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2007 35: 1030Toxicol PatholBartz-Schmidt and Ulrich Schraermeyer

Peter Heiduschka, Petra Blitgen-Heinecke, Aysegül Tura, Despina Kokkinou, Sylvie Julien, Sabine Hofmeister, Karl Ulrichvivo

Melanin Precursor 5,6-Dihydroxyindol: Protective Effects and Cytotoxicity on Retinal Cells in vitro and in  

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Toxicologic Pathology, 35:1030–1038, 2007Copyright C© by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230701831358

Melanin Precursor 5,6-Dihydroxyindol: Protective Effectsand Cytotoxicity on Retinal Cells in vitro and in vivo

PETER HEIDUSCHKA,1,2 PETRA BLITGEN-HEINECKE,1,2 AYSEGUL TURA,3 DESPINA KOKKINOU,1 SYLVIE JULIEN,1SABINE HOFMEISTER,1 KARL ULRICH BARTZ-SCHMIDT,3 AND ULRICH SCHRAERMEYER1,2

1Section for Experimental Vitreoretinal Surgery, University Eye Hospital Tubingen, Schleichstr. 12/1, D-72076 Tubingen, Germany2Steinbeis Transfer Centre for Pathology and Toxicology of the Eye, Schleichstr. 12/1, D-72076 Tubingen, Germany

3University Eye Hospital Dept. I, Schleichstr. 12, D-72076 Tubingen, Germany

ABSTRACT

5,6-Dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, theaim of this study was to assess possible toxic effects of DHI on cells related to the eye, such as human ARPE-19 cells and mouse retinal explants.Moreover, DHI was tested on its effects on retinal function in vivo using electroretinography. We found cytotoxicity of DHI against ARPE-19 cellsat 100 µM, but not at 10 µM. 10 µM DHI exhibited a slight, though not significant protective activity against UV-A damage in ARPE-19 cells.We found cytoprotection in cultured mouse retinas by 50 µM DHI or its diacetylated derivative 5,6-diacetoxyindole (DAI), respectively. In ERGmeasurements in vivo, amplitudes were decreased only slightly by 100 µM DHI compared to saline, whereas a better preservation of amplitudes wasvisible at 10 µM DHI, in particular with respect to cones. In histological sections, more cones were found at 10 µM DHI than at 100 µM DHI. Asa conclusion, DHI shows a slight protective effect at 10 µM both in vitro and in vivo. At 100 µM, it shows a strong cytotoxicity in vitro, which isstrongly reduced in vivo.

Keywords. Electroretinography; toxicity; retina; 5,6-dihydroxyindole; ARPE-19 cells.

INTRODUCTION

Melanin is found in high concentrations in the retinalpigment epithelium and the uvea, which consists of thechoroid, the ciliary body and the iris of eyes (Freeman, 1950;Peters and Schraermeyer, 2001). In the melanisation pathway,dopachrome is converted into 5,6-dihydroxyindole (DHI),and DHI is oxidised to 5,6-dihydroxyquinone (Korner et al.,1982). The oxidation reaction is generally catalysed by ty-rosinase (monophenol, 3,4-dihydroxyphenylalanine:oxygenoxidoreductase, EC 1.14.18.1).

The possible physiological and pharmacological signifi-cance of the diffusible DHI has been so far widely over-looked. In vitro, the antioxidant DHI is a free radical scav-enger (Schmitz et al., 1995) and a potent inhibitor of lipidperoxidation (Memoli et al., 1997). Therefore, a potentialprotective role of DHI is possible.

On the other hand, DHI showed cytotoxicity against mouseCloudman melanoma cells and L fibroblasts (Pawelek andLerner, 1978). The apparent DHI cytotoxicity reflects itsinstability in the culture medium (Urabe et al., 1994) and

Address correspondence to: Dr. Peter Heiduschka, Section for Ex-perimental Vitreoretinal Surgery, University Eye Hospital Tubingen,Schleichstr. 12/1, D-72076 Tubingen, Germany; e-mail: p.heiduschka@uni-tuebingen.de

Abbreviations: ARMD: age-related macular degeneration; ARPE cells:amelanotic retinal pigment epithelial cells; BSS: balanced salt solution;DAI: 5,6-diacetoxyindole; DHI: 5,6-dihydroxyindole; DMEM: Dulbecco’sModified Eagle Medium; EDTA: ethylenediamine tetraacetic acid; ERG:electroretinography, electroretinogram; LDH: lactate dehydrogenase; PBS:phosphate buffered saline; PNA: peanut agglutinin; PI: propidium iodide;RPE: retinal pigment epithelium; UV: ultra violet.

the generation of H2O2 during its autooxidation involvingits semiquinone in the univalent transfer of electrons to O2(Nappi and Vass, 1996). However, there have been no stud-ies determining possible physiological and pharmacologicaleffects of DHI in the eye. Consequently, the aim of this studywas to elucidate such possible effects in ocular cells in vitroor in vivo.

