j ourna l homepage: www.e lsev ie r.com/ locate / f reeradb iomed
Original Contribution
Antioxidants rescue photoreceptors in rd1 mice: Relationship with thiol metabolism
María Miranda a, Emma Arnal b, Satpal Ahuja c, Raquel Alvarez-Nölting a, Rosa López-Pedrajas a,Per Ekström c, Francisco Bosch-Morell a,b, Theo van Veen c,d, Francisco J. Romero a,b,⁎a Department of Physiology, University CEU–Cardenal Herrera, 46113 Moncada, Valencia, Spainb Fundacion Oftalmologica del Mediterraneo, Bifurcacion Pio Baroja-General, 46015 Valencia, Spainc Department of Ophthalmology, Lund University, 22184 Lund, Swedend Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
We have previously shown that the use of a combination of antioxidants delayed the degeneration processin rd1 mouse retina. In an effort to understand the mechanism of action of these substances (zeaxanthin,lutein, α-lipoic acid, glutathione, and Lycium barbarum extract) the changes in the levels of several proteinsand oxidative stress markers in the rd1 retina have been studied. The treatment increased glutathioneperoxidase activity and glutathione levels and decreased cystine concentrations in rd1 retinas. Consideringall the results obtained from treated and untreated animals, a high correlation was present betweenglutathione concentration and glutathione peroxidase activity, and there was a negative correlation betweenglutathione retinal concentration and number of TUNEL-positive cells. No difference was observed betweenthe numbers of nNOS- and NADPH-diaphorase-positive cells in treated and untreated rd1 mice. Thiolcontents and thiol-dependent peroxide metabolism seem to be directly related to the survival ofphotoreceptors in rd1 mouse retina.
Retinitis pigmentosa is a group of inherited disorders character-ized by progressive photoreceptor degeneration leading to nightblindness, peripheral vision loss, and subsequently central vision loss.
The rd1/rd1 mouse has an insertion of viral DNA in the β-subunit ofthe cGMP phosphodiesterase gene [1]. The mutation leads to toxicaccumulation of the secondmessenger cGMPand subsequent abnormallyhighCa2+ levels in the rd1photoreceptors [2,3]. This leads to anapoptotic-like rod cell death [4,5], followed by amutation-independent cone death.Amutation in the samegenehasbeen found inhuman formsof autosomalrecessive retinitis pigmentosa, making the rd1 mouse retina an idealmodel for experimental analysis of human retinal dystrophies [6].
Recently, oxidative stress has been implicated in the pathogenesisof retinitis pigmentosa. In previous studies we have shown that theuse of a combination of antioxidants (zeaxanthin, lutein,α-lipoic acid,and glutathione) drastically reduced the number of rod photorecep-tors displaying oxidatively damaged DNA and delayed the degener-ation process significantly [7]. Recently Komeima et al. showed thatinjecting another combination of antioxidants (α-tocopherol, ascorbicacid, Mn(III) tetrakis (4-benzoic acid) porphyrin, and α-lipoic acid)decreased cone photoreceptor cell death in different mouse models ofretinitis pigmentosa [8].
Reactive oxygen species (ROS) are involved in numerous cellularevents in the nervous system, including the retina [9]. Underunfavorable circumstances, ROS may cause tremendous oxidative
ll rights reserved.
stress upon neurons, affect intracellular macromolecules, and lead toneuronal death in the central nervous system(CNS) [10]. The retina is apart of the CNS, perceiving and processing visual information. Butretinal photoreceptors are highly susceptible to oxidation [11], becausetheyare exposed to a rangeof light intensities, andmay contain cellulardefense mechanisms against ROS elevation. Glutathione (GSH) and itsrelated enzymes are part of this antioxidant defense [12].
GSH is a peptide composed of three aminoacids, cysteine, glutamate,and glycine, and protects against oxidative stress by scavenging freeradicals and other reactive species. GSH depletion occurs in severalforms of cell death, also in the retina [13,14]. Nitric oxide (NO) has alsobeen implicated in neurodegenerative diseases [15]. NO is generated inthe CNS by three isoforms of nitric oxide synthase (NOS) located in theendothelial cells, astroglial cells, and a few neurons [16]. NO plays animportant role in cell-to-cell modulation and vasodilatation viaactivation of NO-sensitive guanyl cyclase and the generation of cGMP[16]. NO may interact with oxygen, superoxide anion, and thiolcompounds, generating reactive nitrogen species, peroxynitrite, and S-nitrosothiols including S-nitrosoglutathione (GSNO) [17].
These NO-derived species may have biological functions eithersimilar or opposite to that of NO. For example, peroxynitrite maycause oxidative stress and possibly neurotoxicity [18]. It has beenproposed that GSNO may be an endogenous NO reservoir that canrelease NO [19] but also protects against oxidative stress in theendothelium, myocardium, brain tissue, and other cells [20].
