Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether hydrogen peroxide (H2O2), a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells. METHODS: To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50-200 mM H2O2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-rho-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting. RESULTS: Caspase-3 activity significantly increased in cells exposed to H2O2. Also, the expression of bcl-2 in cells treated with 200 microM H2O2 was diminished, whereas expression of p53 and p21waf-1 was increased compared to the controls. CONCLUSIONS: Exposure of retinal pigment epithelial cells to concentrations of H2O2 that cause in vitro mitochondrial DNA damage also promotes apoptosis.

The color difference in orbital fat.

OBJECTIVE: To identify and quantify carotenoids found in white and yellow orbital fat. METHODS: Specimens of nasal (white) and preaponeurotic (yellow) orbital fat were obtained from patients during upper eyelid blepharoplasty. Carotenoids and retinoids were extracted and subjected to spectral and high-performance liquid chromatography analyses. RESULTS: The chromophore content of extracts from unsaponified fat, as measured by absorbance at 425 nm per gram of fat, was 2- to 4-fold higher in preaponeurotic fat than in nasal fat. High-performance liquid chromatography analysis from enzymatically digested fat revealed large amounts of lutein, beta-carotene, and retinol and small amounts of other unidentified carotenoids. The amount of beta-carotene and lutein in preaponeurotic fat was approximately 4-fold higher than in nasal fat. CONCLUSIONS: The higher carotenoid content of preaponeurotic fat might cause it to be more yellow than other orbital fat, and lutein and beta-carotene might be selectively absorbed from plasma by preaponeurotic fat. CLINICAL RELEVANCE: The results provide baseline information for studies of the physiological features of orbital fat in normal and diseased conditions.

Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium.

PURPOSE: To investigate the interrelationships between DNA damage, mitochondrial activity, and apoptosis in retinal pigment epithelial cells (RPE) after exposure to rod outer segments (ROS). METHODS: After incubation of cultured human RPE with ROS, mitochondrial redox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidyl serine (PS) using fluorescence microscopy. The expression of the pro-apoptotic proteins, p53 and p21(waf-1), and DNA repair enzymes, apurinic/apyrimidinic endonuclease (APE(ref-1)) and DNA polymerase beta (beta-pol) were quantitatively determined by Western blotting analysis. RESULTS: Mitochondrial function decreased by 20 +/- 5% and annexin V immunofluorscent binding was enhanced after exposure of cells to physiological levels of ROS (3.8 x 10(6)cm(-2)) for 4 h. MtDNA was preferentially damaged after exposure to ROS with increased lesion frequencies of 1.49 +/- 0.37 and 2.2 +/- 0.14 per 10 kb base pairs (bp), respectively after 5 and 7 h contact, compared to untreated controls (zero class damage). APE(ref-1)expression increased more than 340% above controls after exposure to ROS for 7 and 24 h. The expression of beta-pol in cultures increased 110% above controls after 24 h contact with the ROS. The expression of p53 and p21 in cells increased 100 and 38% above controls after 24 h exposure to the ROS. CONCLUSIONS: Exposure of ROS to ROS induced mtDNA damage and dysfunction and activated nDNA repair pathways, which did not prevent apoptosis.

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase.

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na+-K+-ATPase activity as an index. Beta-carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gas-liquid chromatography and high performance liquid chromatography. The Na+-K+-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of beta-carotene oxidative breakdown products, beta-Apo-10'-carotenal and retinal. Most of the beta-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na+-K+-ATPase activity by 50% (IC50) was equivalent to 10 microM non-degraded beta-carotene, whereas the IC50 for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 microM. Carotenoid oxidation products are more potent inhibitors of Na+-K+-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or beta-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.

Recognizing an index case of tuberous sclerosis.

Tuberous sclerosis is the most common neurocutaneous syndrome after neurofibromatosis. Dermatologic manifestations may be the only clues the family physician has to the diagnosis of the disorder, which is also marked by childhood seizures and mental retardation. Characteristic signs of tuberous sclerosis vary widely in severity and can include hypopigmented "ash-leaf spots," fibrous plaques on the forehead, angiofibromas on the face (adenoma sebaceum), a shagreen patch on the lower back and fibromas of the nails. Computed tomographic scanning or magnetic resonance imaging reveal subependymal nodules or cortical "tubers" in the brain. Associated cardiac, retinal, renal and pulmonary pathology can increase morbidity and mortality. Genetic counseling is helpful but has limited use because of the variation in genetic expression and the frequency of new gene mutations that cause this disorder.

