A. E. Dontsov

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.

Comparative study of the dark and light-induced toxicity of lipofuscin granules from human retinal pigment epithelium and their chromophore A2E on the cardiolipin liposome model

The damaging effect of lipofuscin granules from the human retinal pigment epithelium and fluorophore A2E was studied on models of calcein- and ascorbate-loaded cardiolipin liposomes and outer segments of the bovine eye photoreceptor cells in dark and under visible light irradiation. In dark fluorophore A2E induces the release of calcein from calcein-loaded liposomes and reduces the lifetime of the artificial bilayer lipid membrane prepared from dioleyl phosphatidilcholine. A similar detergent-like action A2E exhibits towards ascorbate-loaded liposomes, significantly accelerating the release of ascorbate in dark. In the presence of A2E, irradiation with the full visible light (390–700 nm) stimulates both the release of ascorbate from liposomes and accelerates the destruction of the bilayer lipid membrane. Retinal pigment epithelium lipofuscin granules also accelerate the release of ascorbate from ascorbate-loaded liposomes under visible light irradiation; the blue light (457.9 nm) was twice as more efficient as the green light (514.5 nm). The preliminary irradiation of A2E with the visible light decreases its detergent-like action on the cardiolipin liposomal membranes under the dark conditions and the photosensitizing effect on the lipid peroxidation of the outer segments of photoreceptor cells. Unlike A2E, the visible light irradiation of a suspension of lipofuscin granules under similar conditions does not noticeably decrease their sensitizing activity towards lipid peroxidation. It is assumed that the phototoxicity of retinal pigment epithelium lipofuscin granules is related not only to A2E in their composition, but depends mainly on the content of other photosensitizers (chromophores) in the granules.

Effect of UV radiation and hydrogen peroxide on the antiradical and antioxidant activities of DOPA-melanin and melanosomes from retinal pigment epithelial cells

The effect of continuous UV radiation and hydrogen peroxide on destruction and antioxidant properties of synthetic DOPA-melanin (prepared by oxidation of 3,4-dihydroxyphenylalanine (DOPA)) and melanosomes isolated from cells of the retinal pigment epithelium (RPE) was investigated. The kinetics of melanin destruction was recorded based on the accumulation of fluorescent low-molecular-weight reaction products, the antiradical activity of melanin was determined by chemiluminescence method, the concentration of free radical products was measured by electron paramagnetic resonance, and the antioxidant activity of melanins was estimated by their inhibitory effect on lipid peroxidation. It was shown that UVC—UVA irradiation (up to 5 hours) of DOPA-melanin and melanosomes of retinal pigment epithelium decreased neither the latency period of luminol chemiluminescence nor the inhibitory action of pigments on Fe2+- and UV-induced peroxidation of cardiolipin liposomes. However, very long UV irradiation gave rise to fluorescent destruction products, decreased the concentration of paramagnetic centers in the pigment (especially light-dependent ones), and decreased the antiradical and antioxidant activities. For example, UV irradiation of DOPA-melanin during 52 h resulted in approximately a 2-fold decrease in the concentration of paramagnetic centers and decline of antiradical and antioxidant activities. However, even with such a hard irradiation the pigment retained significant inhibitory activity against lipid peroxidation. The oxidative destruction of DOPA-melanin in the presence of hydrogen peroxide in the dark resulted in complete destruction of the polymer and loss of its protective properties. It is assumed that destruction of RPE cell melanin is caused mainly by oxidative processes.

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.

Preparation and Characterization of Alphitobius diaperinus Melanin

Melanin with a high antioxidant and sorption activity comparable to that of synthetic dioxyphenylalanine (DOPA)-melanin was isolated from the biomass of the darkling beetle Alphitobius diaperinus. The pigment was extracted with a solution of potassium hydroxide, followed by precipitation with concentrated hydrochloric acid and hydrolysis of the resulting precipitate with the same acid. The electron paramagnetic resonance (EPR) signal of melanin was characteristic of eumelanins with a spin concentration of 4.9 × 1017 spin per 1 g of dry weight. The melanin concentration that induced 50% inhibition of peroxidation was 9.2 μg/mL (the analogous concentration of DOPA-melanin was 8.0 μg/mL). The maximum of methylene-blue binding to the beetle melanin was 700 mg of dye per 1 g of dry weight of the preparation. The lipid-free melanin preparation exhibited antiradical activity.

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.

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.

Antiradical and photoprotective activity of oxibiol, a novel water-soluble heteroaromatic antioxidant

Abstract5-Hydroxybenzo[d]thiazol-2-ylammonium N-acetyl-l-cysteinate (oxibiol) was synthesized and studied. This novel water-soluble azaheterocyclic antioxidant is a white crystalline solid (m.p. 148–150 °C) soluble in ethanol and water. The UV spectrum of oxibiol shows two absorption bands at 220±1 and 265±1 nm. Its antiradical activity was examined using a homogeneous hydrophilic chemiluminescent system. The emission quenching constant of oxibiol is (2.4±0.4)·105 L mol−1, which is nearly seven times that of the antioxidant mexidol widely used in current medical practice. Oxibiol efficiently inhibits the free radical oxidation of photoreceptor cells that is induced by iron(II) ions and UV and visible light in the presence of photosensitizers. Oxibiol is especially effective in suppressing the photoinduced lipid peroxidation sensitized by lipofuscin granules from human retinal pigment epithelial cells. Oxibiol is highly resistant when exposed to UV light. Being a novel, more efficient and photoresistant antioxidant, oxibiol can also find other medical applications and replace the widely used agent mexidol.

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 DOPAmelanin 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 Fe2+-ascorbateinduced 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.