In an effort to better understand the mechanisms of antioxidantactions in the degenerated retina, we have in this work analyzed
217M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
the changes in the levels of several proteins and oxidative stressmarkers in the rd1 retina when treated with a new combination ofantioxidants: zeaxanthin, lutein, α-lipoic acid (ALA), and GSH plus anextract obtained from Lycium barbarum. Previous results have shownthat the effectiveness of the administration of a combination ofantioxidants is better than the administration of these antioxidantsindividually [21]; in fact, the mammalian antioxidant defense systemis a complex network and comprises several enzymatic andnonenzymatic entities [22]. We have decided to add a new compoundto the combinations of antioxidants previously used, to try to increasethe protection exerted by the mixture with L. barbarum. Wolfberry(fruit of L. barbarum Linn, in the family Solanaceae) is a traditionaloriental medicine. It contains functional components such ascarotenoids, flavonoids, and polysaccharides, including zeaxanthin,β-carotene, betaine, cerebroside, β-sitosterol, p-coumaric, and vari-ous vitamins [23]. L. barbarum is well known for nourishing the liverand, in turn, improving the eyesight [24]. It has been reported to haveneuroprotective effects and to reduce oxidative stress in aged miceand in streptozotocin-induced diabetic rats [24].
Methods
Animal care and protocols were in accordance with and approvedby the Animal Ethics Committee of the institution and conformed tothe ARVO Statement for the Use of Animals in Ophthalmic and VisionResearch as well as Spanish law regulating animal experiments.
Mice of rd1 origin were treated daily from postnatal day 3 (PN3,postnatal day 3) by oral infusion with a mix of antioxidants (zeaxanthin,lutein,ALA)dissolved inoliveoil andGSH(inwater) andwith theaqueousand lipid extract from L. barbarum until PN10. The antioxidant concentra-tions usedwere as follows: zeaxanthin and lutein, both 0.67mg /kg bodywt; GSH and ALA both 10 mg /kg body wt; and L. barbarumpolysaccharides 175 mg /kg body wt. Control mice received vehiclealone. At PN11 one eye from each mouse was dissected and the retinaswere fixed, cryoprotected, and cryosectioned. The retinas were fixed for1 h by immersion in 4% paraformaldehyde, washed four times with 3%sucrose, and subsequently put overnight for cryoprotection in 25%sucrose, all of which were in Sörensen's phosphate buffer. Twelve-micrometer cross sections of the retinas were then cut on a cryostat(Microm Laborgeräte GmbH, Walldorf, Germany; Microm HM 560) andstored at−20 °C until used.
Terminal deoxynucleotidyl transferase biotin–dUTP nick endlabeling (TUNEL)
TheTUNEL assaywasperformedusing an in situ cell deathdetectionkit conjugatedwith TMR red (RocheDiagnostics,Mannheim, Germany;12156792910), according to the manufacturer's instructions.
Avidin staining
Struthers et al. [25] and recently our group have shown that avidincanbeused to identify oxidatively damagedDNA [7], because the structureof 8-oxoguanine is similar to that of biotin, i.e., the conventional ligand foravidin. We, therefore, used avidin to test whether oxidatively damagedDNA could be detected in rd1 rod photoreceptors with Texas red-conjugated avidin (1:200) (Molecular Probes; A-820, Eugene, OR, USA).
Biochemical assays
The retina from the other eye was dissected and homogenized inprechilled 0.2 M potassium phosphate buffer, pH 7.0. This homoge-nate was used to assay GSH peroxidase (GPx) and glutathionedisulfide reductase (GSSG-R) activity and the concentrations ofmalondialdehyde (MDA), GSH, and other thiol derivatives. Sampleswere kept frozen (−80 °C) until biochemical assays were performed.
The concentration of MDA, a lipid peroxidation product, wasmeasured by liquid chromatography according to a modification of themethod of Richard et al. [26] as previously described [27]. Briefly, 0. 1 mlof sample (or standard solutions prepared daily from 1,1,3,3-tetra-methoxypropane; Sigma, St. Louis, MO, USA) and 0.75 ml of workingsolution (thiobarbituric acid 0.37%, Sigma, and perchloric acid 6.4%,Panreac, Barcelona, Spain, 2:1, v/v) were mixed and heated to 95 °C for1 h. After cooling (10 min in ice-water bath), the flocculent precipitatewas removed by centrifugation (3200 g, 10 min). The supernatant wasneutralized and filtered (0.22 μm) before injection on an ODS 5-μmcolumn(250×4.6mm;Spheryc-5; Brownlee-Colums). Themobile phaseconsisted in 50mMphosphate buffer, pH6.0 (Fluka, Buchs, Switzerland):methanol (Riedel de Häen, Seelze, Germany) (58:42, v/v). Isocraticseparation was performed at 1.0 ml /min flow (HPLC System 325;Kontron,Germany) anddetection at 532nm(UV/VisHPLCDetector 332;Kontron, Germany). Calibration curves were run daily.
GPx activity was assayed, as reported by Lawrence et al. [28],toward hydrogen peroxide. The oxidation of NADPH was followedspectrophotometrically at 340 nm. The reaction mixture consisted of240 mU/ml glutathione disulfide reductase (Boehringer Mannheim,Germany), 1 mM GSH (Boehringer Mannheim, Germany), 0.15 mMNADPH (Boehringer Mannheim, Germany) in 0.1 M potassiumphosphate buffer, pH 7.0 (Fluka, Buchs, Switzerland), containing1 mM EDTA (Boehringer Mannheim, Germany) and 1 mM sodiumazide (Merck, Rahway, NJ, USA); 50-μl samples were added to thismixture and allowed to equilibrate at 37 °C for 3 min. Reaction wasstarted by the addition of 1.5 mM hydrogen peroxide (Fluka, Buchs,Switzerland) to adjust the final volume of the assay mixture to 1 ml.
GSSG-R activity was assayed according to Pinto and Bartley [29].The disappearance of NADPH was followed spectrophotometrically at340 nm. The experimental cuvette contained 0.1 M potassiumphosphate buffer, pH 7.4, with EDTA, 1.5 mM NADPH, 25 mM GSSG,and retinal homogenate.
The content of GSH and other thiol derivatives in the eyehomogenate was quantified by the method of Reed et al. [30]. Briefly,samples were homogenized in prechilled medium containing phos-phate buffer (Fluka, Buchs, Switzerland) (pH 7.0) and perchloric acid(Panreac). Suspensions were centrifuged at 14,000 g and the resultingsupernatants were collected and stored at −80 °C. The samples weremixedwith a solution of iodoacetic acid (Sigma, St Louis,MO, USA) andSanger reagent (1-fluor-2,4-dinitrobencene; Sigma). These productsare quickly separated by HPLC (Gilson, Detector UV/Vis 156,Middleton, WI, USA), which allows the quantification of nanomolarlevels of GSH, GSSG, cysteine, and cystine (Cyss).
Protein content was measured using the Lowry method [31].
Immunohistochemistry
The activity of NOSwas detected byNADPHdiaphorase (NADPH-d)histochemistry. Retinas were incubated at 37 °C for 1 h in 10 ml 0.1M Tris–HCl (pH 8.0) containing 10 mg NADPH (Sigma–Aldrich, StLouis, MO, USA), 2. 5 mg nitroblue tetrazolium (Sigma–Aldrich, StLouis, MO, USA; dissolved in 50 μl dimethyl sulfoxide), and 20 μlTriton X-100. After being rinsed in Tris buffer, the retinas were flatmounted, air dried, dehydrated, and coverslipped. Then, the numberof NADPH-diaphorase-positive cells in the retina was counted.
Expression of neuronal NOS (nNOS) was examined by standardfluorescence immunostaining. Briefly, cryosections were first incu-bated for 60 min in blocking solution: phosphate-buffered salinecontaining 1% bovine serum albumin, 5% normal serum, and 0.5%Triton X-100. This was followed by overnight incubation with thediluted primary antibody at 4 °C (Santa Cruz Biotechnology; SantaCruz, CA, USA). Binding of the primary antibodies was detected usingwashings and application of secondary antibodies conjugated to Alexafluorophores (Molecular Probes, Eugene, OR, USA). As a control, theprimary antibody was omitted, and in all cases no staining was
218 M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
observed. The slidesweremountedwith Vectashield antifademedium(Vector Laboratories, Burlingame, CA, USA) and viewed under a NikonEclipse E800 microscope fitted with fluorescence optics.
Nitrotyrosine detection
Nitrotyrosine was determined in hippocampus homogenateusing an ELISA kit from Deltaclon (Madrid, Spain) according to themanufacturer's instructions.
Buthionine sulfoximine (BSO) treatment
A group of mice received an intraperitoneal (ip) injection of BSO(1.5 g/kg body wt) once daily for 8 consecutive days starting frompostpartum day 3. In addition, another group of pups received ipinjections of BSO and the combination of antioxidants orally. One ofthe retinas was used to determined GSH concentration and the otherwas cryosectioned and used for TUNEL and avidin determination.
Statistical analysis
The results are presented as mean values±standard deviationfrom at least four animals in each group. Statistical significance wasassessed by means of the Student t test. The level of significance wasset at pb0.05.
Results
TUNEL and avidin staining
We performed TUNEL assays to determine the amount of dyingcells in the retina at PN11 and avidin staining to detect DNA oxidation.Cryostat cross sections through the center of the retina (includingoptic nerve head) were used.When administered individually none ofthe antioxidants produced a significant decrease in the number ofTUNEL- or avidin-positive cells at PN11 (Table 1). At PN11 a signi-ficant decrease in the number of TUNEL-positive cells could beobserved in the outer nuclear layer (ONL) of the retina of animalstreated with lutein, zeaxanthin, ALA, GSH, and L. barbarum from PN3(Fig. 7). The number of avidin-positive cells was considerablydecreased upon treatment with the combination of the antioxidants(Fig. 8). At this age (PN11) the number of photoreceptor cell rows inthe ONL is approximately the same in both wild-type and rd1 retinas(data not shown). A statistically significant positive correlation wasestablished between the number of TUNEL-positive cells and thenumber of avidin-positive cells in all retinas (Fig. 2a).
Markers of oxidative stress
MDA was measured to assess oxidative stress in the retina(Fig. 1a). MDA concentration in treated rd1 retinas was slightly
Table 1Percentage of decrease in TUNEL- and avidin-positive cells after treatment with eachantioxidant individually or with the combination of all the antioxidants
Treatment % decrease inTUNEL-positive cells
% decrease inavidin-positive cells
rd1 control 0 0Lutein 10.66 13.89Zeaxanthin 19.86 13.56α-Lipoic acid 4.57 10.86Glutathione 12.67 3.38Lycium barbarum 10.76 15.67All the antioxidants 38.76 20.61
Only the combination of all the antioxidants produced a significant decrease in TUNEL-and avidin-positive cells in the outer nuclear layer.
Fig. 1. Oxidative stress markers in the rd1 retina from untreated mice and mice treatedwith antioxidants. (a) MDA concentration, (b) GPx activity, (c) GSSG-R activity, (d)GSH concentration, and (e) Cyss concentration in retinas from PN11 untreated andtreated rd1 mice. Values are means±SD from at least four animals in each group.⁎pb0.05 versus rd1, two-tailed Student t test.
decreased compared to that in untreated mice. We could observe ahigh positive correlation between the number of avidin-positive cellsand the MDA concentration in retina from all animals (Fig. 2b).
Fig. 2. Correlation between oxidative stress markers and number of TUNEL- and avidin-positive cells. We could observe a high correlation between GSH concentration and GPxactivity and a negative correlation between GSH retinal concentration and number of TUNEL-positive cells. (a) Correlation between number of TUNEL-positive cells and number ofavidin-positive cells. (b) Correlation between retinal MDA concentration and number of avidin-positive cells. (c) Correlation between TUNEL-positive cells and retinal GSHconcentration. (d) Correlation between retinal GSH concentration and retinal GPx activity.
219M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
GPx and GSSG-R activities were assayed in retina homogenates(Figs. 1b and c). GPx is the key enzymatic activity metabolizingcytosolic and mitochondrial hydrogen peroxide. GPx activity wassignificantly increased in retina from treated rd1 mice at PN11
Fig. 3. Immunohistochemical staining of NADPH diaphorase in retina from rd1 mice.Positive staining appears mainly in the INL and GCL. No differences were observedbetween untreated and treated rd1 mice. (a) Cryostat section of retina from PN11 rd1mice treated with the combination of antioxidants, stained by NADPH diaphorase assay.(b) ComparisonofNADPH-diaphorase-positive cells in the retina of untreated and treatedrd1mice at PN11. Values are means±SD from at least four animals in each group.
compared with untreated mice. There was no difference in GSSG-Ractivity in the retina of untreated or antioxidant-treated mice.
GSH and Cyss levels were increased and decreased, respectively, inthe treated rd1 retinas at PN11 compared to untreated mice (Figs. 1dand e). No differences were observed in GSSG and cysteineconcentration nor in the GSH/GSSG and cysteine/cystine ratiosbetween groups (data not shown). A high correlation was established
Fig. 4. Immunostaining of retina with nNOS antibody. (a) Immunohistochemicalstaining of retina from PN11 rd1 mice treated with the combination of antioxidants fornNOS. White arrows show positive cells. (b) Comparison of nNOS-positive cells in theretina of untreated and treated rd1 mice at PN11. Values are means±SD from at leastfour animals in each group.
Fig. 5. Nitrotyrosine concentration. No differences were observed in the concentrationof nitrotyrosine in untreated and antioxidant-treated rd1 mice.
220 M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
between GSH concentration and GPx activity and, interestingly, therewas a negative correlation between GSH retinal concentration andnumber of TUNEL-positive cells (Figs. 2c and d).
NADPH diaphorase
The present results and our previous findings show thatantioxidants protect the retina from apoptosis. NO plays a criticalrole in photoreceptor cell death and some of the antioxidants used inthis study are effective scavengers of peroxynitrites, so it wasinteresting to study the effect of this treatment on NO synthesis. Toreveal the distribution of NOS, we examined NADPH diaphoraseactivity in the retina. NOS is ubiquitously distributed in the CNS,including the retina [32], and colocalizes with NADPH diaphoraseactivity. The NADPH diaphorase histochemical reaction can be used asa simple and reliable way to identify cells synthesizing NO [33,34].Positive staining of NADPH-d appeared in blue in all retinas. NADPH-diaphorase-positive cells were neuronal in appearance (round or ovalcell body with neurites) in the retina. In the rd1 retina NADPHdiaphorase histochemistry revealed large intensely stained neurons,with distal dendrites that were less intensely labeled than the somaand the proximal dendrites (Fig. 3). Cells were located in the innernuclear layer (INL), with their dendrites extending into the innerplexiform layer (IPL), and were present throughout the retina. A fewrare cells in the ganglion cell layer (GCL) were NADPH-d positive. In
Fig. 6. GSH concentration and GSH/GSSG ratio in the retinas of mice injected with BSOfrom PN3 to PN11. (a) GSH concentration in retinas from PN11 untreated mice andtreated mice injected with BSO. (b) GSH/GSSG ratio in retinas from PN11 untreatedmice and treated mice injected with BSO. Values are means±SD from at least fouranimals in each group. ⁎pb0.05 versus rd1, two-tailed Student t test.