Lutein and zeaxanthin are associated with photoreceptors in the human retina.

PURPOSE: Previous studies showed that lutein and zeaxanthin, the major human retinal carotenoids, are concentrated in the macula. In this study, the carotenoids in human macular and peripheral retina and the retinal pigment epithelium (RPE) were analyzed. They were also determined in the rod outer segments (ROS) before and after removal of extrinsic membrane proteins. METHODS: Carotenoids were extracted from the macular and peripheral sections of human retina and RPE with hexane in dim light and analyzed by high performance liquid chromatography (HPLC). ROS samples equivalent to the amount in a single retina were also analyzed. RESULTS: Retinal carotenoid amounts were similar to previous reports, but only low levels were detected in the RPE. Regional ratios of lutein:zeaxanthin were similar in the retina and RPE. Approximately 25% of the total retinal carotenoids were found in the ROS, indicating that a substantial portion of peripheral retinal carotenoids are present in the ROS. However, after removal of the extrinsic membrane proteins and subsequent analysis, carotenoids were not detected. CONCLUSIONS: Most of the carotenoids in the human peripheral retina are present in the ROS. These ROS carotenoids are associated with soluble or salt-dependently bound proteins.

The sensitivity of bovine corneal epithelial lyso-PAF acetyltransferase to cyclooxygenase and lipoxygenase inhibitors is independent of arachidonate metabolites.

Lyso-platelet-activating factor (PAF) acetyltransferase is a critical regulatory step in PAF synthesis. PAF accumulates in the cornea in response to injury and is a potent inflammatory mediator that stimulates corneal cyclooxygenase but not lipoxygenase reactions. 12(S)-hydroxyeicosatetraenoic (HETE) acid, a major lipoxygenase product of mammalian corneas, is also generated during injury. In the bovine corneal epithelium, both PAF acetyltransferase and 12-lipoxygenase are microsomal enzymes. A potential interaction between these two lipid mediators was, therefore, examined. PAF acetyltransferase activity was assayed by determining radioactivity in the trichloroacetic acid-precipitated complex of [3H]PAF bound to albumin, formed after incubation of corneal epithelial microsomes with lyso-PAF and [3H]acetyl CoA. Lipoxygenase metabolism by bovine corneal epithelial microsomes was also studied. While 12(S)-HETE did not activate lyso-PAF acetyltransferase, PAF synthesis was decreased when microsomes were treated with lipoxygenase inhibitors. The IC50 values for nordihydroguaiaretic acid (NDGA), and baicalein were 75 and 105 microM, respectively. The IC50 value for CDC, a more potent inhibitor of platelet 12-lipoxygenase, was greater than 200 microM. The results indicate that concentrations suppressing lyso-PAF acetyltransferase activity exceed those required to inhibit lipoxygenases from bovine corneal epithelia. Similarly, concentrations of aspirin and indomethacin, cyclooxygenase inhibitors that decrease PAF formation, were greater than those reported to block prostaglandin generation. A number of other compounds, some structurally similar to the lipoxygenase inhibitors that suppress lyso-PAF acetyltransferase, and others unrelated chemically but known as anti-oxidant and cationic-chelators, also inhibited lyso-PAF acetyltransferase. This suggests that lyso-PAF acetyltransferase activity is inhibited by mechanisms independent of lipoxygenase and cyclooxygenase metabolites.

Platelet-activating factor preferentially stimulates the phospholipase A2/cyclooxygenase cascade in the rabbit cornea.

