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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

Both HO-1 and CORM-2 protect against CE-induced ROS production.(A) BREA-2B cells pretreated with 10 μM ZnPP or 10 μM CORM-2 were either unlabeled or labeled with 20 μM DCFDA for 30 minutes and then cultured for an additional 3 hours with or without 150 ppm or 300 ppm CE according to the protocol. Cells were then analyzed on flow cytometry. The mean fluorescence intensity (MFI) of DCFDA expression by no DCFDA (gray-filled histogram), with DCFDA no CE (Dashed line histogram) or with DCFDA and with CE treatment (Solid line histogram) were compared and shown in the histogram. Data shown are representative of three experiments. (B) For comparison, the MFI ratio (MFIR) was calculated by dividing the MFI of (+) CE treatments (solid line) by the MFI of the (-) CE with DCFDA (dashed line), respectively. The MFIR of DCFDA staining is shown as mean ± SD of 3 donors. ** p < 0.01 vs control (+) CE 150 ppm; ## p < 0.01 vs control (+) CE 300; && p < 0.01 vs (+) CE 150 ppm in the presence of ZnPP; §§ p < 0.01 vs (+) CE 150 ppm in the presence of CORM-2 by a one-way ANOVA with HSD test.
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pone.0122275.g004: Both HO-1 and CORM-2 protect against CE-induced ROS production.(A) BREA-2B cells pretreated with 10 μM ZnPP or 10 μM CORM-2 were either unlabeled or labeled with 20 μM DCFDA for 30 minutes and then cultured for an additional 3 hours with or without 150 ppm or 300 ppm CE according to the protocol. Cells were then analyzed on flow cytometry. The mean fluorescence intensity (MFI) of DCFDA expression by no DCFDA (gray-filled histogram), with DCFDA no CE (Dashed line histogram) or with DCFDA and with CE treatment (Solid line histogram) were compared and shown in the histogram. Data shown are representative of three experiments. (B) For comparison, the MFI ratio (MFIR) was calculated by dividing the MFI of (+) CE treatments (solid line) by the MFI of the (-) CE with DCFDA (dashed line), respectively. The MFIR of DCFDA staining is shown as mean ± SD of 3 donors. ** p < 0.01 vs control (+) CE 150 ppm; ## p < 0.01 vs control (+) CE 300; && p < 0.01 vs (+) CE 150 ppm in the presence of ZnPP; §§ p < 0.01 vs (+) CE 150 ppm in the presence of CORM-2 by a one-way ANOVA with HSD test.

Mentions: Having established that CE exposure induces apoptosis of epithelial cells, we next investigated the underlying molecular mechanisms causing cell death. Overproduction of ROS can cause apoptosis by inducing mitochondrial dysfunction and subsequent release of pro-apoptotic factors [16,17] and ROS directly induces caspase-3-dependent apoptosis [58]. Intracellular ROS generation was evaluated by pre-incubating BEAS-2B cells with a fluorescent probe, DCFDA, which can be oxidized by H2O2. ROS generation was augmented after 3 hours of exposure to CE in a dose-dependent manner (Fig. 4). Similar experiments were performed on gill cells from zebrafish after loading with 2’7’-dichlorofluorescein (DCF, Invitrogen, Grand Island, NY). The augmentation of ROS production was similar to the results obtained from the DCFDA staining (data not shown). We also found that treatment with HO-1 inhibitor, ZnPP, significantly enhanced CE-induced apoptosis (Fig. 2A). Our results suggest that ROS production and oxidative stress lead to apoptosis in epithelial cells exposed to CE.


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)

Both HO-1 and CORM-2 protect against CE-induced ROS production.(A) BREA-2B cells pretreated with 10 μM ZnPP or 10 μM CORM-2 were either unlabeled or labeled with 20 μM DCFDA for 30 minutes and then cultured for an additional 3 hours with or without 150 ppm or 300 ppm CE according to the protocol. Cells were then analyzed on flow cytometry. The mean fluorescence intensity (MFI) of DCFDA expression by no DCFDA (gray-filled histogram), with DCFDA no CE (Dashed line histogram) or with DCFDA and with CE treatment (Solid line histogram) were compared and shown in the histogram. Data shown are representative of three experiments. (B) For comparison, the MFI ratio (MFIR) was calculated by dividing the MFI of (+) CE treatments (solid line) by the MFI of the (-) CE with DCFDA (dashed line), respectively. The MFIR of DCFDA staining is shown as mean ± SD of 3 donors. ** p < 0.01 vs control (+) CE 150 ppm; ## p < 0.01 vs control (+) CE 300; && p < 0.01 vs (+) CE 150 ppm in the presence of ZnPP; §§ p < 0.01 vs (+) CE 150 ppm in the presence of CORM-2 by a one-way ANOVA with HSD test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383564&req=5

pone.0122275.g004: Both HO-1 and CORM-2 protect against CE-induced ROS production.(A) BREA-2B cells pretreated with 10 μM ZnPP or 10 μM CORM-2 were either unlabeled or labeled with 20 μM DCFDA for 30 minutes and then cultured for an additional 3 hours with or without 150 ppm or 300 ppm CE according to the protocol. Cells were then analyzed on flow cytometry. The mean fluorescence intensity (MFI) of DCFDA expression by no DCFDA (gray-filled histogram), with DCFDA no CE (Dashed line histogram) or with DCFDA and with CE treatment (Solid line histogram) were compared and shown in the histogram. Data shown are representative of three experiments. (B) For comparison, the MFI ratio (MFIR) was calculated by dividing the MFI of (+) CE treatments (solid line) by the MFI of the (-) CE with DCFDA (dashed line), respectively. The MFIR of DCFDA staining is shown as mean ± SD of 3 donors. ** p < 0.01 vs control (+) CE 150 ppm; ## p < 0.01 vs control (+) CE 300; && p < 0.01 vs (+) CE 150 ppm in the presence of ZnPP; §§ p < 0.01 vs (+) CE 150 ppm in the presence of CORM-2 by a one-way ANOVA with HSD test.
Mentions: Having established that CE exposure induces apoptosis of epithelial cells, we next investigated the underlying molecular mechanisms causing cell death. Overproduction of ROS can cause apoptosis by inducing mitochondrial dysfunction and subsequent release of pro-apoptotic factors [16,17] and ROS directly induces caspase-3-dependent apoptosis [58]. Intracellular ROS generation was evaluated by pre-incubating BEAS-2B cells with a fluorescent probe, DCFDA, which can be oxidized by H2O2. ROS generation was augmented after 3 hours of exposure to CE in a dose-dependent manner (Fig. 4). Similar experiments were performed on gill cells from zebrafish after loading with 2’7’-dichlorofluorescein (DCF, Invitrogen, Grand Island, NY). The augmentation of ROS production was similar to the results obtained from the DCFDA staining (data not shown). We also found that treatment with HO-1 inhibitor, ZnPP, significantly enhanced CE-induced apoptosis (Fig. 2A). Our results suggest that ROS production and oxidative stress lead to apoptosis in epithelial cells exposed to CE.

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