treated rd1mice we could observe fewer NADPH-diaphorase-positivecells in the retina, mainly in the cells that had migrated to the GCL,although this decrease was not statistically significant.
nNOS staining
In the rd1 retina nNOS staining showed intensely stained neuronslocated in the INL with their dendrites extending into IPL throughoutthe retina; some nNOS-positive cells were also observed in the GCL(Fig. 4). We could not observe any difference between the numbers ofnNOS-positive cells in treated and untreated rd1 mice. No differencesin nitrotyrosine concentration were observed in retina from treatedand untreated rd1 mice (Fig. 5).
BSO treatment
BSO treatment decreased GSH as well as the ratio GSH/GSSG in rd1retinas. The administration of antioxidants to BSO-treated animalsproduced a small but non significant increase in bothparameters (Fig. 6).When BSOwas administered daily antioxidants failed to increase retinalglutathione concentration (Fig. 6) or to decrease the number of TUNEL-positive cells (Fig. 7) or the number of avidin-positive cells (Fig. 8).
Discussion
ROS can be generated by several cellular sources and are reactive tocellular macromolecules (lipids, proteins, and nucleic acids). Mamma-liancells have evolvedavariety of enzymes todetoxify theROSproduced
Fig. 7. TUNEL-positive cells in rd1 retina. TUNEL assay of cryostat sections of retina fromPN11 rd1 mice (a) untreated, (b) treated with the combination of antioxidants, (c)injected with BSO, and (d) injected with BSO and treated with antioxidants. (e)Comparison of TUNEL-positive cells in the rd1 control, antioxidant-treated, BSO-injected, and BSO-injected plus antioxidant-treated mice at PN11. Values are means±SD from at least four animals in each group. There was a significant decrease inTUNEL-positive cells in the ONL of mice treated with the antioxidants at PN11, whereasanimals that were treated with BSO and antioxidants failed to show a decrease in thenumber of TUNEL-positive cells. ⁎pb0.05 versus control, two-tailed Student t test.
Fig. 8. Avidin staining in rd1 retina. Avidin-stained cryostat sections of retina from PN11rd1 mice (a) untreated, (b) treated with the combination of antioxidants, (c) injectedwith BSO, and (d) injected with BSO and treated with antioxidants. (e) Comparison ofavidin-positive cells in the rd1 control, antioxidant-treated, BSO-injected, and BSO-injected plus antioxidant-treated mice at PN11. Values are means±SD from at leastfour animals in each group. There was a significant decrease in avidin-positive cells inthe ONL of mice treated with the antioxidants at PN11, whereas animals that weretreated with BSO and antioxidants failed to show a decrease in the number of avidin-positive cells. ⁎pb0.05 versus control, two-tailed Student t test.
221M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
during normal metabolism and/or by pathophysiological processes[35,36]. Among these cellular compounds GSH, GPx, and GSSG-R havereceivedextensive attention.GPxcatalyzes thedecompositionofH2O2 towater. In the reaction catalyzed by GPx, GSH is oxidized to GSSG, whichcan be reduced back to GSH via the action of GSSG-R [37,38]. In thisstudy, we used the same combination of antioxidants as previously [7],with the addition of L. barbarum extract. When administered individ-ually none of the antioxidants produced a significant decrease in thenumber of TUNEL- or avidin-positive cells at PN11. This result couldindicate that the combination or a great amount of antioxidants isnecessary to achieve a significant photoreceptor rescue.
When all the antioxidants were administered at the same time asignificant decrease in TUNEL- and avidin-positive cells was observed.Moreover, we observed an increase in GPx activity and GSHconcentration in the retinas of rd1 animals treated with theantioxidant combination compared with retinas from untreatedmice, whereas no change was observed in GSSG-R activity. This canbe explained by the fact that one of the components of ourcombination was GSH, but it is also known that in vivo ALA is reducedto dihydrolipoic acid, which is a strong reductant, regeneratingoxidized antioxidants such as glutathione [39]. Finally, our group hasalso demonstrated that lutein is able to increase GSH concentrationand GPx activity in diabetic retina [40]. Herein we report a decrease inCyss in treated rd1 retinas. Extracellular Cys/Cyss redox status hasbeen implicated in cell growth and apoptosis, with more proliferativecells under reducing conditions [41], whereas cells grown underoxidizing conditions are more sensitive to oxidant-induced apoptosis
[42]. Considerable evidence is available showing that thiols protectagainst oxidant-induced apoptosis, and this has often been linked tomaintenance of cellular GSH pools [43].
