Apocynin prevents vascular effects caused by chronic exposure to low concentrations of mercury

Abstract

Mercury increases the risk of cardiovascular disease and oxidative stress and alters vascular reactivity. This metal elicits endothelial dysfunction causing decreased NO bioavailability via increased oxidative stress and contractile prostanoid production. NADPH oxidase is the major source of reactive oxygen species (ROS) in the vasculature. Our aim was to investigate whether treatment with apocynin, an NADPH oxidase inhibitor, prevents the vascular effects caused by chronic intoxication with low concentrations of mercury. Three-month-old male Wistar rats were treated for 30 days with a) intramuscular injections (i.m.) of saline; b) HgCl(2) (i.m. 1(st) dose: 4.6 µg/kg, subsequent doses: 0.07 µg/kg/day); c) Apocynin (1.5 mM in drinking water plus saline i.m.); and d) Apocynin plus HgCl(2). The mercury treatment resulted in 1) an increased aortic vasoconstrictor response to phenylephrine and reduced endothelium-dependent responses to acetylcholine; 2) the increased involvement of ROS and vasoconstrictor prostanoids in response to phenylephrine, whereas the endothelial NO modulation of such responses was reduced; and 3) the reduced activity of aortic superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased plasma malondialdehyde (MDA) levels. Treatment with apocynin partially prevented the increased phenylephrine responses and reduced the endothelial dysfunction elicited by mercury treatment. In addition, apocynin treatment increased the NO modulation of vasoconstrictor responses and aortic SOD activity and reduced plasma MDA levels without affecting the increased participation of vasoconstrictor prostanoids observed in aortic segments from mercury-treated rats.

Conclusions: Mercury increases the vasoconstrictor response to phenylephrine by reducing NO bioavailability and increasing the involvement of ROS and constrictor prostanoids. Apocynin protects the vessel from the deleterious effects caused by NADPH oxidase, but not from those caused by prostanoids, thus demonstrating a two-way action.

Figure 1. Effect of apocynin treatment on systolic and diastolic blood pressure.

Values of (A) systolic (SBP) and (B) diastolic blood pressure (DBP, mmHg) in the aorta of rats untreated (n = 9) or treated with mercury (HgCl₂, n = 8), apocynin (Apo, n = 9) or apocynin plus mercury (ApoHg, n = 9). The results are expressed as the mean±SEM, t-test *P<0.05 vs. Untreated.

Figure 2. Effect of apocynin treatment on the vascular relaxation response to acetylcholine and sodium nitroprusside.

Concentration-response curves to (A) acetylcholine (ACh) and (B) sodium nitroprusside (SNP) in the aortas of rats untreated, treated with mercury (HgCl₂) or apocynin plus mercury (ApoHg) pre-contracted with phenylephrine. The results (mean±SEM) are expressed as a percentage of the response to phenylephrine. The number of animals used is indicated in parentheses. Two-Way ANOVA *P<0.05.

Figure 3. Effect of apocynin treatment on the vasoconstrictor response to phenylephrine.

Concentration-response curve to phenylephrine (Phe) in the aortas of rats untreated, treated with mercury (HgCl₂), and apocynin plus mercury (ApoHg). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of animals is indicated in parentheses. Two-Way ANOVA *P<0.05.

Figure 4. Effect of apocynin treatment on endothelial modulation of the vasoconstrictor response to phenylephrine.

Concentration-response curve to phenylephrine (Phe) in intact (Control) and endothelium removal (E-) aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of animals is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curves (dAUC) in endothelium denuded and intact segments of the four experimental groups. * P<0.05 vs. Untreated and ^# vs. HgCl₂-treated by t-test.

Figure 5. Effect of apocynin treatment on NO modulation of the vasoconstrictor response to phenylephrine.

Concentration-response curve to phenylephrine (Phe) in aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg) in the absence (Control) and the presence of the NO synthase inhibitor L-NAME (100 µM). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of rats is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curve to phenylephrine (dAUC) in aortic segments in the presence and the absence of L-NAME of the four experimental groups. * P<0.05 vs. Untreated and ^# vs. HgCl₂-treated by t-test.

Figure 6. Effect of apocynin treatment on ROS modulation of the vasoconstrictor response to phenylephrine.

Concentration-response curve to phenylephrine (Phe) in aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg) in the absence (Control) and the presence of the NADPH oxidase inhibitor Apocynin (0.3 mM). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of rats is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curve to phenylephrine (dAUC) in aortic segments incubated in the absence and the presence of apocynin of the four experimental groups. * P<0.05 vs. Untreated and ^# vs. HgCl₂-treated by t-test.

Figure 7. Effect of apocynin treatment on lipid peroxidation and thiol groups in plasma.

Values of (A) TBARS and (B) thiol (SH) groups in the plasma of rats untreated (n = 7) and treated with mercury (HgCl₂, n = 7), apocynin (Apo, n = 6) and apocynin plus mercury (ApoHg, n = 9). Data are expressed as the mean±SEM, t-test *P<0.05 vs. Untreated and ^#P<0.05 vs. HgCl₂-treated.

Figure 8. Effect of apocynin treatment on SOD and GPx activity.

Values of (A) SOD and (B) GPx activities in the aortas of rats untreated (n = 6) and treated with mercury (HgCl₂, n = 6), apocynin (Apo, n = 6) and apocynin plus mercury (ApoHg, n = 6). Data are expressed as the mean±SEM. t-test *P<0.05 vs. Untreated and ^#P<0.05 vs. HgCl₂-treated.

Figure 9. Effect of apocynin treatment on prostanoid modulation of the vasoconstrictor response to phenylephrine.

Concentration-response curve to phenylephrine (Phe) in aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg) in the absence (Control) and the presence of the non-selective COX inhibitor Indomethacin (1 µM). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of rats is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curve to phenylephrine (dAUC) in aortic segments in the absence and the presence of indomethacin of the four experimental groups. * P<0.05 vs. Untreated by t-test.