Overexpression of Cu/Zn-superoxide dismutase and/or catalase accelerates benzo(a)pyrene detoxification by upregulation of the aryl hydrocarbon receptor in mouse endothelial cells

Abstract

A reduction in endogenously generated reactive oxygen species in vivo delays benzo(a)pyrene (BaP)-accelerated atherosclerosis, as revealed in hypercholesterolemic mice overexpressing Cu/Zn-superoxide dismutase (SOD) and/or catalase. To understand the molecular events involved in this protective action, we studied the effects of Cu/Zn-SOD and/or catalase overexpression on BaP detoxification and on aryl hydrocarbon receptor (AhR) expression and its target gene expression in mouse aortic endothelial cells (MAECs). Our data demonstrate that overexpression of Cu/Zn-SOD and/or catalase leads to an 18- to 20-fold increase in the expression of AhR protein in MAECs. After BaP exposure, the amount of AhR binding to the cytochrome P450 (CYP) 1A1 promoter was significantly greater, and the concentrations of BaP reactive intermediates were significantly less in MAECs overexpressing Cu/Zn-SOD and/or catalase than in wild-type cells. In addition, the BaP-induced CYP1A1 and 1B1 protein levels and BaP-elevated glutathione S-transferase (GST) activity were significantly higher in these transgenic cells, in parallel with elevated GSTp1, CYP1A1, and CYP1B1 mRNA levels, compared to wild-type MAECs. Moreover, knockdown of AhR with RNA interference diminished the Cu/Zn-SOD and catalase enhancement of CYP1A1 expression, GST activity, and BaP detoxification. These data demonstrate that overexpression of Cu/Zn-SOD and/or catalase is associated with upregulation of AhR and its target genes, such as xenobiotic-metabolizing enzymes.

Fig. 1

Overexpression of Cu/Zn-SOD and/or catalase reduces BaP-induced ROS. MAECs obtained from wild-type (WT) and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C) were incubated with 1 μM BaP for 30 min or culture medium alone as a control. The level of O₂^•− and the release of H₂O₂ in MAECs were measured as described under Materials and methods. Values represent the means±SEM of four separate experiments in which MAECs were pooled from four mice. *P<0.01 vs the same genotype cells without BaP treatment, and ^†P<0.01 vs BaP-treated wild-type cells.

Fig. 2

Overexpression of Cu/Zn-SOD and/or catalase upregulates AhR expression. The AhR protein levels were measured by Western blot analysis and were expressed as a ratio of their immunoblot intensity relative to β-actin. (A) MAECs obtained from wild-type (WT) mice were incubated with 1 μM BaP for the indicated hours. (B–E) MAECs obtained from WT mice and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C) were treated with1 μM BaP for 4 h. (B) Representative images of Western blots. (C, D, and E) AhR protein levels in the whole-cell lysate and cytosolic and nuclear fractions, respectively. Values represent the means±SEM of five separate experiments in which MAECs were pooled from four mice. *P < 0.05 vs the same genotype cells without BaP treatment, ^†P < 0.01 vs WT cells without BaP treatment, and ^#P < 0.01 vs BaP-treated WT cells.

Fig. 3

BaP increases the DNA-binding activity of AhR. MAECs obtained from wild-type (WT) mice and transgenic mice overexpressing Cu/Zn-SOD (SOD), catalase (CAT), or both Cu/Zn-SOD and catalase (S/C) were treated with 1 μM BaP for 4 h. The binding activity of AhR to the CYP1A1 promoter region was measured with ChIP analysis using two pairs of PCR primers: (a) a primer pair probing the two AhR binding sites in the enhancer region of the CYP1A1 promoter and (b) a primer pair probing the AhR binding site adjacent to the transcription starting site. Values represent the means±SEM of four separate experiments in which MAECs were pooled from four mice. *P < 0.01 vs the same genotype cells without BaP treatment, ^†P < 0.05 vs BaP-treated WT cells.

