Karlis Briviba

Glutathione Peroxidase Protects against Peroxynitrite-mediated Oxidations A NEW FUNCTION FOR SELENOPROTEINS AS PEROXYNITRITE REDUCTASE

There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 × 106 m −1 s−1. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of “classical” glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 μmsteady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.

Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein

Antioxidant activity of carotenoids in multilamellar liposomes assayed by inhibition of formation of thiobarbituric acid‐reactive substances was in the ranking: lycopene>α‐tocopherol>α‐carotene>β‐cryptoxanthin>zeaxanthin=β‐carotene>lutein. Mixtures of carotenoids were more effective than the single compounds. This synergistic effect was most pronounced when lycopene or lutein was present. The superior protection of mixtures may be related to specific positioning of different carotenoids in membranes.

SINGLET OXYGEN MEDIATES THE UVA-INDUCED GENERATION OF THE PHOTOAGING-ASSOCIATED MITOCHONDRIAL COMMON DELETION

Mutations of mitochondrial (mt) DNA accumulate during normal aging. The most frequent mutation is a 4,977-base pair deletion also called the common deletion, which is increased in photoaged skin. Oxidative stress may play a major role in the generation of large scale mtDNA deletions, but direct proof for this has been elusive. We therefore assessed whether the common deletion can be generated in vitro through UV irradiation and whether reactive oxygen species are involved in this process. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion employing a semiquantitative polymerase chain reaction technique. There was a time/dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide evidence for the involvement of reactive oxygen species in the generation of aging-associated mtDNA lesions in human cells and indicate a previously unrecognized role of singlet oxygen in photoaging of human skin.

Protection against peroxynitrite

Peroxynitrite formed in vivo from superoxide and nitric oxide can mediate oxidation, nitration, or nitrosation reactions, leading to impaired function, toxicity, and alterations in signaling pathways. Protection against peroxynitrite is important for defense of normal tissue, especially during inflammation. Biological protection against peroxynitrite is organized in three categories: prevention, interception, and repair. Prevention is the control of the formation of peroxynitrite precursors, nitric oxide and superoxide. Interception is by direct reaction with peroxynitrite, leading to non‐toxic products. In this regard, organoselenium compounds, metalloporphyrin derivatives, and peroxidases (e.g. glutathione peroxidase and myeloperoxidase) exhibit high second‐order rate constants with peroxynitrite. Ebselen, like glutathione peroxidase, protects in a catalytic fashion utilizing glutathione as reductant in the peroxynitrite reductase reaction. Protection by metalloporphyrins can be maintained through glutathione or ascorbate. Repair processes remove damaged products and restitute intact biomolecules.

Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts.

Reactive oxygen species (ROS) have been shown to be important messenger molecules in the induction of several genes. In human dermal fibroblasts the herbicide paraquat (PQ2+) was used to induce intracellular oxidative stress that was modulated by the inhibition of copper, zinc superoxide dismutase (Cu,ZnSOD), glutathione peroxidase (GSHPx), catalase, and blocking of the Fenton reaction. Interstitial collagenase (MMP-1) mRNA increased time dependently for up to 72 h following paraquat treatment. A correlation with the translation of MMP-1 could, however, only be detected up to 24 h, indicating an uncoupling of transcription and translation. Interleukin-1 alpha and beta mRNA showed two peaks at 6 h and 72 h. The inhibition of catalase by aminotriazol (ATZ), inhibition of GSHPx by buthionine sulfoximine (BSO), and blocking the Fenton reaction by the iron chelator desferrioxamine (DFO) in concert led to an increase in steady-state MMP-1 mRNA levels, possibly dependent on intracellular H2O2 increase. This combined treatment potentiated MMP-1 mRNA induction up to 6.5-fold compared to paraquat treated controls. Furthermore, exogenously added H2O2 caused an increase in MMP-1 mRNA levels. In contrast, inhibition of Cu,ZnSOD by diethyldithiocarbamate (DDC), leading to diminished H2O2 production from O2.-, decreased MMP-1 mRNA induction. Collectively, our data provide evidence that H2O2 is an important intermediate in the downstream signalling pathway finally leading to the induction of increased steady state MMP-1 mRNA levels. The synthesis of MMPs may contribute to connective tissue damage in vivo related to photoaging, inflammatory diseases, and tumor invasion.

Singlet oxygen induces collagenase expression in human skin fibroblasts

Singlet oxygen generated in a dark reaction by thermodissociation of an endoperoxide (NDPO2) elicits an increase in mRNA of interstitial collagenase (MMP‐1) in cultured human fibroblasts. The effect is enhanced in deuterium oxide‐based medium and is abolished in the presence of non‐toxic doses of sodium azide. In contrast, the mRNA level of the tissue inhibitor of metalloproteinases (TIMP‐1) remains unaltered under these experimental conditions. These observations support the suggestion that an unbalanced synthesis of collagenase and TIMP reported to occur following UV‐A irradiation or during inflammatory conditions may be mediated by singlet oxygen.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL‐1α, IL‐1β, and IL‐6. These effects are enhanced in deuterium oxide‐based medium and are diminished in the presence of non‐toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal‐coated resin particles with visible light (λ>450 nm) results in the induction of interstitial collagenase, IL‐1 and IL‐6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL‐1 and IL‐6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

SINGLET MOLECULAR OXYGEN PRODUCTION IN THE REACTION OF PEROXYNITRITE WITH HYDROGEN PEROXIDE

Peroxynitrite and hydrogen peroxide are mediators of cytotoxicity. This study shows that the peroxynitrite anion reacts with hydrogen peroxide to release oxygen accompanied by emission of chemiluminescence (CL). Direct characterization of this light emission attributes it to the transition of singlet molecular oxygen to the triplet ground state. Chemiluminescence was monitored: (i) by dimol light emission in the red spectral region (> 610 nm) using a red‐sensitive photomultiplier; and (ii) by monomol light emission in the infrared (1270 nm) with a liquid nitrogen‐cooled germanium diode. These properties of photoemission and the enhancing effect of deuterium oxide on CL intensity as well as the quenching effect of sodium azide are diagnostic of molecular oxygen in the excited singlet state. For comparison, singlet molecular oxygen arising from the thermolysis of the water‐soluble endoperoxide of 3,3′‐(1,4‐naphthylidene)dipropionate or from the hypochlorite/H2O2 system was also monitored. These novel observations identify a potential singlet oxygen‐dependent mechanism contributing to cytotoxicity mediated by peroxynitrite and hydrogen peroxide.

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation.

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen‐activated protein kinases (MAPKs) in WB‐F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 μM steady‐state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein‐bound 3‐nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 μM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium‐containing proteins.

Ultraviolet B Wavelength Dependence for the Regulation of Two Major Matrix-Metalloproteinases and Their Inhibitor TIMP-1 in Human Dermal Fibroblasts

Abstract— The wavelength dependence for the regulation of two major matrix‐metalloproteinases, interstitial collagenase (MMP‐1) and stromelysin‐1 (MMP‐3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP‐1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP‐1 and MMP‐3 mRNA steady‐state levels and the secretion of the corresponding proteins up to 4.4‐fold, whereas almost no increase was observed at wavelengths <290 nm. In contrast, the synthesis of TIMP‐1 increased only marginally. This unbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP‐1 and MMP‐3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earths surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.