To circumvent possible cytotoxicity of DHI, we also stud-ied effects of the diacetylated derivative of DHI, termed 5,6-diacetoxyindole (DAI) which is quite stable until taken upinto cells whereupon it may be cleaved by endogenous es-terases into DHI and acetic acid (Urabe et al., 1994). Further-more, 5,6-diacetoxyindole (DAI) is a substrate of tyrosinase(Riley, 1967).

Firstly, we determined the cytotoxicity of DHI against thehuman retinal pigment epithelial cell line ARPE-19. Near-ultraviolet light peaking at 365 nm can have lethal actionon retinal pigment epithelial cells in vitro (Liu et al., 1995).Therefore, we checked whether DHI protects ARPE-19 cellsagainst ultraviolet A (UV-A) radiation induced cytotoxicity.

Secondly, we evaluated effects of DHI and DAI on viabil-ity of isolated mouse retinas and of normal untreated con-trol retinal explants after in vitro short-term culture. Retinaldamage was quantified by a biochemical assay, measuringthe release of lactate dehydrogenase (LDH) from leaky cells,and by light microscopic morphology. Additionally, the via-bility of cells can be assessed by propidium iodide exclusion(Giraudi et al., 2005).

Thirdly, we also checked the influence of DHI on reti-nal function in vivo. For this purpose, a solution of DHI wasinjected intravitreally, and the function of the retina was eval-uated by electroretinography. Finally, the animals were enu-cleated, and the retinas were inspected histologically.

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Vol. 35, No. 07, 2007 EFFECTS OF 5,6-DIHYDROXYLINDOLE ON RETINAL CELLS 1031

METHODS

Compounds5,6-Dihydroxyindole (DHI) was prepared according to

Wakamatsu and Ito (1988). The melting point of the fi-nal product was 137◦C. Quality control of the final prod-uct was achieved by 1H- and 13C-NMR spectrometry (datanot shown). 5,6-Diacetoxyindole (DAI, CAS number 15069-79-1) was obtained from TCI Europe N.V., Zwijndrecht,Belgium.

Cytotoxicity of 5,6-Dihydroxyindole Against ARPE-19 CellsARPE-19, a commercially available human retinal pig-

ment epithelial cell line, was cultured at 37◦C under 5% CO2in complete medium consisting of a 1:1 mixture of DMEMand Nutrient Mixture F12 Medium supplemented with 10%fetal bovine serum, 100 kU/L of penicillin, 100 mg/L of strep-tomycin and 2 mM L-glutamine. ARPE-19 cells were seededat a density of 3 × 105 cells in a 24-well plate and allowed togrow to confluence. Afterwards, in the presence of differentconcentrations of 5,6-dihydroxyindole (0, 10 or 100 µM),the ARPE-19 cells were cultured for a further 4 hours inphosphate buffered saline (PBS, pH 7.4, 3 wells per concen-tration). After medium change, the cells were incubated for5 days at 37◦C in complete medium and then were assessedby manual cell counting as described below.

Exposure of ARPE-19 Cells to UV LightThe ARPE-19 cells were cultured in 24-well plates

(30,000 cells/well). After incubation of confluent monolay-ers of the cells for 4 hours in PBS (pH 7.4) with 10 µM5,6-dihydroxyindole and a succeeding wash in PBS, the cellswere placed in fresh PBS and subjected to acute exposure for30 minutes to light using a Bio-Link BLX instrument (Vil-ber Lourmat, Torcy, France) with T-8.L tubes (365 nm) asan ultraviolet light source. This instrument is equipped withan UV energy programming system (in J/cm2) with a timeintegrator, which monitors the UV light emission. The irradi-ation stops automatically when the energy received matchesthe programmed energy. Cells receiving no light served asthe control. The light exposure did not heat the medium. Thepower was calibrated with a power meter. The energy densitywas 0.45 J/cm2. After illumination, the cells were washed,complete medium was added, and cultures were maintainedfor 24 hours at 37◦C with 5% CO2 until cell counting.

