Ommochromes from the Compound Eyes of Insects: Physicochemical Properties and Antioxidant Activity.

The objective of this study was screening of ommochromes from the compound eyes of insects and comparison of their antioxidant properties. Ommochromes were isolated in preparative quantities from insects of five different families: Stratiomyidae, Sphingidae, Blaberidae, Acrididae, and Tenebrionidae. The yield of ommochromes (dry pigment weight) was 0.9-5.4% of tissue wet weight depending on the insect species. Isolated pigments were analyzed by high-performance liquid chromatography and represented a mixture of several ommochromes of the ommatin series. The isolated ommochromes displayed a pronounced fluorescence with the emission maxima at 435-450 nm and 520-535 nm; furthermore, the emission intensity increased significantly upon ommochrome oxidation with hydrogen peroxide. The ommochromes produced a stable EPR signal consisting of a singlet line with g = 2.0045-2.0048, width of 1.20-1.27 mT, and high concentration of paramagnetic centers (> 1017 spin/g dry weight). All the investigated ommochromes demonstrated high antiradical activity measured from the degree of chemiluminescence quenching in a model system containing luminol, hemoglobin, and hydrogen peroxide. The ommochromes strongly inhibited peroxidation of the photoreceptor cell outer segments induced by visible light in the presence of lipofuscin granules from the human retinal pigment epithelium, as well as suppressed iron/ascorbate-mediated lipid peroxidation. The obtained results are important for understanding the biological functions of ommochromes in invertebrates and identifying invertebrate species that could be used as efficient sources of ommochromes for pharmacological preparations to prevent and treat pathologies associated with the oxidative stress development.

Loss of Melanin by Eye Retinal Pigment Epithelium Cells Is Associated with Its Oxidative Destruction in Melanolipofuscin Granules.

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.

Antiglycation activity of melatonin.

For the first time, it was found that the hormone melatonin exhibited antiglycation activity in vitro. It was shown that melatonin significantly slowed down the accumulation of fluorescent Schiff adducts formed as a result of BSA modification in the presence of high concentration of fructose. It was noted that, unlike the fructosylation reaction, melatonin did not affect the process of modification of BSA by methylglyoxal. We assume that melatonin is able to inhibit the development of the Maillard reaction but does not affect the process of BSA modification by reactive carbonyls.

Paramagnetics melanin and Mn2+ in black soldier fly Hermetia illucens.

Larva, prepupa (last instar larva), pupa, and an empty shell of pupa after hatching of the black soldier fly Hermetia illucens contain eumelanin, an active synthesis of which is observed at the prepupal stage, which is probably due to the release of prepupa from the feed substrate thickness to the open space for pupation. It was shown for the first time that prepupa contains high quantities of the magnetically active form of manganese Mn2+. This fact indicates that Mn2+ stimulates the copper-containing tyrosinase-the key enzyme in the synthesis of melanin in the period of migration and adaptation of the insect to the solar radiation.

Lipofuscin component A2E does not reduce antioxidant activity of DOPA-melanin.

We studied the capacity of DOPA-melanin (natural eumelanin analog) to bind chromophore A2E of retinal pigmented epithelial cell lipofuscin granule into complexes. DOPA-melanin bound up to 200 nm A2E per 1 mg polymer; antioxidant activity of the resultant complexes was evaluated. Luminol chemiluminescence quenching in the presence of hydrogen peroxide showed that the chemiluminescence latency/concentration constants were virtually the same for DOPA-melanin and its A2E complexes. Comparison of the inhibitory effects of DOPA-melanin and DOPA-A2E complexes by rate of UV-induced peroxidation of the outer segments of photoreceptor cells showed higher inhibitory activity of the complexes in comparison with pure DOPA-melanin. Antioxidant activity of DOPA-A2E complexes towards Fe(2+)-ascorbate-induced peroxidation of the outer segments of photoreceptor cells was also higher than that of DOPA-melanin. The results indicated that chromophore A2E of lipofuscin granules in the studied concentrations did not attenuate the antioxidant effects of DOPA-melanin and even potentiated it. This suggested that A2E excess in retinal pigmented epithelium cells could be bound by melanosome melanin and lose its toxicity.

Photobiology of lipofuscin granules in the retinal pigment epithelium cells of the eye: norm, pathology, age.

Lipofuscin granules (LGs) are accumulated in the retinal pigment epithelium (RPE) cells. The progressive LG accumulation can somehow lead to pathology and accelerate the aging process. The review examines composition, spectral properties and photoactivity of LGs isolated from the human cadaver eyes. By use of atomic force microscopy and near-field microscopy, we have revealed the fluorescent heterogeneity of LGs. We have discovered the generation of reactive oxygen species by LGs, and found that LGs and melanolipofuscin granules are capable of photoinduced oxidation of lipids. It was shown that A2E, as the main fluorophore (bisretinoid) of LGs, is much less active as an oxidation photosensitizer than other fluorophores (bisretinoids) of LGs. Photooxidized products of bisretinoids pose a much greater danger to the cell than non-oxidized one. Our studies of the fluorescent properties of LGs and their fluorophores (bisretinoids) showed for the first time that their spectral characteristics change (shift to the short-wavelength region) in pathology and after exposure to ionizing radiation. By recording the fluorescence spectra and fluorescence decay kinetics of oxidized products of LG fluorophores, it is possible to improve the methods of early diagnosis of degenerative diseases. Lipofuscin ("aging pigment") is not an inert "slag". The photoactivity of LGs can pose a significant danger to the RPE cells. Fluorescence characteristics of LGs are a tool to detect early stages of degeneration in the retina and RPE.

[Antiradical and NO-inhibiting activities of bet-hydroxy(ethoxy) derivatives of nitrous heterocycles].

The antiradical and NO-inhibiting activities of beta-hydroxy(ethoxy) derivatives of nitrous heterocycles (3-hydroxypyridine, 5-hydroxybenzimodazole, and 6-hydroxy(alkoxy)-benzothiazole) have been studied. The antiradical activity has been studied using a homogeneous hydrophilic chemiluminescent system, and the quenching constants (Ki) have been determined. For the most reactive compound, 4-methylthiobenzimidazolyl-3-hydroxypyridine, Ki = 4.5 x 105 M(-1). The NO-inhibiting activity was estimated on a model of the endotoxin shock of experimental animals using a spin trap of nitric oxide radicals based on complexes of iron with sodium diethyldithiocarbamate. It was found that the compounds at doses of 0.25-1 mmol/kg have both the inhibitory and stimulating action on the production of nitric oxide in the liver of animals. The results obtained suggest that some derivatives of nitrous heterocycles can be used as effective antioxidant preparations.

Studying the photoprotective activity of a new class of heteroaromatic antioxidants.

Photoprotective activity of heteroaromatic compounds (derivatives of 3-hydroxypyridine, amino-6-hydroxybenzothiazole, and 5-hydroxybenzimidazole) was studied in the system of UV-induced cardiolipin peroxidation. Although all three compounds had the antioxidant effect during free radical oxidation of luminol, only derivatives of amino-6-hydroxybenzothiazole and 5-hydroxybenzimidazole inhibited the process of UV-induced lipid peroxidation. The 3-hydroxypyridine derivative did not inhibit UV-induced cardiolipin peroxidation, which was probably related to degradation of this compound under the influence of UV light and formation of degradation products that cannot inhibit free radical processes.

[Effect of fluorinated silicone oil on the rabbit retinal antioxidant status].

The high-grade fluorinated silicone oil made in Russia was tested for its effect on the rabbit retinal antioxidant status. Experiments were made on 36 rabbits (72 eyes) undergoing replacement of the excised vitreous body with sterile 0.9 NaCl solution or with one of the study implants (fluorinated silicon oil, silicon oil, or perfluorodecahydronaphthalene) after subtotal vitrectomy. The impact of the study implants on the rabbit retinal antioxidant status was postoperattively investigated. The experimental findings suggest that the high-grade fluorinated silicon oil made in Russia may be used as an implant for short-term vitreous chamber tamponade.

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes.

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.

Antioxidative activity of synthetic melanins. Cardiolipin liposome model.

The inhibiting effect of melanin synthesized from dihydroxyphenylalanine (DOPA), dopamine, adrenaline and adrenolutin on the ultraviolet- or the Fe(2+)-ascorbic acid-induced peroxidation of cardiolipin liposomes has been studied. All these melanins are able to inhibit both the ultraviolet- and the Fe(2+)-ascorbic acid-induced lipid peroxidation. Antioxidative activity of melanins enhances in the order: dopamine-melanin less than melanin synthesized from dopamine in the presence of Cu(2+) less than DOPA--melanin less than melanin synthesized from adrenaline in the presence of Cu(2+) approximately equal to adrenolutin-melanin, and correlates with their ability to scavenge superoxide anion radical. The optical screening effect of the investigated melanins in the inhibition of lipid peroxidation was not higher than 15% for the most active melanins.

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids.

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.

An antioxidative role of ocular screening pigments.

The action of ocular screening pigments of vertebrates (melanins) as well as those of invertebrates (ommochromes) on lipid peroxidation has been studied. Lipid peroxidation has been induced by one of the following systems: Fe2+ + ascorbic acid; Fe2+ + NADPH + liver microsomes; xanthine + xanthine oxidase; u.v. illumination; intense visible light, high concentration of O2. Measurements of the lipid peroxidation rate, as estimated from the accumulation of malonic dialdehyde, showed a sharp decrease of the lipid peroxidation rate in the presence of either melanosomes or ommochromes. Synthetic DOPA melanin was also found to exert a strong inhibiting effect on lipid peroxidation. A comparative study of lipid peroxidation in retinal pigment epithelium (RPE) of pigmented and albino rabbits demonstrated that the latter tissue is more sensitive to the effect of the above mentioned prooxidant systems. Apparently this finding is related to the presence of melanin-containing granules in the pigmented tissue rather than to differences in efficiency of other endogenous antioxidant systems. The activities of superoxide dismutase and glutathione peroxidase are practically equal in the RPE of pigmented and albino rabbits whereas the alpha-tocopherol content is higher in albinos. Possible mechanisms of inhibition of lipid peroxidation by melanosomes and ommochromes are discussed. It is proposed that their antioxidant function is one of the most important physiological features of melanins (vertebrate eye) and ommochromes (invertebrate eye).

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors.

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.

Phytosorbent prepared from sunflower seed husks prevents mercuric chloride accumulation in kidney and muscle of adult rabbits.

In the present study, the effect of a melanin-containing phytosorbent, "Victoria," on mercury accumulation in rabbits' tissues was studied. This phytosorbent is derived from black sunflower seed husks. Domestic rabbits were administered either one single nontoxic low-level dose of mercuric chloride (i.e., 50 microg/1 kg body weight [control group]) or combinations of mercury and the phytosorbent "Victoria" (i.e., experimental group). Mercury and phytosorbent were administered per os daily for 12 d. Mercury in tissues was determined by cold-vapor atomic absorption spectroscopy. Mercury in kidney and muscle of the experiment group was, on average, 25.8 and 4.7 times less, respectively, than in the control group. The authors concluded that the phytosorbent prevented accumulation of mercury in the kidney and muscle tissues and exerted a protective effect against mercury toxicity.

[Photo-oxidative processes in eye structures. Protective function of the lens and shielding pigments]. / Fotookislitel'nye protsessy v strukturakh glaza. Zashchitnaia funktsiia khrustalika i ékraniruiushchikh pigmentov.

The results of visual cell photoreceptor membrane light damage mechanism research and some mechanisms of optical and chemical protection of eye structure from photo-sensibilized oxidation are reviewed.

[Opposite effect of lipofuscin granules and melanosomes from human retinal pigment epithelium of eye on photooxidation of cardiolipin]. / Raznonapravlennost' deistviia lipofustsinovykh granul i melanosom iz retinal'nogo pigmentnogo épiteliia glaza cheloveka pri fotookislenii kardiolipina.

The influence of lipofuscin granules and melanosomes from human retinal pigment epithelium on the light-induced photooxidation of cardiolipin liposomes and the generation of superoxide radicals was studied. Lipofuscin granules were able to stimulate, while melanosomes inhibited, the cardiolipin photooxidation. The visible light irradiation of both melanosomes and lipofuscin granules generated superoxide radicals with mean rates of 1.5 nmole/min/10(7) and 38 nmole/min/10(7) granules, accordingly. However, melanosomes but not lipofuscin granules reacted readily with superoxide radicals. Moreover, the rate constant of degradation of superoxide radicals in the presence of melanosomes was about five orders of magnitude higher than the rate constant of its photogeneration. Therefore, we propose that melanosomes in retinal pigment epithelium cells have a photoprotective role whereas lipofuscin granules may stimulate photodestructive reactions.

[Quenching of singlet molecular oxygen by screening pigments-- melanins and ommochromes]. / Tushenie singletnogo molekuliarnogo kisloroda ékraniruiushchimi pigmentami--melaninami i ommokhromami.

Synthetic DOPA-melanin and natural screening pigments--sepiomelanin and ommochromes are shown to quench the luminescence of singlet molecular oxygen (1O2) in aqueous (D2O, pD = 7.5-8.1) solutions. The rate constants of 1O2 quenching are found to be equal to (1.2 +/- 0.6) 10(8) M-1 s-1 for monomeric units in DOPA-melanin and to (3 +/- 1) 10(6) M-1 s-1 for ommochromes. The data suggest that screening is not the only function of melanins, which may play a role of inhibitors of photodynamic damage in living tissues.

[Study of the binding of iron ions by melanoprotein granules of the eye pigment epithelium using gamma-resonance spectroscopy]. / Issledovanie sviazyvaniia ionov zheleza melanoproteinovymi granulami pigmentnogo épiteliia glaza metodom gamma-rezonansnoi spektroskopii.

Interaction between Fe2+ ions and melanoproteid granules (MPG) of bovine eye pigment epithelium was studied by gammaresonance spectroscopy. MPG was found to form complexes with bi- and three-valent ferrum ions. MPG can both directly bind Fe2+ ions and oxidize them Fe3+ inactive in prooxidant state and bind the latter. The activity of ferrum ion binding increases when suspension is illuminated with visible light and pH of the incubation solution increases. The protein part of MPG does not participate in the complex formation with ferrum ions. The complex formation proceeds mainly by carboxyl, amino- and imino-groups of melanin polymer.

[Binding of iron ions by shielding eye pigments of vertebrates and invertebrates--melanosomes and ommochromes]. / Sviazyvanie ionov zheleza ékraniruiushchimi pigmentami glaza pozvonochnykh i bespozvonochnykh zhivotnykh--melanosomami i ommokhromami.

Melanosomas of eye pigment epithelium tissue of vertebrates, synthetic DOPA-melanin and ommochromas from eye of invertebrates bind effectively bivalent iron ions. Constants of the binding process are calculated. Endogenic iron concentration in melanosomas of the frog eye pigment epithelium is determined. A conclusion is drawn that mechanism of antioxidation action of shielding eye pigments may include Fe2+ ion binding into inactive components.