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Melanin Precursor 5,6-Dihydroxyindol: Protective Effects and Cytotoxicity on Retinal Cells in vitro and in vivo
1 Section for Experimental Vitreoretinal Surgery, University Eye Hospital Tübingen, Schleichstr. 12/1, D-72076 Tübingen, Germany Correspondence: Address correspondence to: Dr. Peter Heiduschka, Section for Experimental Vitreoretinal Surgery, University Eye Hospital Tübingen, Schleichstr. 12/1, D-72076 Tübingen, Germany; e-mail:p.heiduschka{at}uni-tuebingen.de
5,6-Dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, the aim 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 cells at 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 ERG measurements in vivo, amplitudes were decreased only slightly by 100 µM DHI compared to saline, whereas a better preservation of amplitudes was visible 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. As a 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 is strongly reduced in vivo.
Key Words: Electroretinography • toxicity • retina • 5,6-dihydroxyindole • ARPE-19 cells 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’s Modified 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
Melanin is found in high concentrations in the retinal pigment epithelium and the uvea, which consists of the choroid, 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 tyrosinase (monophenol, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1). The possible physiological and pharmacological significance of the diffusible DHI has been so far widely overlooked. In vitro, the antioxidant DHI is a free radical scavenger (Schmitz et al., 1995) and a potent inhibitor of lipid peroxidation (Memoli et al., 1997). Therefore, a potential protective role of DHI is possible. On the other hand, DHI showed cytotoxicity against mouse Cloudman melanoma cells and L fibroblasts (Pawelek and Lerner, 1978). The apparent DHI cytotoxicity reflects its instability in the culture medium (Urabe et al., 1994) and the generation of H2O2 during its autooxidation involving its semiquinone in the univalent transfer of electrons to O2 (Nappi and Vass, 1996). However, there have been no studies determining possible physiological and pharmacological effects of DHI in the eye. Consequently, the aim of this study was to elucidate such possible effects in ocular cells in vitro or in vivo. To circumvent possible cytotoxicity of DHI, we also studied effects of the diacetylated derivative of DHI, termed 5,6-diacetoxyindole (DAI) which is quite stable until taken up into cells whereupon it may be cleaved by endogenous esterases into DHI and acetic acid (Urabe et al., 1994). Furthermore, 5,6-diacetoxyindole (DAI) is a substrate of tyrosinase (Riley, 1967). Firstly, we determined the cytotoxicity of DHI against the human retinal pigment epithelial cell line ARPE-19. Nearultraviolet light peaking at 365 nm can have lethal action on retinal pigment epithelial cells in vitro (Liu et al., 1995). Therefore, we checked whether DHI protects ARPE-19 cells against ultraviolet A (UV-A) radiation induced cytotoxicity. Secondly, we evaluated effects of DHI and DAI on viability of isolated mouse retinas and of normal untreated control retinal explants after in vitro short-term culture. Retinal damage was quantified by a biochemical assay, measuring the release of lactate dehydrogenase (LDH) from leaky cells, and by light microscopic morphology. Additionally, the viability of cells can be assessed by propidium iodide exclusion (Giraudi et al., 2005). Thirdly, we also checked the influence of DHI on retinal function in vivo. For this purpose, a solution of DHI was injected intravitreally, and the function of the retina was evaluated by electroretinography. Finally, the animals were enucleated, and the retinas were inspected histologically.
Compounds 5,6-Dihydroxyindole (DHI) was prepared according to Wakamatsu and Ito (1988). The melting point of the final product was 137°C. Quality control of the final product was achieved by 1H- and 13C-NMR spectrometry (data not 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 Cells
Exposure of ARPE-19 Cells to UV Light
Counting of Viable ARPE-19 Cells After DHI Treatment and UV-A Irradiation
Male NMRI mice (8–9 weeks old, Harlan-Winkelmann, Borchen, Germany) were sacrificed, the eyes were enucleated, collected into 0.1% (w/v) glucose-phosphate buffered saline (PBS), and the retinae dissociated from the retinal pigment epithelium were mounted onto cellulose nitrate filters (0.45 µm pore size, Sartorius, Göttingen, Germany) with the ganglion cell layer facing upwards (n = 6 independent experiments, each with 3–4 retinas). The retinal whole mounts were incubated in 1 ml of DMEM/F-12 (without phenol red and serum; Gibco, Invitrogen, Carlsbad, CA) with or without 50 µM DHI at 37°C and with 5% CO2 in a humidified atmosphere. In this and the following experiments, the DHI concentration was chosen according to the magnitudes applied in the literature, in particular Pawelek and Lerner, 1978.
