Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke

Abstract

Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP(-/-) mice. Aortic rings isolated from tobacco smoke-exposed GSTP(-/-) mice showed greater attenuation of ACh-evoked relaxation than those from GSTP(+/+) mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP(+/+) mice, modification of some proteins by acrolein was increased in the aorta of GSTP(-/-) mice. Aortic rings prepared from GSTP(-/-) mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP(+/+) mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.

Fig. 1.

Tissue abundance of glutathione-S-transferase (GST) isoform P (GSTP) protein. A: lung and aortic distribution of GSTP protein in male naïve wild-type (WT) and GSTP-null mice. Positive immunohistological staining was present in the WT lung (most intense in the pseudostratified columnar epithelium nuclei, arrows) and aorta but was absent in GSTP-null tissues. B: Western blots with anti-GSTP antibody using lysates obtained from the aorta, urinary bladder, stomach, small intestine, lung, liver, kidney, and heart from WT (+) and GSTP-null (−) mice. C: catalytic activity of GST in the mouse lung and aorta. Total GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) in the lung (n = 3 WT and 3 null) and whole aorta (n = 4 WT and 4 null) homogenates of WT and GSTP-null mice. *P < 0.05.

Fig. 2.

GSTP deficiency increased the susceptibility to tobacco smoke-induced endothelial dysfunction. Aortas were removed from WT and GSTP-null mice exposed for 3 days to cigarette smoke (CS) as described in the text, and their contractile and relaxant responses to cumulative concentrations of phenylephrine (PE; A and C) and ACh (B and D) were measured (n = 4 WT and 4 null). Control mice (n = 4 WT and 3 null) were exposed to air alone. *P < 0.05.

Fig. 3.

CS exposure (3 days) induced lung and liver antioxidant proteins. A: FGF-regulated protein (FR)-1 was induced in the lung of WT mice and was downregulated in GSTP-null mice. B: basal hepatic expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) was greater in GSTP-null mice compared with WT mice. CS exposure downregulated hepatic NQO1 expression in GSTP-null mice. C: basal hepatic expression of cytochrome P-450 (CYP)1A1/1A2 was greater in GSTP-null mice compared with WT mice. CS induced hepatic CYP1A1 expression in WT mice. D: catalytic activity of GST in the mouse lung. Total GST and GSTP activities in lung homogenates of WT and GSTP-null mice exposed to air or CS were measured using CDNB or ethacrynic acid (EA), respectively. Whole lung homogenate or hepatic microsomal proteins (25–100 μg) were blotted with polyclonal antibodies, and band density was normalized to total amido black staining. Protein expression was calculated as a percentage of WT air-exposed samples. *P < 0.05, significant difference compared with WT air-exposed mice (n = 3 mice/group).

Fig. 4.

Exposure to tobacco smoke results in the systemic delivery of acrolein. A–D: Western blots showing protein-acrolein adducts in the lung (A), plasma (B), and aorta (C and D) of mice exposed to air or CS. Adult male WT and GSTP-null mice were exposed for 5 h to tobacco smoke or air and euthanized immediately after exposure. Protein-acrolein adducts were detected in lysates of tissues frozen immediately after exposure using purified IgG rabbit polyclonal anti-keyhole limpet hemocyanin (KLH)-acrolein antibody. M, marker. Summary data of major protein-acrolein adducts in tissues were normalized to total amido black-stained protein. n = 3 mice/group in A, C, and D; n = 4 mice/group for B. *P < 0.05; †0.10 > P > 0.05.

Fig. 5.

GSTP-dependent endothelial dysfunction of inhaled acrolein. WT and GSTP-null mice were exposed to air or acrolein (1 ppm) by inhalation for 3 days (5 h/day) and killed 16 h after the final exposure. Isolated thoracic aortic (∼5 mm) sensitivities to PE (A and B) and ACh (C and D) were measured to assess contractility and endothelium-dependent relaxation, respectively. n = 4 WT mice (4 mice/group) and 8 null mice (8 mice/group). *P < 0.05.

Fig. 6.

Exposure to acrolein results in the systemic delivery of acrolein. A–D: Western blots showing protein-acrolein adducts in the lung (A), plasma (B), and aorta (C and D) of mice exposed to air or acrolein. Adult male WT and GSTP-null mice were exposed for 5 h to acrolein (5 ppm) or air and then euthanized immediately after exposure. Protein-acrolein adducts were detected in lysates of tissues frozen immediately after exposure using purified IgG rabbit polyclonal anti-KLH-acrolein antibody. Summary data of major protein-acrolein adducts in tissues were normalized to total amido black-stained protein. n = 3 mice/group for A, C, and D; n = 4 mice/group for B. *P < 0.05; †0.10 > P > 0.05.

Fig. 7.

Acrolein toxicity in WT and GSTP-null mice. A: ACh-stimulated relaxation of PE-precontracted aortas of male naïve C57BL/6 mice treated with acrolein. Naïve aortic rings (n = 3–5) were exposed to normal buffer (control) or acrolein (10, 40, or 80 μM) for 90 min before responses to ACh were measured in PE-precontracted aortas. B: effects of N-acetylcysteine (NAC) on ACh-induced relaxation in acrolein-pretreated aortas of naïve WT and GSTP-null mice. n = 4 mice/group. *P < 0.05 vs. GSTP-null control; †P < 0.05 vs. acrolein treatment in WT mice.