A reduction in the cellular GSH has the potential to increase theconcentration of peroxides in the cell without an increase in its rate ofproduction, thereby causing cellular oxidative stress. Moreover, GSHdepletion has been reported to induce apoptosis in certain cellsystems [44,45]; our study may confirm this hypothesis, as we haveobserved a decrease in TUNEL-positive cells when GSH content wasincreased in retina (Figs. 3 and 4b).
We decided to investigate whether the treatment with antiox-idants could affect NOS. NOS is ubiquitously distributed in the retina[32] and colocalizes with NADPH-d activity [33,34]. Although nostatistically significant difference could be established in nNOS orNADP diaphorase staining between treated and untreated animals, aclear tendency to NADP-diaphorase-staining decrease was observedin the GCL of treated rd1 mouse retinas.
It is possible that the isolated decrease or increase in GSHconcentration is able to alter the effects of NO without affecting theexpression of NOS. GSH forms adducts with NO: S-nitrosoglutathione[46]. GSNO is approximately 100 times more potent than the classicalantioxidant GSH. It has been demonstrated that GSNO and NO protectbrain dopamine neurons from hydroxyl-radical-induced oxidative stressin vivo by (i) inhibiting iron-stimulated hydroxyl radical formation, i.e.,the Fenton reaction; (ii) terminating lipid peroxidation; (iii) augmentingthe antioxidative potency of GSH; (iv) mediating neuroprotective actionof brain-derived neurotrophin; and (v) inhibiting cysteinyl proteases[47]. Another study showed that alterations in GSH levels change theeffects of NO in midbrain cultures from neurotrophic to neurotoxic [48].Under these conditions, NO triggers a programmed cell death withmarkers of both apoptosis and necrosis, the kind of death that is seen inthis retinitis pigmentosa model. So, restoring GSH levels can help NO toexert its beneficial effects, whereas the decrease in GSH levels allowsmore NO to be free and commit neurotoxic actions.
BSO is an inhibitor of γ-glutamylcysteine synthetase and, conse-quently lowers tissue glutathione [49]. The administration of BSO to rd1mice depleted the retina of GSH and the administration of our mixtureof antioxidants could not increase it. Moreover when the mice wereadministered BSO and antioxidants at the same time, the antioxidantsfailed to prevent photoreceptor apoptosis as shown by the TUNEL assay(Fig. 7). In view of this result, active GSH synthesis seems necessary toallow the antioxidant mixture to exert a sort of “GSH-sparing” effect onthe retina of treated animals. According to Meister [50], BSO inducescataracts, as well as having many other effects, that were prevented notby the administration of GSH but of GSH-methyl ester, showing theinability of extracellular GSH alone to prevent this effect.
Both the present results and previous findings consistentlyconfirm that antioxidants reduce cell death in rd1 retina. Herein wedemonstrate the importance of maintaining thiol homeostasis toprotect against retinal cell death.
Acknowledgments
We thank Birgitta Klefbohm and Hodan Abdalle for excellenttechnical assistance. This work was partially supported by funds fromthe Fundación San Pablo and the Copernicus/Santander Program,SAF2007-66801, from the Ministerio de Innovación, Madrid, Spain, toF.J.R.; Conselleria Educació Generalitat Valenciana to M.M.; andFoundation Fighting Blindness to T.v.V.
References
[1] Bowes, C.; Li, T.; Danciger, M.; Baxter, L. C.; Applebury, M. L.; Farber, D. B. Retinaldegeneration in the rd mouse is caused by a defect in the beta subunit of rodcGMP-phosphodiesterase. Nature 347:677–680; 1990.
[2] Farber, D. B.; Lolley, R. N. Cyclic guanosine monophosphate: elevation in degeneratingphotoreceptor cells of the C3H mouse retina. Science 186:449–451; 1974.
222 M. Miranda et al. / Free Radical Biology & Medicine 48 (2010) 216–222
[3] Fox, D. A.; Poblenz, A. T.; He, L. Calcium overload triggers photoreceptor apoptoticcell death in chemical-induced and inherited retinal degenerations. Ann. N. Y.Acad. Sci. 893:282–285; 1999.
[4] Chang, G. Q.; Hao, Y.;Wong, F. Apoptosis: final common pathway of photoreceptordeath in rd, rds, and rhodopsin mutant mice. Neuron 11:595–605; 1993.
[5] Portera-Cailliau, C.; Sung, C. H.; Nathans, J.; Adler, R. Apoptotic photoreceptor celldeath in mouse models of retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 91:974–978; 1994.
[6] McLaughlin, M. E.; Ehrhart, T. L.; Berson, E. L.; Dryja, T. P. Mutation spectrum ofthe gene encoding the β subunit of rod phosphodiesterase among patientswith autosomal recessive retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 92:3249–3253; 1995.
[7] Miranda, M.; Johnson, L. E.; Ahuja, S.; Ekstrom, P. A.; Romero, J.; van Veen, T.Significant photoreceptor rescue by treatment with a combination of antioxidantsin an animal model for retinal degeneration. Neuroscience 145:1120–1129; 2007.
[8] Komeima, K.; Rogers, B. S.; Lu, L.; Campochiaro, P. A. Antioxidants reduce cone celldeath in a model of retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 103:11300–11305; 2006.
[9] Halliwell, B. Reactive oxygen species and the central nervous system. J. Neurochem.59:1609–1623; 1992.
[10] Halliwell, B. Reactive oxygen species in living systems: source, biochemistry, androle in human disease. Am. J. Med. 91:14S–22S; 1991.
[11] Tanito, M.; Nishiyama, A.; Tanaka, T.; Masutani, H.; Nakamura, H.; Yodoi, J.; Ohira,A. Change of redox status andmodulation by thiol replenishment in retinal photo-oxidative damage. Invest. Ophthalmol. Visual Sci. 43:2392–2400; 2002.
[12] Ganea, E.; Harding, J. J. Glutathione-related enzymes and the eye. Curr. Eye Res. 31:1–11; 2006.
[13] Roh, Y. J.; Moon, C.; Kim, S. Y.; Park, M. H.; Bae, Y. C.; Chun, M. H.; Moon, J. I.Glutathione depletion induces differential apoptosis in cells of mouse retina, invivo. Neurosci. Lett. 417:266–270; 2007.
[14] Maher, P.; Hanneken, A. Flavonoids protect retinal ganglion cells from oxidativestress-induced death. Invest. Ophthalmol. Visual Sci. 46:4796–4803; 2005.
[15] Boll, M. C.; Alcaraz-Zubeldia, M.; Montes, S.; Rios, C. Free copper, ferroxidase andSOD1 activities, lipid peroxidation and NO(x) content in the CSF: a differentmarker profile in four neurodegenerative diseases. Neurochem. Res. 33:1717–1723; 2008.
[16] Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric oxide: physiology, pathophys-iology, and pharmacology. Pharmacol. Rev. 43:109–142; 1991.
[17] Hogg, N.; Singh, R. J.; Kalyanaraman, B. The role of glutathione in the transport andcatabolism of nitric oxide. FEBS Lett. 382:223–228; 1996.
[18] Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Apparenthydroxyl radical production by peroxynitrite: implications for endothelial injuryfrom nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87:1620–1624; 1990.
[19] Nikitovic, D.; Holmgren, A. S-nitrosoglutathione is cleaved by the thioredoxinsystem with liberation of glutathione and redox regulating nitric oxide. J. Biol.Chem. 271:19180–19185; 1996.
[20] Rauhala, P.; Mohanakumar, K. P.; Sziraki, I.; Lin, A. M.; Chiueh, C. C. S-nitrosothiolsand nitric oxide, but not sodium nitroprusside, protect nigrostriatal dopamineneurons against iron-induced oxidative stress in vivo. Synapse 23:58–60; 1996.
[21] Stahl, W.; Sies, H. Bioactivity and protective effects of natural carotenoids. Bio-chim. Biophys. Acta 1740:101–107; 2005.
[22] Organisciak, D. T.; Darrow, R. M.; Barsalou, L.; Kutty, R. K.; Wiggert, B.Susceptibility to retinal light damage in transgenic rats with rhodopsinmutations.44:486–492; 2003.
[23] Inbaraj, B. S.; Lu, H.; Hung, C. F.; Wu, W. B.; Lin, C. L.; Chen, B. H. Determination ofcarotenoids and their esters in fruits of Lycium barbarum Linnaeus by HPLC-DAD-APCI-MS. J. Pharm. Biomed. Anal. 47:812–818; 2008.
[24] Chang, R. C.; So, K. F. Use of anti-aging herbal medicine, Lycium barbarum, againstaging-associated diseases. What do we know so far? Cell. Mol. Neurobiol. 28:643–652; 2008.
[25] Struthers, L.; Patel, R.; Clark, J.; Thomas, S. Direct detection of 8-oxodeoxyguanosineand 8-oxoguanine by avidin and its analogues. Anal. Biochem. 225:20–31; 1988.
[26] Richard, M. J.; Guiraud, P.; Meo, J.; Favier, A. High performance liquidchromatography separation of malondialdehyde thiobarbituric acid adduct in
biological materials (plasma and human cell) using a commercially availablereagent. J. Chromatogr. 577:9–18; 1992.
[27] Romero,M. J.; Bosch-Morell, F.; Romero, B.; Rodrigo, J. M.; Serra, M. A.; Romero, F. J.Serum malondialdehyde: possible use for the clinical management of chronichepatitis C patients. Free Radic. Biol. Med. 25:993–997; 1998.
