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Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

Li FJ, Duggal RN, Oliva OM, Karki S, Surolia R, Wang Z, Watson RD, Thannickal VJ, Powell M, Watts S, Kulkarni T, Batra H, Bolisetty S, Agarwal A, Antony VB - PLoS ONE (2015)

Bottom Line: CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production.Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis.Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States of America.

ABSTRACT
The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

No MeSH data available.


Related in: MedlinePlus

Upregulation of HO-1 and NOX4 in response to CE stimulation.Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** p < 0.01 vs control by a one-way ANOVA with HSD test.
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pone.0122275.g005: Upregulation of HO-1 and NOX4 in response to CE stimulation.Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** p < 0.01 vs control by a one-way ANOVA with HSD test.

Mentions: To test the hypothesis that CE induced NOX4 expression, we characterized the NOX4 expression in gills from zebrafish and BEAS-2B epithelial cells. By IHC analysis, we found that expression of NOX4 increased when zebrafish were exposed to CE (Fig. 5B) but no significant changes occur in NOX1, NOX2 and NOX3 expression (data not shown). Western blot analysis also confirmed that NOX4 protein levels increased significantly when BEAS-2B cells were treated with CE at concentrations of 50 to 150 ppm for 4 hours (Fig. 5C). Therefore, generation of ROS on exposure to CE may be, in part a consequence of upregulation of NOX4.


Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

Li FJ, Duggal RN, Oliva OM, Karki S, Surolia R, Wang Z, Watson RD, Thannickal VJ, Powell M, Watts S, Kulkarni T, Batra H, Bolisetty S, Agarwal A, Antony VB - PLoS ONE (2015)

Upregulation of HO-1 and NOX4 in response to CE stimulation.Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** p < 0.01 vs control by a one-way ANOVA with HSD test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383564&req=5

pone.0122275.g005: Upregulation of HO-1 and NOX4 in response to CE stimulation.Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** p < 0.01 vs control by a one-way ANOVA with HSD test.
Mentions: To test the hypothesis that CE induced NOX4 expression, we characterized the NOX4 expression in gills from zebrafish and BEAS-2B epithelial cells. By IHC analysis, we found that expression of NOX4 increased when zebrafish were exposed to CE (Fig. 5B) but no significant changes occur in NOX1, NOX2 and NOX3 expression (data not shown). Western blot analysis also confirmed that NOX4 protein levels increased significantly when BEAS-2B cells were treated with CE at concentrations of 50 to 150 ppm for 4 hours (Fig. 5C). Therefore, generation of ROS on exposure to CE may be, in part a consequence of upregulation of NOX4.

Bottom Line: CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production.Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis.Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States of America.

ABSTRACT
The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

No MeSH data available.


Related in: MedlinePlus