Platelet-activating factor (PAF) is formed in the cornea after injury as well as by infiltrating inflammatory cells. We have studied the effects of PAF on the release and metabolism of arachidonic acid (AA) in the rabbit cornea. Corneal lipids were labeled in vivo by injecting [3H]AA and subsequently incubated in vitro with 100 nM PAF in the presence or absence of 10 microM BN50727, a PAF antagonist. The AA and eicosanoids released by incubated corneas were analyzed by high-performance liquid chromatography (HPLC). Tissue lipids were examined by mono- and bidimensional thin-layer chromatography (TLC). Within 5 min, PAF stimulated AA release to 76% above control levels. BN50727 inhibited the AA release elicited by PAF at all time points studied. The decreased content of [3H]AA in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) following PAF exposure and the lack of stimulation by PAF on the release of [3H] linoleic acid suggest that the cytosolic phospholipase A2 was activated. PAF also stimulated depletion of AA from the inositol lipids, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-biphosphate (PIP2) and increased content of [3H]AA into diacylglycerol (DAG) and phosphatidic acid (PA). This reaction indicates that PAF could also mediate activation of other phospholipases in the cornea. In addition, PAF preferentially stimulated the cyclooxygenase pathway. The PAF antagonist BN50727 mainly suppressed the PAF-stimulated release of PGE2. The antagonist did not inhibit lipoxygenase activity even after 30 min of PAF stimulation. These results suggest that PAF activate a phospholipase A2/cyclooxygenase pathway in the cornea via a PAF-receptor mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the phospholipase/cyclooxygenase cascade in the rabbit cornea by platelet-activating factor is challenged by PAF receptor antagonists.

Platelet-activating factor (PAF) is a potent lipid inflammatory mediator which is generated in the cornea after injury. Its activity is regulated by interaction with specific receptors. The binding of PAF to its receptors initiates biochemical sequences that cluminate in the release of additional lipid mediators. An arachidonoyl-dependent phospholipase A2 is activated to release arachidonic acid from membrane phospholipids, especially phosphatidylcholine and ethanolamine. Arachidonic acid is then predominantly metabolized by the cyclooxygenase pathway to prostaglandins F2 alpha, E2 and D2, whereas the lipoxygenase pathway is not influenced by PAF. The release of arachidonic acid and prostaglandins stimulated by PAF is challenged by the PAF receptor antagonists BN 50727 and BN 50730. PAF acting intracellularly may also induce the synthesis of cyclooxygenase, presumably the 'inducible' isoform PGHS2, which has been implicated in the inflammatory response. Thus, the therapeutic use of PAF receptor angatonists could be potentially beneficial in the management of ocular inflammatory disease.

The incorporation and release of 12(S)-hydroxyeicosatetraenoic acid and its major metabolite, 8(S)-hydroxyhexadecatrienoic acid, from rabbit corneal lipids.

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) is the predominant corneal lipoxygenase metabolite formed after injury. To investigate the metabolic fate of this eicosanoid in the tissue, [3H]12(S)-HETE was injected intracamerally into rabbits. Corneas were removed 1 to 18 hr after labeling. In some experiments, either entire corneas or the constituent tissues (epithelium, endothelium and stroma) were then incubated in oxygenated Ames' medium for different times. Eighteen hours after injection, the radioactivity was mainly incorporated into the membrane phospholipids, phosphatidyl choline (49%) and phosphatidyl ethanolamine (40%). Free 12(S)-HETE represented less than 1% of the total label. Analysis of the products after phospholipase A2 treatment indicated that the label was acylated in the sn-2 position. HPLC analysis of extracts from tissue and medium showed the presence of 12(S)-HETE and a more polar metabolite established as 8(S)-hydroxyhexadecatrienoic acid [8(S)-OH-16:3] by gas-chromatography-mass-spectrometry. Within 1 hr of injection, 27% of the tissue label was recovered as 8(S)-OH-16:3 and at 8 hr the ratio of incorporated 8(S)-OH-16:3 to 12(S)-HETE was 2:1. 8(S)-OH-16:3 was released into the medium faster than 12(S)-HETE. Metabolism was highest on the epithelial corneal surface. The mitochondrial beta-oxidation inhibitor, 4-pentenoic acid, did not inhibit the formation of 8(S)-OH-16:3 which suggested a peroxisomal beta oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the acyl composition of the platelet-activating factor (PAF) precursor and other choline phosphoglycerides of the rabbit retinal rod outer segments and neural retina.

