Quenching of singlet molecular oxygen by screening pigments-- melanins and ommochromes

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.     

Inhibition of the lysis of fungi by melanins

Evidence is presented that the resistance of Aspergillus nidulans hyphae to lysis by a β-(1→3) glucanase-chitinase mixture results from the presence of melanin in the fungal walls. The resistance of the walls to digestion was directly correlated with the melanin content of the mycelium. A melanin-less mutant of A. nidulans was highly susceptible to hydrolysis by the enzyme mixture. Preincubation of a synthetic melanin with the glucanase, chitinase, and a protease, before addition of the substrate, resulted in a marked inhibition of the rate of substrate hydrolysis. Melanin also appeared to combine with and protect at least certain substrates from decomposition, as indicated by the direct relationship between the extent of inhibition of casein hydrolysis by a bacterial protease and the length of time the protein was incubated with the melanin prior to addition of the enzyme. Melanin was found to be highly resistant to microbial degradation, a likely requirement for the polyaromatic to be effective in protecting fungal structures from lysis or decomposition by natural communities of microorganisms.     

Effect of melanins on lipid peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe(2+)-induced, Fe(2+)-ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O2). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.     

Inhibition of tetraphenylphosphonium-induced NMR-visible lipid accumulation in human breast cells by chlorpromazine

The effects of chlorpromazine (CPZ) on the lipid accumulation induced by the cationic lipophilic compound tetraphenylphosphonium chloride (TPP) were examined using proton nuclear magnetic resonance spectroscopy (NMR), lipid extraction and thin layer chromatography (TLC), and electron microscopy (EM). Chlorpromazine at concentrations of 12 or 25 microM significantly reduced the NMR-visible lipid accumulation induced by a 48-h treatment with 6.25 microM TPP in the human breast cell line, HBL-100, without affecting cell viability. TPP caused threefold increases in whole-cell triglyceride levels that were attenuated by the addition of CPZ. Electron micrographs of TPP-treated HBL-100 cells showed that the destruction of mitochondria was accompanied by the accumulation of lipid droplets and myelinoid bodies. The addition of CPZ to TPP-treated cells reduced the occurrence of lipid droplets but not of mitochondrial destruction. Treatment with CPZ, in the presence or absence of TPP, resulted in large cytoplasmic inclusions indicating the inhibition of lysosomal metabolism. The induction and attenuation of NMR-visible lipids in conjunction with concomitant changes in both intracellular lipid droplets and whole-cell triglyceride levels provides evidence that NMR-visible lipids arise from cytoplasmic neutral lipid droplets. The observation that CPZ, a known inhibitor of lysosomal and cytosolic lipid metabolism, attenuates the formation of neutral triglycerides indicates that lysosomal processing may be a major step in the accumulation of NMR-visible lipid in breast cell lines.     

Potentiation of lipid peroxides by ischemia in rat brain

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate: pyruvate and glutathione (oxidized): glutathione (reduced), as well as increased total ubiquinone content and loss of -tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.     

Prostaglandin biosynthesis can be triggered by lipid peroxides.

Studies of ferriheme cyclooxygenase, using two different assay systems, show that a variety of peroxides can trigger a rapid acceleration of cyclooxygenase activity to produce prostaglandins. Lipid hydroperoxides formed by lipoxygenase were the most potent activators tested, followed by prostaglandin G₂, which was slightly less potent. Peroxides nonspeciflcally generated during arachidonate autoxidation were as potent as the enzymatically formed lipid peroxides. These findings have important implications for cell function since any process which generates peroxides may activate the cyclooxygenase. Thus the balance between formation and removal of cellular lipid peroxides sets a peroxide tone that can regulate the rate of prostaglandin formation in cells.     

