The β-Blocker Carvedilol Prevents Benzo(a)pyrene-Induced Lung Toxicity, Inflammation and Carcinogenesis

Abstract

The current study evaluated the effects of the β-blocker carvedilol on benzo(a)pyrene (B(a)P) and its active metabolite benzo(a)pyrene diol epoxide (BPDE)-induced lung toxicity, inflammation and carcinogenesis and explored the potential mechanisms. Carvedilol blocked the BPDE-induced malignant transformation of human bronchial epithelial cells BEAS-2B. In BEAS-2B cells, B(a)P strongly activated ELK-1, a transcription factor regulating serum response element (SRE) signaling, which was attenuated by carvedilol. Carvedilol also inhibited the B(a)P-induced AhR/xenobiotic responsive element (XRE) and mRNA expression of CYP1A1 and attenuated B(a)P-induced NF-κB activation. In a B(a)P-induced acute lung toxicity model in CD-1/IGS mice, pretreatment with carvedilol for 7 days before B(a)P exposure effectively inhibited the B(a)P-induced plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration and histopathologic abnormalities in the lung, and upregulated the expression of GADD45α, caspase-3 and COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol treatment for 20 weeks did not affect body weight but significantly attenuated tumor multiplicity and volume. These data reveal a previously unexplored role of carvedilol in preventing B(a)P-induced lung inflammation and carcinogenesis by inhibiting the cross-talk of the oncogenic transcription factors ELK-1, AhR and NF-κB.

Keywords: benzo(a)pyrene; carvedilol; lung cancer; tobacco smoking; β-blocker.

Figure 1

Effects of carvedilol on BPDE-induced lung epithelial transformation. (A) The active metabolite of B(a)P, benzo[a]pyrene diol epoxide (BPDE), was used to induce transformation of BEAS-2B cells. The cells were pretreated with carvedilol for 2 h and then treated with 0.2 μM of BPDE for 1 h, and the cells were cultured in the presence of carvedilol for 7 days. The cells were then seeded in soft agar in 96-well plate (2000 cells/well) with carvedilol in the top layer of agar. Cell colonies were counted under the microscope after 10 days of incubation. (B) Representative images were taken using GelCount™ of the wells containing colonies growing on agar after 10 days incubation. (C) Cell viability of BEAS-2B after being treated with carvedilol at various doses for 24, 48 or 72 h based on MTT assay. Data plotted are mean ± SD; n = 6. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. ****: p < 0.0001.

Figure 2

Effects of carvedilol on B(a)P-induced activation of ELK/SRE, NF-κB and AhR/XRE and expression of CYP1A1 mRNA. (A) Effects of carvedilol on B(a)-induced activation of ELK-1/SRE promoter. The BEAS-2B cells expressing exogenously transfected ELK-1/SRE luciferase reporter and the Renilla control reporter (10:1) were treated with vehicle, 10 µM B(a)P, with or without carvedilol for 24 h before dual luciferase assay. (B) Effect of carvedilol on B(a)P-induced NF-κB promoter activity. The BEAS-2B cells expressing exogenously transfected NF-κB luciferase reporter and Renilla control reporter (10:1) were treated with vehicle, 10 µM B(a)P, with or without carvedilol for 24 h before dual luciferase assay. (C) Effects of carvedilol on B(a)P-induced AhR/XRE promoter activity. The BEAS-2B cells expressing exogenously transfected AhR/XRE luciferase reporter and Renilla control reporter (10:1) were treated with vehicle, 10 µM B(a)P, with or without carvedilol for 24 h before dual luciferase assay. (D) Effect of carvedilol on B(a)P-induced mRNA expression of CYP1A1 determined using qRT-PCR. The BEAS-2B cells were treated with B(a)P (10 µM), with or without carvedilol (5.0 µM), for 6 h, before RNA isolation. Data plotted are mean +/− SD. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. *: p < 0.05; **: p < 0.01; ***: p <0.001; ****: p < 0.0001. ns: non-significant.

Figure 3

Effects of carvedilol on B(a)P-induced lung toxicity. (A) Experimental design. The CD-1/IGS mice were pretreated with carvedilol (20 mg/kg/day) or curcumin (100 mg/kg/day) by oral gavage for 7 days before B(a)P exposure. The mice were euthanized 24 h after B(a)P exposure. (B) Effects of carvedilol and curcumin on B(a)P-induced plasma levels of malondialdehyde (MDA), a marker to assess lipid peroxidation (LPO). (C) Effects of carvedilol and curcumin on B(a)P-induced plasma levels of lactate dehydrogenase (LDH), a marker for tissue damage. (D) Effects of carvedilol on B(a)P-induced histopathologic changes. H&E staining of mouse lung sections of the lung (×20). Scale bar: 100 μm. (E) IHC analysis for expression of COX-2, caspase-3 and GADD45α (×20). Scale bars: 100 μm. Data plotted are mean + SD. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 4

Effects of carvedilol on B(a)P-induced lung carcinogenesis. (A) Experimental design. The A/J mice were divided into five groups: (1) negative control group without B(a)P exposure (n = 5); (2) B(a)P-only group (n = 17); (3) B(a)P and treated with gefitinib (n = 16); (4) B(a)P and treated with 3.2 mg/kg carvedilol (n = 16); and (5) B(a)P and treated with 20 mg/kg carvedilol (n = 16). The drug treatment started three weeks before the B(a)P exposure. The dose of gefitinib was 400 mg/kg, given by oral gavage once a week. Carvedilol was given in the drinking water. (B) Body weight changes over the course of the studies. (C) Count of tumor numbers on the lung surface. (D) The tumor volume at the surface of the lung. Data are expressed as mean ± SD. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5

Effects of carvedilol on B(a)P-induced lung carcinogenesis. (A) Representative H&E-stained cross-sections of lungs from each treatment group (×4). Scale bars: 500 μm. (B) Number of tumors in H&E-stained cross-sectional areas of each block. Data are expressed as mean ± SD. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 6

Effects of carvedilol on B(a)P-induced Ki-67 expression in lung tumors. (A) Representative cross-section images of lung after immunohistochemical staining with Ki-67 antibody (×20). Scale bars: 100 μm. (B) Number of Ki-67 positive cells in lung tumors per microscopic field. Data are expressed as mean ± SD. An ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was used to assess statistical differences. **: p < 0.01, ****: p < 0.0001.