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New anti-RNS and -RCS products for cosmetic treatment
Abstract
Synopsis
Oxidants and free radicals are known to be a very important factor in skin aging, taking an active part in lipidic peroxidation, breakage of proteins and DNA, etc. The most well‐known are reactive oxygen species (ROS), for example, superoxide radical anion, or more commonly called, superoxide (O ), hydroxyl radical (OH • ) or hydrogen peroxide (H 2 O 2 ). Both free radicals and other oxidants can be generated by metabolic activity within the cell and by other environmental challenges,. In addition, other dangerous species are known such as reactive nitrogen species (RNS) and reactive carbonyl species (RCS). Some of the most important RNS are peroxynitrite (ONOO ⁻ ), nitrogen dioxide radical ( • NO 2 ) and the nitronium ion (NO ). For RCS, some of the most important are 4‐hydroxynonenal (HNE), acrolein (ACR), malondialdehyde (MDA) or glyoxal (GXL). Both compounds (RNS and RCS) are thought to play an important role in many diseases and in skin aging, for example, collagen cross‐linking, DNA damage, protein tyrosine nitration, etc. This work investigates two new specific chemicals: Lipochroman‐6 ® – an anti‐RNS which shows good results in inhibiting the nitration of tyrosine by peroxynitrite, and Aldenine ® – a tripeptide anti‐RCS which protects cells from reactive carbonyl compounds such as HNE or ACR; it also shows the ability to prevent glycation of proteins, specifically by superoxide dismutase (SOD).
... Peroxynitrite reacts with tyrosine to form 3-nitrotyrosine, leading to irreversible modifications in proteins and compromising the interconversion between the phosphorylated and dephosphorylated states of kinase substrates, a major mechanism of cellular signaling. Additionally, peroxynitrite damages important constituents of the extracellular matrix (ECM) like hyaluronic acid [29]. The data indicate that MeO-MBM is an antioxidant with additional photoantioxidative properties on the cellular level. ...
Avobenzone, one of the most commonly used UV filters in topical sunscreens, is susceptible to photodegradation with a consequential reduction of its UV absorbing properties. This loss of function may lead to skin irritation, photodermatosis, and photoallergic reactions caused by photodegradation byproducts. In this work, we aim to address this issue with a substance named methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, but which converts to avobenzone, a known and approved UVA filter, under mainly UVB light irradiation. The antioxidant and intracellular radical formation properties of MeO-MBM were compared to the ones of avobenzone. The UV irradiation of MeO-MBM led to an increase in UV absorption primarily in the UVA range after conversion, both in vitro and in vivo. HPTLC and UHPLC studies illustrate the conversion of MeO-MBM to avobenzone in vitro after irradiation at 250 kJ/m², reaching a conversion rate of 48.8%. A stable molecular antioxidant activity was observed, since 100-µM MeO-MBM was measured to be 11.2% in the DPPH assay, with a decrease to 9.7% after irradiation. In comparison, the molecular antioxidant activity of 100-µM avobenzone was determined to be 0.8%. In keratinocytes, MeO-MBM reduces the intracellular ROS by 90% and avobenzone by 75% with tBHP as the inducer and by 53% and 57%, respectively, when induced by pyocyanin, indicating the redox scavenging capacity of both these molecules. These results indicate that MeO-MBM functions initially as an antioxidant material and as a photoantioxidant during its conversion process to avobenzone. This research provides insight into the development of active ingredients for topical applications with dynamic functionalities. Using this approach, we demonstrate the possibility to extend the UV protection offered to skin cells while combating cellular stress in parallel.
... GHK also possesses strong antioxidant and anti-inflammatory actions. GHK inactivated damaging free radical by-products of lipid peroxidation, such as 4-hydroxynoneal, acrolein, malondialdehyde, and glyoxal, protecting cultured skin keratinocytes from ultraviolet (UV)-radiation [31]. GHK was shown to completely block Cu(2+)-dependent oxidation of low density lipoproteins (LDL). ...