Inhibition of UV-induced DNA repair at different steps by quinacrine and anthralin

Inhibition of DNA repair can result in accumulation of unrepaired and partially repaired lesions in DNA. Such lesions are important, not only for their primary disruption of information fidelity, but because they may serve as inducers for repair pathways which may be error prone. Inhibition of UV repair by quinacrine and anthralin (50μM each) was detected in ³H thymidine-labeled mouse L1210 cells by sedimentation of nucleoids on neutral sucrose gradients. Quinacrine delayed strand-nicking (and presumably lesion removal) following uv irradiation and anthralin exerted its strongest effects on some other repair step(s) subsequent to strand-incision with accumulation of strand disruptions. Since anthralin is a potent tumor promoter, it will be interesting to examine other promoters to see if they also cause accumulation of repair ‘intermediates’ which could act as inducers of error prone repair.     

Inhibition by melanin of lipid photo-oxidation

Removal of melanosomes from retinal pigment epithelium cells is accompanied by a sharp rise in the rate of VIS light-induced lipid peroxidation. The synthetic DOPA-melanin effectively suppresses the UV light-induced lipid peroxidation of cardiolipin not only through a passive decrease of irradiation (optical screening), but also by active chemical inhibition of the reaction. It is assumed that the observed active inhibition is due to the interaction between DOPA-melanin and the free radical products generated in cardiolipin upon UV illumination. It is concluded that the high photoresistance of melanin-containing cells of retinal pigment epithelium is due to the ability of melanosomes to exert strong inhibition of photo-induced lipid peroxidation.     

Inhibition of lipid peroxidation by S-nitrosoglutathione and copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 μM) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 μM). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Artifact formation during transmethylation of lipid peroxides.

By-product formation during base-catalyzed transesterification of lipid peroxides was observed in model experiments. A partially oxidized linoleic acid methyl ester as well as purified hydroxylinoleates served as test compounds. By-products formed during a simulated transmethylation of purified hydroxylinoleates were separated from the main components by means of thin-layer chromatography. The loss attributable to these by-products amounted to 10%. Oxygenated fatty acid derivatives were completely destroyed by acid-catalyzed transmethylation. Catalytic hydrogenation prior to base-catalyzed transmethylation proved to be a simple means to minimize side-product formation. By using this technique the yield of hydroxystearates from a partially autoxidized linoleic acid methyl ester preparation was improved significantly.     

Inhibition of lipid peroxidation by alpha-tocopherolquinone and alpha-tocopherolhydroquinone

The antioxidant effect of alpha-tocopherolquinone and alpha-tocopherolhydroquinone was studied in liposomes and rat liver submitochondrial particles. Both alpha-tocopherolquinone and alpha-tocopherolhydroquinone inhibit lipid peroxidation induced by ascorbate/Fe2+ in liposomes and by cumene hydroperoxide in submitochondrial particles. Alpha-tocopherolhydroquinone is much more effective than alpha-tocopherolquinone in inhibiting lipid peroxidation. Submitochondrial particles, depleted of ubiquinones and reincorporated with alpha-tocopherolquinone, are protected from lipid peroxidation only in the presence of succinate. Alpha-tocopherolquinone cannot replace endogenous ubiquinones in the respiratory chain function, nevertheless it can be reduced by the mitochondrial respiratory chain substrates, presumably through the reduced ubiquinones.     

Inhibition of lipid oxidation by serum and albumin

The influence of serum and albumin on enzymatic and non-enzymatic lipid oxidation was investigated. Intensity of oxidation was measured as the amount of oxygen consumed by the sample and by quantitation of malonaldehyde formed during breakdown of lipid peroxides. Non-enzymatic lipid oxidation was stimulated by ascorbic acid or ferrous ions and enzymatic by NADPH-dependent oxidase, 15-lipoxygenase and 12-lipoxygenase. Albumin inhibits lipid oxidation only when pure fatty acid (arachidonic or linoleic) is the substrate for this oxidation. Serum was a stronger inhibitor than an equivalent amount of albumin and it also inhibited oxidation of a mixture of lipids from liver microsomes. It is concluded that serum contains two antioxidant factors: albumin which binds fatty acids and probably another factor which is a true antioxidant.