Propidium Iodide Staining After 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 temperature. PI is a nucleic acid dye which cannot penetrate intact cell membranes, and is thus excluded from healthy cells. Retinae were then washed twice in 0.1% glucose-PBS, fixed for 30 minutes in 4% paraformaldehyde-PBS, permeabilised in 0.1% Triton X-100-PBS, counterstained with DAPI (Molecular Probes, Eugene, OR), and analysed by fluorescence microscopy (Olympus, Hamburg, Germany) using the Analysis software (Soft Imaging System, Münster, Germany). Quantification of the stained cells was performed in 10 areas of 0.159 mm2. The extent of cell damage was expressed as the percentage of the number of PI stained cells to the total number of (DAPI-counterstained) cells.
Lactate Dehydrogenase (LDH) Release Assay
Intravitreal Injections The animal experiments were approved by the local authorities and conducted in accordance with institutional and governmental guidelines in the use of animals. We used 11 wild-type mice, strain C57BL/6. In contrast to NMRI mice, these animals show a more stable electroretinographic response, in particular with respect to the light stress by the microscope lamp during the surgery. The animals were anaesthetised by a mixture of ketamine and xylazine (120 mg/kg ketamine, 10 mg/kg xylazine). A small incision was made into the outer corner of the eyes. The eyeball was rotated by grasping the conjunctiva with a pair of fine tweezers and gentle pulling. The conjunctiva was incised to allow direct access to the sclera. A small hole was made into the sclera using 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 a Hamilton syringe with a blunt 33 gauge needle. Assuming a volume of the mouse vitreous of 10 to 12 µl, the final DHI concentration was expected to be approximately 100 or 10 µM, respectively. For simplicity, we refer to the final concentrations in this manuscript. After the injection, the needle remained in the eye for additional two or three seconds to minimise reflux and was then drawn back. The eyeball was brought back into his normal position, and the eye was covered by the antibiotic ointment Gentamytrex (Dr. Mann Pharma, Berlin). DHI solution was injected either into the right or the left eye, and BSS without DHI was injected into the contralateral eye for a control. Sham surgery was performed unilaterally in four animals for an additional control. The whole procedure was performed using a microscope equipped with illumination. The person who performed the injections was not aware if DHI solution or saline were within the syringe.
Electroretinography
Histology
Statistical Analysis
Cytotoxicity of DHI Against Human ARPE-19 Cells The cytotoxicity of the antioxidant 5,6-dihydroxyindole (DHI) on cultured ARPE-19 cells was investigated using concentrations of 10 µM or 100 µM, respectively (Figure 1). DHI showed no significant effect on the cells at a concentration of 10 µM. However, a significant (p < 0.001) cytotoxic effect of DHI on the ARPE-19 cells was observed at a concentration of 100 µM, leading to almost complete loss of the cells.
Effects of DHI on UV-Induced Cytotoxicity Against ARPE-19 Cells ARPE-19 cells were exposed to UV-light in order to induce death 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 quickly due to enhanced non-enzymatic oxidation of DHI. In order to avoid absorption of UV light by the dark DHI oxidation products and hence reduced UV light intensity, we changed the medium immediately before the UV irradiation.
Whereas pre-incubation with 10 µM DHI had no effect on cell survival compared to the control, there was a difference in the cell numbers after UV-A irradiation between control medium and 10 µM DHI indicating a slight, though not significant protective effect of DHI.
Effects of DHI and DAI on Mouse Retinal Explants The explants were exposed to DHI and the antioxidant 5,6-Diacetoxyindole (DAI), each of them at a concentration of 50 µM. Damage of retinal cells was assessed using the lactate dehydrogenase (LDH) assay. After an incubation of 1 hour, DHI provided significant protection (p = 0.02) of isolated retinas against acute damage in cell culture compared to Dulbecco’s Modified Eagle Medium (DMEM) as a control (Figure 3a). After 3 hours, a protective action of DHI still could be seen compared to DMEM (p = 0.02, Figure 3b). Using DAI, no protective effect on mouse retinal explant cultures could be seen neither after 1 hour incubation nor after 3 hours incubation (Figure 3).