Counting of Viable ARPE-19 Cells After DHI Treatmentand UV-A Irradiation

Viable cells were counted 24 hours after UV-A irradiation.Cells were washed twice with PBS (pH 7.4) and incubatedfor 3 minutes with a trypsin/EDTA solution (1 ml/well). Af-terwards, they were washed with 2 ml PBS and centrifugedat 1,000 rpm for 2 min. The resulting pellet was filled upwith complete medium. Cell counting was performed using aNeubauer cell counting chamber (Haemocytometer, neoLab,Heidelberg, Germany). The filled counting chamber was ex-amined on a light microscope (Axionplan, Zeiss, Germany)with a magnification of 40×. Each experiment was repeatedfour times (n = 4), with four wells per experimental groupand repetition.

WHOLE RETINAL EXPLANT CULTURES

Male NMRI mice (8–9 weeks old, Harlan-Winkelmann,Borchen, Germany) were sacrificed, the eyes were enucle-ated, collected into 0.1% (w/v) glucose-phosphate bufferedsaline (PBS), and the retinae dissociated from the retinal pig-ment epithelium were mounted onto cellulose nitrate filters(0.45 µm pore size, Sartorius, Gottingen, Germany) with theganglion cell layer facing upwards (n = 6 independent ex-periments, each with 3-4 retinas). The retinal whole mountswere incubated in 1 ml of DMEM/F-12 (without phenol redand serum; Gibco, Invitrogen, Carlsbad, CA) with or without50 µM DHI at 37◦C and with 5% CO2 in a humidified atmo-sphere. In this and the following experiments, the DHI con-centration was chosen according to the magnitudes appliedin the literature, in particular Pawelek and Lerner, 1978.

ASSESSMENT OF RETINAL CELL DEATH

Propidium Iodide StainingAfter incubation, retinal explants were stained with

0.5 µg/ml propidium iodide (PI; Sigma-Aldrich, St.Louis,MO) in 0.1% glucose-PBS for 15 minutes at room temper-ature. PI is a nucleic acid dye which cannot penetrate intactcell membranes, and is thus excluded from healthy cells. Reti-nae were then washed twice in 0.1% glucose-PBS, fixed for30 minutes in 4% paraformaldehyde-PBS, permeabilised in0.1% Triton X-100-PBS, counterstained with DAPI (Molec-ular Probes, Eugene, OR), and analysed by fluorescence mi-croscopy (Olympus, Hamburg, Germany) using the Analysissoftware (Soft Imaging System, Munster, Germany). Quan-tification of the stained cells was performed in 10 areas of0.159 mm2. The extent of cell damage was expressed as thepercentage of the number of PI stained cells to the total num-ber of (DAPI-counterstained) cells.

Lactate Dehydrogenase (LDH) Release AssayLDH is a cytoplasmic enzyme which is rapidly released

into the culture medium upon damage to the plasma mem-branes of the cells (Decker and Lohmann-Matthes, 1988).Measurement of LDH efflux into the culture supernatant istherefore an alternative to quantify cell death. Whole reti-nas dissociated from the pigment epithelium were carefullytransferred into sterile 96-well plates (1 retina/well) contain-ing 200 µl of DMEM/F-12 pro well and washed briefly. Thewashing medium was removed by a micropipette taking carenot to disturb the retina, replaced with 220 µl of fresh mediumwith or without 50 µM DHI or DAI, respectively, and thefree-floating retinas were incubated at 37◦C with 5% CO2 for1 hour or 3 hours (n = 3 for each treatment). The assay me-dia incubated without retina served as background controls.Viability of retinal cells was measured with a colorimetricLDH assay. LDH activity released from damaged and dyingretinal cells was quantified in cell-free supernatants. To deter-mine the maximum amount of LDH release, additional retinas(n = 3 for each assay) were incubated in 2% Triton X-100in DMEM/F-12 for 1 hour and 3 hours. After incubation,the culture supernatants were carefully removed and diluted1/2 and 1/5 in DMEM/F-12, respectively. One hundred µl ofthe culture media and the dilutions were transferred into new96-well plates and incubated with the reaction mixture of thecytotoxicity detection kit (Roche Diagnostics, Mannheim,

1032 HEIDUSCHKA ET AL. TOXICOLOGIC PATHOLOGY

Germany) according to the manufacturer’s instructions. Theabsorbance of the samples was red at 492 nm with a refer-ence wavelength of 690 nm using a microplate reader (Ear400 ATX, SLT-Labinstruments, Crailsheim, Germany).

