture was boiled at 97°C for 30 min. Spectrophotometric measurements at 535 nm were taken at 20° C. ACE activity assay ACE activity was measured in plasma, heart, aorta, brain, kidney and lung using a fluorometric method adapted from Friedland and Silverstein. 29 Briefly, triplicate tissue and plasma samples (3 μL) were incubated for 15-90 minutes at 37°C with 40 μL of assay buffer containing the ACE substrate 5 mM Hip‑His-Leu (Sigma). The reaction was stopped by the addition of 190 μL of 0.35 M HCl. The generated product, His-Leu, was measured fluorometrically following 10 min of incubation with 100 μL of 2% o-Phthalaldehyde in methanol. Fluorescence measurements were taken at 37°C in a FLUOstar Optima plate reader (BMG Labtech, Offenburg, Germany) with 350 nm excitation and 520 nm emission filters. The fluorescence plate reader was controlled using the FLUOstar Optima Software. Black 96-Well polystyrene microplates (Biogen Cientifica, Madrid, Spain) were used. A calibration curve with ACE from the rabbit lung (Sigma) was included in each plate. Data analysis and statistics The results are expressed as the mean ± SD. All parameters were tested for normality using the one-sample Kolmogorov- 375