In a second run, extent of cell death was determined by propidium iodide (PI) staining. Inspection of the retina for PI stained cells was performed in the ganglion cell layer. Again, mouse retinal explants were exposed to 50 µM DHI or DAI, respectively. After 1 hour of incubation, neither DHI nor DAI showed significant protection of cells in mouse retinal explants against culture dependant damage (Figure 4a). Both DHI (p = 0.03) and DAI (p < 0.01) significantly protected the murine retinal explants against tissue culture damage after 3 hours incubation (Figure 4b). Thereby, the mean protective effect of DHI after 3 hours incubation was 1.8-fold higher than that of DAI (Figure 4b).
Intravitreal Injections and Electroretinography Intravitreal injections of 1 µl of either BSS, 100 µM DHI or 1 mM DHI were tolerated well in the most cases by the animals. One eye showing strong bleeding and one eye with endophthalmitis were excluded from further examination. Consequently, the eyes in this study were distributed into four groups — eyes with sham surgery (n = 4), eyes injected with BSS (n = 6), and eyes injected with DHI to the final concentration 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 decreased in both eyes of the animals. This decrease of amplitudes was visible in both scotopic and photopic ERGs, and also in the oscillatory potentials. The amplitudes of scotopic ERG recovered slowly; however, they did not reach their initial values even two weeks after the intravitreal injections. In contrast, photopic b-wave amplitudes did not recover noteworthy (Figures 5, 6). Photopic 30 Hz Flicker responses were also affected by the injections. Nevertheless, the amplitudes remained relatively stable after injection of 10 µM DHI.
In general, the standard deviations of the ERG amplitudes were rather high, in particular for the animals that received intravitreal BSS injection. The magnitudes of the standard deviations make it difficult to judge about the significance of the observed changes. Consequently, no significant differences could be stated in the amplitudes between the four experimental groups regarding scotopic a- and b-waves and photopic b-waves, respectively, except for the photopic b-waves 1day after the injection and the photopic 30 Hz Flicker amplitudes 7 days after the injection of 10 or 100 µM DHI, respectively. Despite this lack of significance, it was a general trend that the amplitudes after injection of 100 µM DHI were smaller than those obtained after the injection of 10 µM DHI. This is visible in outlines also in Figure 5, where the waveforms obtained after the injection of 10 µM shows higher amplitudes than the others. Furthermore, the latencies of the corresponding waves increased clearly one day after the intravitreal injections and showed a recovery, being almost complete (not shown).
Histology
Age-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 iris colour, fundus pigmentation and ARMD suggest that melanin interacts 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 antioxidative precursors, for example DHI, may have a protective role against ARMD and oxidative stress in general. The melanin-related metabolites 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid and 5-S-cysteinyldopa were found to be more potent inhibitors of lipid peroxidation than ascorbic acid or glutathione (Memoli et al., 1997). Being small diffusible molecules, these compounds are likely to perform their antioxidative role not only in the retinal pigment epithelium (RPE), but also in the adjacent layers of the retina, in particular in the photoreceptor layer, where the oxygen turnover is especially high. On the other hand, based on earlier reports about possible toxicity of melanin precursors, cytotoxicity of DHI was reported in a study by Pawelek and Lerner (1978) when applied in a concentration of 100 µM, whereas 10 µM DHI had no toxic effect. In the cited study, Cloudman S91 melanoma cells and L-cell fibroblasts served as model systems. Our intention was to check cytotoxicity of DHI in cells more related to the eye, such as ARPE-19 cells, retinal explants, and even in whole eyes in vivo. Similar to the findings in Pawelek and Lerner (1978), we found clear cytotoxicity of DHI towards cultured ARPE-19 cells, if applied at a concentration of 100 µM. Toxic effects could not be detected at a DHI concentration of 10 µM, which is also in accordance to Pawelek and Lerner (1978). In our mouse retina explant culture experiments, we have found protective effects of DHI on isolated retinas at a concentration of 50 µM after 1 or 3 hours incubation using LDH release assay and propidium iodide staining, respectively. Statistically significant cytoprotective activity of DHI was verifiable with both retinal cell death bioassays only after 3 hours of incubation. Regarding the protective effect of DHI on retinal explants, it is likely