IN VIVO EXPERIMENTS

Intravitreal InjectionsThe animal experiments were approved by the local au-

thorities and conducted in accordance with institutional andgovernmental guidelines in the use of animals. We used 11wild-type mice, strain C57BL/6. In contrast to NMRI mice,these animals show a more stable electroretinographic re-sponse, in particular with respect to the light stress by themicroscope lamp during the surgery. The animals were anaes-thetised by a mixture of ketamine and xylazine (120 mg/kgketamine, 10 mg/kg xylazine). A small incision was madeinto the outer corner of the eyes. The eyeball was rotatedby grasping the conjunctiva with a pair of fine tweezers andgentle pulling. The conjunctiva was incised to allow directaccess to the sclera. A small hole was made into the sclerausing a sharp 30 gauge needle. 1 µl of a 1 mM or 100 µM,respectively, solution of DHI in balanced salt solution (BSS,Alcon) was injected through the hole intravitreally using aHamilton syringe with a blunt 33 gauge needle. Assuminga volume of the mouse vitreous of 10 to 12 µl, the finalDHI concentration was expected to be approximately 100or 10 µM, respectively. For simplicity, we refer to the finalconcentrations in this manuscript.

After the injection, the needle remained in the eye for ad-ditional two or three seconds to minimise reflux and was thendrawn back. The eyeball was brought back into his normalposition, and the eye was covered by the antibiotic ointmentGentamytrex (Dr. Mann Pharma, Berlin). DHI solution wasinjected either into the right or the left eye, and BSS with-out DHI was injected into the contralateral eye for a control.Sham surgery was performed unilaterally in four animals foran additional control. The whole procedure was performedusing a microscope equipped with illumination. The personwho performed the injections was not aware if DHI solutionor saline were within the syringe.

ElectroretinographyElectroretinograms were recorded from anaesthetised

mice according to standard procedures using the commercialRetiPort32 device from Roland Consult Systems, Germany.After a dark adaptation period of at least 12 hours (i.e.overnight), animals were anaesthetised as described above.The cornea was de-sensitised by a drop of Novesine (Novar-tis Ophthalmics). The pupils were dilated by a drop of Tropi-camide (Novartis Ophthalmics). Gold wire ring electrodesserved as working and reference electrodes that were putonto the cornea of the eyes and into the mouth, respectively.A stainless steel needle electrode was inserted into the tail ofthe animals for grounding. All these manipulations were per-formed under dim red light, without bringing the animal intoambient light after overnight dark adaptation. After additional5 minutes to allow the pupil to dilate, standard electroretino-graphic measurements were performed, with scotopic flashERG, and additional run for scotopic oscillatory potentials,and photopic flash ERG after 10 minutes of light adaptation.The maximum light intensity used for the flashes was 3 cd.

The time of measurement was 160 ms, sample rate 3.2 kHz,and frequency range of 0.5 to 200 Hz for both scotopic andphotopic flash ERG and 50 to 500 Hz for oscillatory poten-tials. The body temperature of the animals was kept on 37◦Cduring the measurement. ERG recordings were performedbefore the intravitreal injection, and then 1, 7 and 14 daysafter the injection, respectively. The person who performedthe measurements did not know which eye received whichinjection.

HistologyThe animals were enucleated after the last ERG measure-

ment 14 days after the surgery, the eyes were fixed in formalinand embedded in paraffin wax, and sections were preparedaccording to standard procedures. In order to check for thenumber of cones after the different treatments, the so-calledPNA staining was performed (Blanks and Johnson, 1984).The retinal sections were de-parrafinised and washed withTris-buffered saline. To label cones, the samples were in-cubated with biotin-labelled lectin (Sigma, L6135, dilution1:200). Bound lectin was visualised using streptavidin con-jugated with alkaline phosphatase (ChemMateTMDetectionKit, DakoCytomation, K 5005). Digital images were taken,and cones per 100 µm length of retinal section were countedmanually. Five to eight sections were evaluated per eye, withfour to six images per section.