Choline phosphoglycerides comprise almost half of vertebrate retinal phospholipids. This lipid pool contains the precursor of the potent lipid mediator, platelet-activating factor. The acyl composition and distribution of the different subclasses of the choline phosphoglycerides (alkylacyl-[or the precursor of platelet-activating factor], alkenylacyl-[or choline plasmalogen] and diacyl-glycero-3-phosphocholine) were studied in intact rabbit retina, neural retina and rod outer segments. Choline phosphoglycerides were isolated by high performance liquid chromatography and derivatized by acetylation after phospholipase C treatment. The derivatives were purified by high performance liquid chromatography and subjected to methanolysis. Fatty acids were analyzed by capillary gas liquid chromatography. In the intact retina and in the neural retina, the alkylacyl-glycero-3-phosphocholine and alkenylacyl-glycero-3-phosphocholine comprise 1.2% and 1.5%, respectively, of the total choline phosphoglycerides, whereas the rod outer segments contain twice the proportion of the precursor of platelet-activating factor and no detectable plasmalogens. On a mole percent basis, arachidonic acid was highest in the neural retinal alkenylacyl-glycero-3-phosphocholine (27%), 18% in the alkylacyl-glycero-3-phosphocholine and only 5% in the diacyl-glycero-3-phosphocholine. However, alkylacyl-glycero-3-phosphocholine from rod outer segments was enriched in docosapentaenoic acid (18%) while arachidonic acid was in the 3-4% range. Our results suggest that, in the neural retina, alkyl-arachidonoyl-glycero-3-phosphocholine is a source of both platelet-activating factor and of arachidonic acid which may be a substrate for both prostaglandins and lipoxygenase metabolites during an inflammatory episode and may contribute to the retinal pathology.

The platelet-activating factor precursor of the injured cornea is selectively implicated in arachidonate and eicosanoid release.

The purpose of this study was to isolate the platelet-activating factor (PAF) precursor and other choline phosphoglycerides (GPC) i.e. the alkenylacyl and diacyl lipids from the rabbit cornea, to analyze their fatty acid content and to determine which pool was the most susceptible to arachidonate depletion when activated corneal tissue released arachidonic acid (AA) and metabolites. Rabbit iridal GPC was also analyzed for comparative purposes. The fatty acid methyl esters of the GPC components extracted from the rabbit cornea and iris-ciliary body, isolated by high performance liquid chromatography (HPLC), were determined by capillary gas liquid chromatography. Rabbit corneas were labelled in vivo by intracameral injection of 3H-AA (1 microCi, specific activity = 218 Ci/mmol) and cryogenically injured 18 h later. Corneas were incubated in vitro and the AA and eicosanoids released into the medium were extracted and separated by HPLC. The GPC was extracted from the tissues and the labeling of the three GPC constituents was quantified by liquid scintillation counting. The corneal and iridal PAF precursor represented 4.1 +/- 0.2% and 2.9 +/- 0.2% respectively of total GPC in those tissues. On a mole basis, the alkyl arachidonoyl species constituted 12.7 +/- 0.7% of the corneal and 38 +/- 0.6% of the iridal PAF precursors respectively. The release of AA and prostaglandins by the cornea was linear until 15 min; whereas 12-HETE levels continuously increased until 60 min. All GPC components lost label but 1-O-alkyl-2-arachidonoyl was the most affected, with its labeled content 50% less than the non-injured control.(ABSTRACT TRUNCATED AT 250 WORDS)

The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury.

Previous work has shown that, shortly after rabbit corneas are injured, arachidonic acid metabolism is activated, and 12-hydroxyeicosatetraenoic acid (12-HETE) is one of the main products formed (Bazan, H. E. P., Birkle, D. L., Beuerman, R., and Bazan, N. G. (1985) Invest. Ophthalmol. & Visual Sci. 26, 474-480; Bazan, H. E. P. (1987) Invest. Ophthalmol. Visual Sci. 28, 314-319). In order to determine whether this metabolite is a lipoxygenase product, anesthetized rabbit corneas injured in vivo, either cryogenically or by 1 M NaOH, were subsequently incubated in vitro with [14C] arachidonic acid in the presence of indomethacin. 12-HETE was the main metabolite produced, as established by gas chromatography-mass spectrometry. The (R)- and (S)-enantiomers of novel naphthoyl-pentafluorobenzoyl derivatives of 12-HETE were resolved by chiral-phase high performance liquid chromatography. The radiolabeled 12-HETE from whole cornea and from isolated epithelium, endothelium, or stroma eluted as a single peak co-chromatographing with the (S)-enantiomer and was detected both by UV absorbance at 234 nm and by radioactivity. In noninjured corneas a smaller peak of radiolabeled (12S)-HETE was also eluted from the chiral column. The stereochemistry was additionally confirmed by liquid chromatography-mass spectrometry. These studies suggest that (12S)-lipoxygenase is activated in the injured rabbit cornea.

