8-hydroxy-2--deoxyguanosine and Dry-Eye-Syndromes

Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Corneal Injuries; Disease Models, Animal; Dry Eye Syndromes; Epithelial Cells; Epithelium, Corneal; Gene Expression Regulation; Glutamate-Cysteine Ligase; Humans; Keratitis; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Rats; Reactive Oxygen Species; RNA, Messenger; Scopolamine; Triterpenes; Wound Healing

An imbalance between free radical generation and radical scavenging antioxidant systems results in oxidative stress, which has been associated with cell injury observed in many age-related diseases. The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase-1 (Sod1) in mice leads to many different phenotypes that resemble accelerated aging. In this study we examined the morphologic features and the secretory functions of the lacrimal glands in Sod1(-/-) mice. Lacrimal glands showed atrophy of acinar units; fibrosis; infiltration with CD4(+) T cells, monocytes, and neutrophils; increased staining with both 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine; increases in apoptotic cells; and the presence of the epithelial-mesenchymal transition in senescent Sod1(-/-) mice. Electron microscopy findings revealed evidence of epithelial-mesenchymal transition, presence of swollen and degenerated mitochondria, and the presence of apoptotic cell death in the lacrimal glands of senescent Sod1(-/-) mice. These alterations were also associated with the accumulation of secretory vesicles in acinar epithelial cells, decreased production of both stimulated and nonstimulated tears, and a decline in total protein secretion from the lacrimal glands. Our results suggest that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated lacrimal gland alterations.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Animals; Apoptosis; Cytokines; Deoxyguanosine; Disease Models, Animal; DNA Damage; Dry Eye Syndromes; Epithelial-Mesenchymal Transition; Fibrosis; Lacrimal Apparatus; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Mitochondria; Oxidative Stress; Superoxide Dismutase; Tears

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cells, Cultured; Cornea; Cyclooxygenase 2; Deoxyguanosine; Disease Models, Animal; Dry Eye Syndromes; Epithelial Cells; Epithelium, Corneal; Gene Expression; Heme Oxygenase-1; Humans; Interleukin-6; Lacrimal Apparatus; Lactoferrin; Male; Matrix Metalloproteinase 9; Ophthalmic Solutions; Oxidative Stress; Rats; Rats, Sprague-Dawley; Selenium

To investigate whether oxidative stress is involved in the etiology of the corneal disorder in blink-suppressed dry eye in a clinically relevant in vivo rat model.. A series of treatments were performed under continuous exposure to low-humidity airflow. Rats were placed on a jogging board (JB) made of a plastic pipe for 7.5 h/d, and for 16.5 hours, they were placed in individual cages without a JB. This protocol was repeated for up to 30 days. Corneal surface alteration was evaluated by the score of punctate fluorescein staining. To assess oxidative stress status, the levels of damaged DNA, and the protein modification by reactive aldehydes in corneal epithelia were detected by immunohistochemistry, using 8-hydroxy-2-deoxyguanosine, 4-hydroxynonenal- and malondialdehyde-specific antibodies.. Significant increases in the fluorescein staining score were observed from days 1 to 30 compared with the initial value. The average score for the dry eye group was significantly increased compared with that for the nontreatment group at all time points throughout the experiment. Immunoreactivity of all oxidative stress markers increased in the dry eye treatment. Quantitative analysis of the positive-stained cells showed a significant increase in the number of positive cells after 10 and 30 days in the dry eye treatment group compared with the nontreatment group.. These results suggest a relationship between the accumulation of oxidative stress and the etiology of corneal epithelial alterations in blink-suppressed dry eye.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Apoptosis; Biomarkers; Blinking; Cell Differentiation; Deoxyguanosine; Disease Models, Animal; Dry Eye Syndromes; Epithelium, Corneal; Female; Immunoenzyme Techniques; In Situ Nick-End Labeling; Malondialdehyde; Microscopy, Fluorescence; Oxidative Stress; Oxidoreductases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction