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. 2013 Oct 15;272(2):345-55.
doi: 10.1016/j.taap.2013.06.025. Epub 2013 Jul 9.

The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

Ruijin Zheng  1 , Iris Po, Vladimir Mishin, Adrienne T Black, Diane E Heck, Debra L Laskin, Patrick J Sinko, Donald R Gerecke, Marion K Gordon, Jeffrey D Laskin
Affiliations

The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

Ruijin Zheng et al. Toxicol Appl Pharmacol. .

Abstract

The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5 J/cm(2)) or nitrogen mustard (100 nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10h with 30 μM 4-HNE or 6h with 10 μM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard.

Keywords: 4-Hydroxynonenal; 9-Nitrooleic acid; Cornea; Nitrogen mustard; Nitrosative stress; UVB.

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Figures

Figure 1
Figure 1. Morphology of rabbit corneas treated with UVB or nitrogen mustard
Cornea organ cultures were exposed to control, UVB (0.5 J/cm2) or nitrogen mustard (NM, 100 nmol). Black arrows indicate areas of separation of epithelium from the stroma following UVB or NM treatment. After 3 hr and 6 hr, histological sections were prepared and stained with hematoxylin and eosin. Original magnification x 400
Figure 2
Figure 2. Effects of UVB on 4-HNE formation in rabbit corneas
Cornea organ cultures were exposed to control or UVB (0.5 J/cm2). After 3 hr and 6 hr, histological sections were prepared and the central portions of the corneas were analyzed for 4-HNE using mouse monoclonal 4-HNE primary antibody and Alexa-Fluor 488 labeled secondary antibody. Nuclei were visualized using DAPI staining. White arrows and arrowheads indicate areas of 4HNE adduct formation on the apical epithelial surface and basal epithelial surface, respectively. Original magnification x 400
Figure 3
Figure 3. Effects of nitrogen mustard on 4-HNE formation in rabbit corneas
Cornea organ cultures were treated with control or 100 nmol nitrogen mustard. After 3 hr and 6 hr, histological sections were prepared and the central portions of the corneas were analyzed for 4-HNE using mouse monoclonal primary 4-HNE antibody and Alexa-Fluor 488-labeled secondary antibody. Nuclei were visualized using DAPI staining. White arrows and arrowheads indicate areas of 4-HNE adduct formation on the apical epithelial surface and basal epithelial surface, respectively. Original magnification x 400
Figure 4
Figure 4. Effects of UVB on HO-1 expression in rabbit corneas
Cornea organ cultures were exposed to control or UVB (0.5 J/cm2). After 3 hr and 6 hr, histological sections were prepared and the central portions of corneas analyzed for HO-1 expression using mouse monoclonal primary HO-1 antibody and Alexa-Flour 488-labeled secondary antibody. Nuclei were visualized using DAPI staining. White arrows and arrowheads indicate areas of HO-1 formation on the apical epithelial surface and basal epithelial surface, respectively. Original magnification x 400
Figure 5
Figure 5. Effects of nitrogen mustard on HO-1 expression in rabbit corneas
Cornea organ cultures were treated with control or 100 nmol nitrogen mustard. After 3 hr and 6 hr, histological sections were prepared and the central portions of corneas analyzed for HO-1 expression using mouse monoclonal HO-1 antibody and Alexa-Flour 488-labeled secondary antibody. Nuclei were visualized using DAPI staining. White arrows and arrowheads indicate areas of HO-1 formation on the apical epithelial surface and basal epithelial surface, respectively. Original magnification x 400
Figure 6
Figure 6. 4-HNE metabolism in human corneal epithelial cells
Cell suspensions (2 × 106/ml) were incubated with 100 μM 4-HNE. At the indicated time points, reactions were stopped by the addition of an equal volume of acetonitrile/acetic acid (96:4, v/v). Top panel: HPLC analysis of 4-HNE metabolism. After stopping the reaction, cells were pelleted and clear supernatants analyzed by HPLC. Middle panel: Kinetics of 4-HNE metabolism. Bottom panel: Cells were extracted and analyzed by HPLC after treatment with 100 μM 4-HNE for 5, 15, 30, 60 and 120 min.
Figure 7
Figure 7. Formation of 4-HNE-protein adducts in human corneal epithelial cells
Cells were treated with vehicle control (C) or 4-HNE (30 μM) for 0, 15, 30, 60 and 90 min. Cell lysates were then prepared, and protein analyzed by western blotting using mouse monoclonal antibody to 4-HNE. Each lane contained 10 μg of corneal epithelial protein.
Figure 8
Figure 8. Effects of electrophilic lipid peroxidation products on HO-1 expression in human corneal epithelial cells
Cells were treated with vehicle control (C), 30 μM 4-HNE (left panels) or 10 μM 9-nitrooleic acid (9-NO, right panels). mRNA or protein was extracted from the cells at the indicated times and analyzed by real time PCR (upper panels) and western blotting (lower panels), respectively. β-actin was used as a protein loading control. Data are presented as the mean ± SE (n = 3). *Significantly different from control (P < 0.05).
Figure 9
Figure 9. Role of MAP kinases and PI3/Akt kinase signaling in 4-HNE-induced HO-1 expression in human corneal epithelial cells
Panel A: Effects of 4-HNE on expression of MAP kinases and PI3K/Akt kinase. Cells were treated with control (C) or 30 μM 4HNE for 15, 30, 60 and 90 min. Cell lysates were prepared and analyzed for total and phosphorylated p38, JNK, Erk1/2, or PI3/Akt by western blotting. Panel B: Effects of MAP kinase and PI3K/Akt inhibitors on 4-HNE-induced HO-1 expression. Cells were pre-incubated with inhibitors to p38 (SB203580,10 μM), JNK (SP600125, 20 μM), Erk1/2 (PD98059, 10 μM) or PI3K (wortmannin, 0.1 μM) for 3 hr and then treated with 30 μM 4-HNE for additional 6 hr or 10 hr. Total cell lysates were prepared and analyzed for HO-1 protein expression by western blotting.
Figure 10
Figure 10. Role of MAP kinases and PI3/Akt kinase signaling in 9-nitrooleic acid-induced HO-1 expression in human corneal epithelial cells
Panel A: Effects of 9-nitrooleic acid on expression of MAP kinases and PI3K/Akt kinases. Cells were treated with control (C) or 10 μM 9-nitrooleic acid for 15, 30, 60 and 90 min. Cell lysates were prepared and analyzed for total and phosphorylated p38, JNK, Erk1/2, or PI3/Akt using western blotting. Panel B: Effects of MAP kinase and PI3/Akt inhibitors on 9-nitrooleic acid-induced HO-1 expression. Human epithelial cells were pre-incubated with inhibitors to p38 (SB203580,10 μM), JNK (SP600125, 20 μM), Erk1/2 (PD98059, 10 μM) or PI3/Akt kinase (wortmannin, 0.1 μM) for 3 hr and then treated with 10 μM 9-nitrooleic acid for additional 6 hr. Total cell lysates were then prepared and analyzed for HO-1 protein expression by western blotting.
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