Effects of ebselen on arachidonate metabolism by ocular and non-ocular tissues.

The formation of cyclooxygenase products in rabbit and rat ocular and non-ocular tissues in vitro, detected by radio-thin-layer chromatography, was inhibited in a concentration-dependent manner by ebselen (PZ 51), an anti-inflammatory seleno-organic compound which has glutathione peroxidase and anti-oxidant activities. The exception was prostaglandin F2 alpha (PGF2 alpha) formation in the rabbit irisciliary body which was stimulated by ebselen in the concentration range 2-10 microM. These observations were confirmed by gas chromatography-mass spectrometry. The concentration that inhibited 50% of prostaglandin biosynthesis (IC50) in the rabbit iris-ciliary body was 9.3 microM. Ebselen also inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) in rabbit and rat ocular tissues and rabbit platelets. The IC50 in the rabbit cornea was 4 microM, whereas higher concentrations were generally required to achieve similar inhibition in other tissues. The formation of 12-HETE by rabbit spleen, however, was not decreased by ebselen at concentrations that were inhibitory in other tissues.

Oxidant and anti-oxidant effects on arachidonate metabolism by rabbit ocular tissues.

Ocular tissues have the capacity to metabolize arachidonate to prostaglandins and related materials, such as hydroxy arachidonate derivatives (HETEs) which are potent mediators of the inflammatory response. Reactive oxygen species are also present during the inflammatory response, resulting in an altered oxidative environment within the eye. This study was designed to evaluate the possible impact of the oxidant hydrogen peroxide and anti-oxidants, ascorbic acid and glutathione, upon arachidonate metabolism. It was found that hydrogen peroxide was observed to potently inhibit arachidonate metabolism in the cornea, but not in the iris-ciliary body. This might be related to a more efficient detoxification of hydrogen peroxide by iris-ciliary body. Ascorbate appeared to have a general stimulatory influence upon arachidonate metabolism in the iris-ciliary body. In the cornea, ascorbate selectively reduced metabolism to HETE while enhancing the products generated by the cyclo-oxygenase pathway. In both cornea and iris-ciliary body reduced glutathione suppressed conversion of arachidonate to its active metabolites. These observations indicate that arachidonate metabolism in ocular tissues is sensitive to the oxidative environment.

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets.

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.

Biochemical studies on a 12-lipoxygenase in human uterine cervix.

Human uterine cervix possesses a high 12-lipoxygenase activity; this enzyme has been isolated in a purified form from the squamous epithelial region of human cervix and its major properties have been investigated. Enzyme activity was present in all subcellular fractions obtained by centrifugation; the highest specific activity was associated with the microsome fraction (160,000 X g pellet). Purification of the enzyme was achieved by acetone precipitation, ion exchange chromatography on CM-cellulose and affinity chromatography on linoleyl-aminoethyl-Sepharose. The product from the incubation of sodium [1-14C]arachidonate with crude enzyme extracts co-chromatographed with authentic 12-hydroxyeicosatetraenoic acid, but the purified enzyme gave a product that behaved like the 12-hydroperoxy derivative. The enzyme had optimum activity at pH 6.5, a Km of 15 microM for arachidonic acid and was stimulated by ATP and Ca2+. Enzyme activity was inhibited by esculetin, nordihydroguaiaretic acid, eicosatetraynoic acid, detergents at concentrations greater than 0.1% (w/v) and preincubation of substrate with GSH and GSH peroxidase. The occurrence of a high 12-lipoxygenase activity is discussed in relation to the specific physiological functions of this tissue.