[28] Lawrence, R. A.; Parkhill, L. K.; Burk, R. F. Hepatic cytosolic non-seleniumdependent glutathione peroxidase activity: its nature and the effect of seleniumdeficiency. J. Nutr. 108:981–987; 1978.
[29] Pinto, R. E.; Bartley, W. The effect of age and sex on glutathione reductase andglutathione peroxidase activities and on aerobic glutathione oxidation in rat liverhomogenates. Biochem. J. 112:109–115; 1969.
[30] Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W.W.; Potter, D. W. High-performance liquid chromatography analysis of nanomole levels of glutathione,glutathione disulfide, and related disulfides. Anal. Biochem. 106:55–62; 1980.
[31] Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.
[32] Palmer, R. M.; Ashton, D. S.; Moncada, S. Vascular endothelial cells synthesizenitric oxide from L-arginine. Nature 333:664–666; 1988.
[33] Vincent, S. R.; Kimura, H. Histochemical mapping of nitric oxide synthase in therat brain. Neuroscience 46:755–784; 1992.
[34] Dawson, T. M.; Bredt, D. S.; Fotuhi, M.; Hwang, P. M.; Snyder, S. H. Nitric oxidesynthase and neuronal NADPH diaphorase are identical in brain and peripheraltissues. Proc. Natl. Acad. Sci. U.S.A. 88:7797–7801; 1991.
[35] Freeman, B. A.; Crapo, J. D. Free radicals and tissue injury. Lab. Invest. 47:412–426;1982.
[36] Kehrer, J. P. Free radicals as mediators of tissue injury and disease. Crit. Rev.Toxicol. 23:21–48; 1993.
[37] Fang, Y. Z.; Yang, S.; Wu, G. Free radicals, antioxidants, and nutrition. Nutrition 18:872–879; 2002.
[38] Lei, X. G. In vivo antioxidant role of glutathione peroxidase: evidence fromknockout mice. Methods Enzymol. 347:213–225; 2002.
[39] Packer, L.; Witt, E.; Tritschler, H. J.; Wessel, K.; Ulrich, H. Antioxidant propertiesand clinical implications of alpha-lipoic acid. In: Packer, L., Cadenas, E. (Eds.),Biothiols in Health and Disease. Dekker, New York, pp. 479–484; 1995.
[40] Muriach, M.; Bosch-Morell, F.; Alexander, G.; Blomhoff, R.; Barcia, J.; Arnal, E.;Almansa, I.; Romero, F. J.; Miranda, M. Lutein effect on retina and hippocampus ofdiabetic mice. Free Radic. Biol. Med. 41:979–984; 2006.
[41] Jonas, C. R.; Ziegler, T. R.; Gu, L. H.; Jones, D. P. Extracellular thiol/disulfide redoxstate affects proliferation rate in a human colon carcinoma (Caco2) cell line. FreeRadic. Biol. Med. 33:1499–1506; 2002.
[42] Jiang, S.; Moriarty-Craige, S. E.; Orr, M.; Cai, J.; Sternberg, P.; Jones, D. P. Oxidant-induced apoptosis in human retinal pigment epithelial cells: dependence onextracellular redox state. Invest. Ophthalmol. Visual Sci. 46:1054–1061; 2005.
[43] Hall, A. G. The role of glutathione in the regulation of apoptosis. Eur. J. Clin. Invest.29:238–245; 1999.
[44] Fernandes, R.; Cotter, T. Apoptosis or necrosis: intracellular levels of glutathioneinfluence mode of cell death. Biochem. Pharmacol. 48:675–681; 1994.
[45] Jenner, P.; Olanow, C. Oxidative stress and the pathogenesis of Parkinson'sdisease. Neurology 47:s161–s170; 1996.
[46] Prasad, A.; Andrews, N. P.; Padder, F. A.; Husain, M.; Quyyumi, A. A. Glutathionereverses endothelial dysfunction and improves nitric oxide bioavailability. J. Am.Coll. Cardiol. 34:507–514; 1999.
[47] Chiueh, C. C.; Rauhala, P. The redox pathway of S-nitrosoglutathione, glutathioneand nitric oxide in cell to neuron communications. Free Radic. Res. 31:641–650;1999.
[48] Canals, S.; Casarejos, M. J.; de Bernardo, S.; Rodríguez-Martín, E.; Mena, M. A.Glutathione depletion switches nitric oxide neurotrophic effects to cell death inmidbrain cultures: implications for Parkinson's disease. J. Neurochem. 79:1183–1195; 2001.
[49] Schuette, M.; Werner, P. Redistribution of glutathione in the ischemic rat retina.Neurosci. Lett. 246:53–56; 1998.
[50] Meister, A. Glutathione deficiency produced by inhibition of its synthesis, and itsreversal; applications in research and therapy. Pharmacol. Ther. 51:155–194;1991.