Statistical AnalysisValues are given as mean ± standard deviation. Statistical

evaluation was based on Student’s t-test for two populations.A double-sided p-value of less than 0.05 was considered sta-tistically significant.

RESULTS

Cytotoxicity of DHI Against Human ARPE-19 CellsThe cytotoxicity of the antioxidant 5,6-dihydroxyindole

(DHI) on cultured ARPE-19 cells was investigated using con-centrations of 10µM or 100µM, respectively (Figure 1). DHIshowed no significant effect on the cells at a concentration of10 µM. However, a significant (p < 0.001) cytotoxic effect ofDHI on the ARPE-19 cells was observed at a concentrationof 100 µM, leading to almost complete loss of the cells.

Effects of DHI on UV-Induced Cytotoxicity AgainstARPE-19 Cells

ARPE-19 cells were exposed to UV-light in order to inducedeath of approximately 20% of the cell population (Figure 2).We first irradiated the cells in the presence of diluted DHI.However, the medium became dark brownish very quicklydue to enhanced non-enzymatic oxidation of DHI. In orderto avoid absorption of UV light by the dark DHI oxidationproducts and hence reduced UV light intensity, we changedthe medium immediately before the UV irradiation.

Whereas pre-incubation with 10 µM DHI had no effect oncell survival compared to the control, there was a differencein the cell numbers after UV-A irradiation between controlmedium and 10 µM DHI indicating a slight, though not sig-nificant protective effect of DHI.

Vol. 35, No. 07, 2007 EFFECTS OF 5,6-DIHYDROXYLINDOLE ON RETINAL CELLS 1033

FIGURE 1.—5,6-dihydroxyindole (DHI) shows cytotoxic effects on humanretinal pigment epithelial ARPE-19 cells in vitro if applied at a concentrationof 100 µM (p < 0.001). The cells were incubated for 4 hours in the presenceof DHI and, after medium exchange, without DHI for a further 5 days. In alldiagrams, statistical significance is indicated by asterisks, with * - p ≤ 0.05,** - p ≤ 0.01, and *** - p ≤ 0.001.

Effects of DHI and DAI on Mouse Retinal ExplantsIn this experiment, mouse retinal explants were used. As

preparation of the retinas includes cut of the optic nerve andhence axotomy of the retinal ganglion cells, it is likely thatretinal ganglion cells constitute the cell population dying dur-ing the first hours after the preparation of the retinal explants.

The explants were exposed to DHI and the antioxidant5,6-Diacetoxyindole (DAI), each of them at a concentrationof 50 µM. Damage of retinal cells was assessed using thelactate dehydrogenase (LDH) assay. After an incubation of1 hour, DHI provided significant protection (p = 0.02) ofisolated retinas against acute damage in cell culture comparedto Dulbecco’s Modified Eagle Medium (DMEM) as a control(Figure 3a). After 3 hours, a protective action of DHI still

FIGURE 2.—Effects of 5,6-dihydroxyindole (DHI) on UV-A induced damageof human retinal pigment epithelial ARPE-19 cells in vitro. The cells were cul-tured for 24 hours after pre-incubation with 10 µM DHI and following irradiationwith UV-A light (365 nm, 0.45 J/cm2). UV-A radiation showed no significantcytotoxicity against the cells (p = 0.21). 10 µM DHI revealed no cytoprotectionagainst UV-A induced damage (p = 0.12). Four runs of the experiment, with 4wells per group and run.

FIGURE 3.—Effects of 50 µM 5,6-dihydroxyindole (DHI) and 50 µM 5,6-diacetoxyindole (DAI) on lactate dehydrogenase (LDH) release from mouseretina explant cultures after 1 hour (a) or 3 hours (b) incubation in vitro. DHI sig-nificantly protected the murine retinas compared to untreated controls (p = 0.02)or DAI (p = 0.01) after 1 hour (a). DHI-mediated inhibition of retinal damagewas found also after 3 hours (p = 0.02) (b).

could be seen compared to DMEM (p = 0.02, Figure 3b).Using DAI, no protective effect on mouse retinal explantcultures could be seen neither after 1 hour incubation norafter 3 hours incubation (Figure 3).

In a second run, extent of cell death was determined bypropidium iodide (PI) staining. Inspection of the retina for PIstained cells was performed in the ganglion cell layer.