Fig. 4

Overexpression of Cu/Zn-SOD and/or catalase enhances BaP-induced CYP1A1 and CYP1B1 expression. MAECs were obtained from wild-type (WT) mice and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C). The cells were incubated with 1 μM BaP for 4 h to determine protein levels and for 2 h to measure mRNA levels. The CYP1A1 and 1B1 protein levels in the whole-cell lysate were measured by Western blot analysis and were expressed as a ratio of their immunoblot intensity relative to β-actin. The CYP1A1 and 1B1 mRNA levels were determined by quantitative real-time RT-PCR and expressed relative to the GAPDH mRNA level. Values represent the means±SEM of five separate experiments in which MAECs were pooled from four mice. *P < 0.01 vs the same genotype cells without BaP treatment, ^†P < 0.01 vs BaP-treated WT cells.

Fig. 5

Overexpression of Cu/Zn-SOD and/or catalase enhances BaP-induced GSTp1 mRNA and total GST activity. MAECs were obtained from wild-type (WT) mice and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C). The cells were incubated with 1 μM BaP for 4 h to determine total GST activity and for 2 h to measure GSTp1 mRNA level. Total GST activity in the whole-cell lysate was determined as described under Materials and methods and expressed as the amount of CDNB conjugated with reduced glutathione. The mRNA level of GSTp1 was determined by quantitative real-time RT-PCR and expressed relative to the GAPDH mRNA level. Values represent the means±SEM of five separate experiments in which MAECs were pooled from four mice. *P < 0.05 vs the same genotype control cells without BaP treatment, ^†P < 0.05 vs BaP-treated WT cells.

Fig. 6

Overexpression of Cu/Zn-SOD and/or catalase reduces BaP reactive intermediates and lipid peroxidation. MAECs obtained from wild-type (WT) mice and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C) were incubated with 1 μM BaP for 8 h. (A) BaP reactive metabolites were determined with a reverse-phase HPLC system with fluorescence detection. The concentrations of (B) F₂-isoprostanes and (C) isofurans in the MAECs were measured using GC/MS. Values represent the means±SEM of five separate experiments in which MAECs were pooled from four mice. *P < 0.05 vs BaP-treated WT cells, ^†P < 0.05 vs the same genotype cells without BaP treatment.

Fig. 7

Knockdown of AhR inhibits BaP detoxification and diminishes BaP-induced gene expression in MAECs overexpressing Cu/Zn-SOD and/or catalase. MAECs obtained from wild-type (WT) mice and transgenic mice overexpressing Cu/Zn-SOD, catalase (CAT), or both Cu/Zn-SOD and catalase (S/C) were transfected with AhR siRNA or control siRNA. The cells were incubated with 1 μM BaP or culture medium alone for 4 h for determining protein levels, 2 h for determining mRNA levels, and 8 h for determining BaP metabolites. (A and B) The AhR protein level in the whole-cell lysate was measured by Western blot analysis and expressed as a percentage of their immunoblot intensity relative to β-actin. The mRNA levels of (C) AhR, (E) CYP1B1, (F) CYP1A1, and (G) GSTp1 were determined by quantitative real-time RT-PCR and expressed relative to GAPDH mRNA level. (H) The total GST activity was determined using a Cayman GST activity assay kit. (D) BaP reactive metabolites, including 9-hydroxy-BaP, 3-hydroxy-BaP, 9,10-dihydroxy-BaP, 7,8-dihydro-BaP, 4,5-dihydroxy-BaP, and 3,6-dihydroxy-BaP, were determined with an HPLC system and expressed in picomoles of total BaP reactive metabolites per milligram of cellular proteins. Values represent the means±SEM of five separate experiments in which MAECs were pooled from four mice. *P < 0.05 vs the same genotype cells transfected with control siRNA, ^†P < 0.05 vs WT cells transfected with control siRNA, and ^#P < 0.05 vs WT cells transfected with AhR siRNA.