that the melanin precursor scavenges reactive oxygen species as a result of its highly oxidisable catechol residue. Presumable as an antioxidant, DHI prolongs survival of mouse retinas in organ culture. Similarly, the vitamins C and E decrease retinal oxidative stress (Fernandez-Robredo et al., 2005). Vitamin E was administered as active ingredient of an ophthalmic solution in a preclinical study (Kojima et al., 1996), and several pharmaceutical companies sell consumer health products containing vitamin E. To investigate whether DHI exerts toxic or protective, respectively, effects on the retina in vivo, solutions of DHI were injected intravitreally into wild-type mice to reach final intravitreal concentrations of 10 or 100 µM, respectively. As seen in Figures 5 and 6, the first consequence of the injection is a drastic decrease of amplitudes in both scotopic and photopic ERGs. We have found such a decrease also in other studies where intravitreal injections have been performed in mice. The reason for this observation is not clear at the moment. Presumably, it is a combination of mechanical trauma and the strong light of the microscope used during the injection. In this context, damage by strong light appears to have the most prominent effect, as the amplitudes went down also after sham surgery. Moreover, the BSS used for control injections and for the dilution of DHI may have a decreasing effect on the amplitudes. An impaired ERG was found after intravitreal irrigation with BSS (Moorhead et al., 1979; Garner et al., 2001) and even an impairment of the blood-retinal barrier (Garner et al., 2001). After the incubation of rabbit retina in physiologic saline, lactated Ringer’s solution, or BSS, an initial decrease of b-wave amplitudes with subsequent partial recovery was found (Negi et al., 1981). The amplitudes recovered gradually during the following days, some of them reaching initial values, in particular in the scotopic ERG, where the rods play the major role. Two main conclusions may be drawn after the ERG measurements. Firstly, there is no significant difference in the amplitudes between the eyes injected with BSS alone or with 100 µM DHI, which shows that DHI does not have an obvious toxic effect on retinal function. Secondly, there appears to be a protective effect of 10 µM DHI on cone function after light damage. In contrast to scotopic ERG, where no clear amplitude difference can be seen between the different kinds of injections, there is a clear advantage of photopic amplitudes after injection of 10 µM DHI compared to BSS or 100 µM DHI, 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 to count the cones. We did not see any differences in the general structure of the retina. However, there have been significant fewer cones after the injection of 100 µM DHI compared to 10 µM. This shows that DHI may act slightly toxic if it is present at a higher concentration, and that cones appear to be particularly susceptible to that toxicity. There is a clear link between strong light and oxidative damage in the retina, because activation of various photosensitive molecules leads to formation of reactive oxygen species (Anderson et al., 1994; Boulton et al., 2001; Glickman, 2002). Furthermore, damage of the photoreceptors caused by strong light can be reduced by various antioxidative substances, e.g. dimethylthiourea (Organisciak et al., 1992), thioredoxin (Tanito et al., 2002), ascorbic acid (Organisciak et al., 1985), and phenyl-N-tert-butylnitrone (Ranchon et al., 2003; Tomita et al., 2005). Moreover, survival of cones is promoted if antioxidative substances are applied (Komeima et al., 2006, 2007). It therefore may be speculated that the antioxidant DHI is able to neutralise oxygen radicals created during the strong light episode during surgery. However, if the concentration of DHI exceeds a certain limit, which appears to be in the range around 100 µM, it becomes toxic, as seen in the data of ARPE-19 cell and cone survival. Although being a precursor of melanin that has some protective effects regarding the incidence of ARMD, it is not very likely that DHI as a precursor of melanin could be used directly for the medication of this disease. Nevertheless, DHI deserves attention due to its strong antioxidative properties, and the effects of DHI on ocular tissues should be further investigated. One possible option would be to add DHI or similar antioxidative compounds to rinsing solutions used in ocular surgery, which would protect the retina from the oxidative effects caused by the strong light of the operation microscope.
This project was supported by the German Research Council (Deutsche Forschungsgemeinschaft, DFG), Project SCHR 436/12-1.
Toxicologic Pathology, Vol. 35, No. 7,
1030-1038 (2007)
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Abstract
Introduction
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