Again, mouse retinal explants were exposed to 50 µM DHIor DAI, respectively. After 1 hour of incubation, neither DHInor DAI showed significant protection of cells in mouse reti-nal explants against culture dependant damage (Figure 4a).Both DHI (p = 0.03) and DAI (p < 0.01) significantly pro-tected the murine retinal explants against tissue culture dam-age after 3 hours incubation (Figure 4b). Thereby, the meanprotective effect of DHI after 3 hours incubation was 1.8-foldhigher than that of DAI (Figure 4b).

FIGURE 4.—Effects of 50 µM 5,6-dihydroxyindole (DHI, n = 3) and 50 µM5,6-diacetoxyindole (DAI, n = 3) on propidium iodide staining of mouse retinaexplant cultures after 1 hour (a) or 3 hours (b) incubation in vitro, respectively.DHI and DAI did not protect the murine retinas compared to untreated DMEMcontrols after 1 hour (a). In contrast, significant inhibition of mouse retina dam-age in cell culture with DHI (p = 0.03) and DAI (p = 0.01), respectively, wasfound after 3 hours (b). The mean protective effect of DHI was 1.8 times higherthan that of DAI.

1034 HEIDUSCHKA ET AL. TOXICOLOGIC PATHOLOGY

Intravitreal Injections and ElectroretinographyIntravitreal injections of 1 µl of either BSS, 100 µM DHI

or 1 mM DHI were tolerated well in the most cases by theanimals. One eye showing strong bleeding and one eye withendophthalmitis were excluded from further examination.Consequently, the eyes in this study were distributed intofour groups — eyes with sham surgery (n = 4), eyes injectedwith BSS (n = 6), and eyes injected with DHI to the final con-centration of 10 µM (n = 5) or 100 µM (n = 5), respectively.

Standard electroretinograms can be recorded in the mice(left column in Figure 5). One day after intravitreal injection,the amplitudes of the recorded ERGs where dramatically de-creased in both eyes of the animals. This decrease of ampli-tudes was visible in both scotopic and photopic ERGs, andalso in the oscillatory potentials. The amplitudes of scotopicERG recovered slowly; however, they did not reach their ini-tial values even two weeks after the intravitreal injections. Incontrast, photopic b-wave amplitudes did not recover note-worthy (Figures 5, 6). Photopic 30 Hz Flicker responses werealso affected by the injections. Nevertheless, the amplitudesremained relatively stable after injection of 10 µM DHI.

In general, the standard deviations of the ERG amplitudeswere rather high, in particular for the animals that receivedintravitreal BSS injection. The magnitudes of the standarddeviations make it difficult to judge about the significanceof the observed changes. Consequently, no significant dif-ferences could be stated in the amplitudes between the fourexperimental groups regarding scotopic a- and b-waves andphotopic b-waves, respectively, except for the photopic b-waves 1 day after the injection and the photopic 30 Hz Flickeramplitudes 7 days after the injection of 10 or 100 µM DHI,respectively.

Despite this lack of significance, it was a general trend thatthe amplitudes after injection of 100 µM DHI were smallerthan those obtained after the injection of 10 µM DHI. This isvisible in outlines also in Figure 5, where the waveforms ob-tained after the injection of 10 µM shows higher amplitudesthan the others.

Furthermore, the latencies of the corresponding waves in-creased clearly one day after the intravitreal injections andshowed a recovery, being almost complete (not shown).

HistologyNo test item-induced changes could be found histologi-

cally in the eyes. Due to the differences seen in photopicERG between the eyes injected with 10 and 100 µM, respec-tively, we decided to evaluate the number of cones survivingthe experimental procedures. For this purpose, we performedthe so-called PNA staining, which labels selectively the cones(Figure 7). The results of cone counting are shown in Figure 8.It can be seen that the numbers of cones in the eyes undergoingsham surgery does not differ significantly from the numberof cones in the BSS-injected eyes. There is also no significantdifference to the number of cones after intravitreal injectionof 10 µM DHI. In contrast, there are significantly less conespresent in the retina after 100 µM than after 10 µM DHI.

DiscussionAge-related macular degeneration (ARMD), the major

cause of blindness of the elderly in the industrial countries,occurs more than twice as often in Caucasians than in black

Africans (Gregor and Joffe, 1978). Associations between iriscolour, fundus pigmentation and ARMD suggest that melanininteracts strongly with the pathways leading to this disease(Young, 1988; Sandberg et al., 1994; Nicolas et al., 2003).Besides melanin itself, also it’s highly diffusible and antiox-idative precursors, for example DHI, may have a protec-tive role against ARMD and oxidative stress in general. Themelanin-related metabolites 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid and 5-S-cysteinyldopawere found to be more potent inhibitors of lipid peroxida-tion than ascorbic acid or glutathione (Memoli et al., 1997).Being small diffusible molecules, these compounds are likelyto perform their antioxidative role not only in the retinal pig-ment epithelium (RPE), but also in the adjacent layers ofthe retina, in particular in the photoreceptor layer, where theoxygen turnover is especially high.

On the other hand, based on earlier reports about possi-ble toxicity of melanin precursors, cytotoxicity of DHI wasreported in a study by Pawelek and Lerner (1978) when ap-plied in a concentration of 100 µM, whereas 10 µM DHI hadno toxic effect. In the cited study, Cloudman S91 melanomacells and L-cell fibroblasts served as model systems. Our in-tention was to check cytotoxicity of DHI in cells more relatedto the eye, such as ARPE-19 cells, retinal explants, and evenin whole eyes in vivo.

Similar to the findings in Pawelek and Lerner (1978), wefound clear cytotoxicity of DHI towards cultured ARPE-19cells, if applied at a concentration of 100 µM. Toxic effectscould not be detected at a DHI concentration of 10 µM, whichis also in accordance to Pawelek and Lerner (1978).

In our mouse retina explant culture experiments, we havefound protective effects of DHI on isolated retinas at a con-centration of 50 µM after 1 or 3 hours incubation using LDHrelease assay and propidium iodide staining, respectively.Statistically significant cytoprotective activity of DHI wasverifiable with both retinal cell death bioassays only after 3hours of incubation.

Regarding the protective effect of DHI on retinal explants,it is likely that the melanin precursor scavenges reactiveoxygen species as a result of its highly oxidisable catecholresidue. Presumable as an antioxidant, DHI prolongs survivalof mouse retinas in organ culture. Similarly, the vitamins Cand E decrease retinal oxidative stress (Fernandez-Robredoet al., 2005). Vitamin E was administered as active ingredientof an ophthalmic solution in a preclinical study (Kojima et al.,1996), and several pharmaceutical companies sell consumerhealth products containing vitamin E.

To investigate whether DHI exerts toxic or protective, re-spectively, effects on the retina in vivo, solutions of DHI wereinjected intravitreally into wild-type mice to reach final in-travitreal concentrations of 10 or 100 µM, respectively. Asseen in Figures 5 and 6, the first consequence of the injec-tion is a drastic decrease of amplitudes in both scotopic andphotopic ERGs. We have found such a decrease also in otherstudies where intravitreal injections have been performed inmice. The reason for this observation is not clear at the mo-ment. Presumably, it is a combination of mechanical traumaand the strong light of the microscope used during the injec-tion. In this context, damage by strong light appears to havethe most prominent effect, as the amplitudes went down alsoafter sham surgery.

Vol. 35, No. 07, 2007 EFFECTS OF 5,6-DIHYDROXYLINDOLE ON RETINAL CELLS 1035

FIGURE 5.—Overview of waveforms obtained in electroretinographic measurements in the animals that received BSS, 10 µM or 100 µM DHI, respectively.Waveforms of the electroretingrams were averaged, i.e. waveforms obtained in all 16 eyes before the injections, and waveforms obtained in the eyes 1, 7 and 14days after the injections according to the kind of injected solution. For clarity, the waveforms obtained after sham surgery were not included. It is obvious that allamplitudes were decreased after the injections. Moreover, the extent of the decrease was similar if BSS or 100 µM DHI had been injected, whereas the decrease wasnot as big in the case of injection of 10 µM DHI. The ERG amplitudes recovered to some extent and where similar in all three groups 14 days after the injections.

1036 HEIDUSCHKA ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 6.—Diagrams showing percentage of changes in amplitudes of scotopic a- and b-waves, photopic b-waves and photopic 30 Hz Flicker response. As alreadyseen in Figure 5, amplitudes decrease sharply 1 day after the injections. Amplitudes recovered during the following days, however, recovery was not complete inmost cases. The error bars represent standard deviations. The asterisks indicate significance of the parameter difference of 10 µM and 100 µM DHI, respectively(∗p ≤ 0.05, ∗∗p ≤ 0.01).

Vol. 35, No. 07, 2007 EFFECTS OF 5,6-DIHYDROXYLINDOLE ON RETINAL CELLS 1037

FIGURE 7.—Results of PNA staining of paraffin sections of mouse eyes. Typical appearance of stained retinal sections after either sham operation, injection ofBSS, injection of 10 µM DHI and injection of 100 µM DHI, as indicated below the images. Each image has a width of 100 µm. Abbreviations: GCL – ganglioncell layer, IPL – inner plexiform layer, INL – inner nuclear layer, OPL – outer plexiform layer, ONL – outer nuclear layer, PR IS – photoreceptor inner segments,PR OS – photoreceptor outer segments, RPE – retinal pigment epithelium.

Moreover, the BSS used for control injections and for thedilution of DHI may have a decreasing effect on the ampli-tudes. An impaired ERG was found after intravitreal irriga-tion with BSS (Moorhead et al., 1979; Garner et al., 2001)and even an impairment of the blood-retinal barrier (Garneret al., 2001). After the incubation of rabbit retina in phys-iologic saline, lactated Ringer’s solution, or BSS, an initialdecrease of b-wave amplitudes with subsequent partial re-covery was found (Negi et al., 1981).

The amplitudes recovered gradually during the followingdays, some of them reaching initial values, in particular in thescotopic ERG, where the rods play the major role. Two mainconclusions may be drawn after the ERG measurements.Firstly, there is no significant difference in the amplitudesbetween the eyes injected with BSS alone or with 100 µMDHI, which shows that DHI does not have an obvious toxiceffect on retinal function. Secondly, there appears to be a

FIGURE 8.—Diagram showing results of cone counting based on histologicalretina sections after PNA staining. The error bars represent standard deviations.There is a significant difference in the number of cones between the eyes afterthe injection of 10 µM or 100 µM DHI, respectively (∗p ≤ 0.05).

protective effect of 10 µM DHI on cone function after lightdamage. In contrast to scotopic ERG, where no clear ampli-tude difference can be seen between the different kinds ofinjections, there is a clear advantage of photopic amplitudesafter injection of 10 µM DHI compared to BSS or 100 µMDHI, which is even significant in two cases.

In the light of the differences in the photopic responses,we performed PNA staining of retinal sections in order tocount the cones. We did not see any differences in the generalstructure of the retina. However, there have been significantfewer cones after the injection of 100 µM DHI compared to10 µM. This shows that DHI may act slightly toxic if it ispresent at a higher concentration, and that cones appear to beparticularly susceptible to that toxicity.

There is a clear link between strong light and oxidativedamage in the retina, because activation of various pho-tosensitive molecules leads to formation of reactive oxy-gen species (Anderson et al., 1994; Boulton et al., 2001;Glickman, 2002). Furthermore, damage of the photoreceptorscaused by strong light can be reduced by various antioxidativesubstances, e.g. dimethylthiourea (Organisciak et al., 1992),thioredoxin (Tanito et al., 2002), ascorbic acid (Organisciaket al., 1985), and phenyl-N -tert-butylnitrone (Ranchon et al.,2003; Tomita et al., 2005). Moreover, survival of cones ispromoted if antioxidative substances are applied (Komeimaet al., 2006, 2007). It therefore may be speculated that theantioxidant DHI is able to neutralise oxygen radicals createdduring the strong light episode during surgery. However, ifthe concentration of DHI exceeds a certain limit, which ap-pears to be in the range around 100 µM, it becomes toxic, asseen in the data of ARPE-19 cell and cone survival.

Although being a precursor of melanin that has some pro-tective effects regarding the incidence of ARMD, it is notvery likely that DHI as a precursor of melanin could be useddirectly for the medication of this disease. Nevertheless, DHIdeserves attention due to its strong antioxidative properties,and the effects of DHI on ocular tissues should be furtherinvestigated. One possible option would be to add DHI or

1038 HEIDUSCHKA ET AL. TOXICOLOGIC PATHOLOGY

similar antioxidative compounds to rinsing solutions used inocular surgery, which would protect the retina from the ox-idative effects caused by the strong light of the operationmicroscope.

ACKNOWLEDGEMENT

This project was supported by the German Research Coun-cil (Deutsche Forschungsgemeinschaft, DFG), Project SCHR436/12-1.

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