The human peptide GHK (glycyl-l-histidyl-l-lysine) has multiple biological actions, all of which, according to our current knowledge, appear to be health positive. It stimulates blood vessel and nerve outgrowth, increases collagen, elastin, and glycosaminoglycan synthesis, as well as supports the function of dermal fibroblasts. GHK’s ability to improve tissue repair has been demonstrated for skin, lung connective tissue, boney tissue, liver, and stomach lining. GHK has also been found to possess powerful cell protective actions, such as multiple anti-cancer activities and anti-inflammatory actions, lung protection and restoration of chronic obstructive pulmonary disease (COPD) fibroblasts, suppression of molecules thought to accelerate the diseases of aging such as NFκB, anti-anxiety, anti-pain and anti-aggression activities, DNA repair, and activation of cell cleansing via the proteasome system. Recent genetic data may explain such diverse protective and healing actions of one molecule, revealing multiple biochemical pathways regulated by GHK.
... GHK protects cultured skin keratinocytes from lethal ultraviolet radiation damage by quenching reactive carbonyl species such as 4-hydroxynonenal, acrolein, malondialdehyde, and glyoxal. The effect was observed with 20 mg/mL or 0.2% concentrations of GHK, which can be safely used in protective cosmetic formulations such as sunscreens [38][39][40]. ...
Topical remedies capable of protecting skin from damage and supporting its regeneration can improve skin’s health as well as its appearance. Small copper peptides have an excellent safety record and are widely used in cosmetic products. The most studied copper peptide is GHK-Cu (glycyl-L-histidyl-L-lysine), a small copper-binding peptide, naturally present in human plasma. Since its discovery in 1973, in vivo and in vitro studies have shown that GHK-Cu possesses a wealth of health-positive actions including improving wound contraction and epithelization, and increasing the production of growth factors and activity of antioxidant enzymes. Recently, gene expression profiling shed new light on diverse biological actions of GHK-Cu. The present paper discusses evidence of GHK-Cu and other small copper peptides possessing potent anti-cancer properties.
... GHK effectively reduces the formation of both acrolein and another product of oxidation, 4-hydroxynonenal. GHK also blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehyde, and glyoxal [64][65][66]. ...
Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function.
... GHK also blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehyde, and glyoxal [37]. ...
The copper binding tripeptide GHK (glycyl-l-histidyl-l-lysine) is a naturally occurring plasma peptide that significantly declines during human aging. It has been established that GHK:Copper(2+) improves wound healing and tissue regeneration and stimulates collagen and decorin production. GHK-Cu also supports angiogenesis and nerve outgrowth, improves the condition of aging skin and hair, and possesses antioxidant and anti-inflammatory effects. In addition, it increases cellular stemness and secretion of trophic factors by mesenchymal stem cells. GHK’s antioxidant actions have been demonstrated in vitro and in animal studies. They include blocking the formation of reactive oxygen and carbonyl species, detoxifying toxic products of lipid peroxidation such as acrolein, protecting keratinocytes from lethal Ultraviolet B (UVB) radiation, and blocking hepatic damage by dichloromethane radicals. In recent studies, GHK has been found to switch gene expression from a diseased state to a healthier state for certain cancers and for chronic obstructive pulmonary disease (COPD). The Broad Institute’s Connectivity Map indicated that GHK induces a 50% or greater change of expression in 31.2% of human genes. This paper reviews biological data demonstrating positive effects of GHK in skin and proposes interaction with antioxidant-related genes as a possible explanation of its antioxidant activity.
... GHK directly blocks the formation of 4-hydroxynonenal and acrolein toxins created by carbonyl radicals that cause fatty acid decomposition [59,60]. GHK also blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4hydroxynoneal, acrolein, malondialdehye, and glyoxal [61]. ...
During human aging there is an increase in the activity of inflammatory, cancer promoting, and tissue destructive genes plus a decrease in the activity of regenerative and reparative genes. The human blood tripeptide GHK possesses many positive effects but declines with age. It improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, and boney tissue), increases collagen and glycosaminoglycans, stimulates synthesis of decorin, increases angiogenesis, and nerve outgrowth, possesses antioxidant and anti-inflammatory effects, and increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Recently, GHK has been found to reset genes of diseased cells from patients with cancer or COPD to a more healthy state. Cancer cells reset their programmed cell death system while COPD patients' cells shut down tissue destructive genes and stimulate repair and remodeling activities. In this paper, we discuss GHK's effect on genes that suppress fibrinogen synthesis, the insulin/insulin-like system, and cancer growth plus activation of genes that increase the ubiquitin-proteasome system, DNA repair, antioxidant systems, and healing by the TGF beta superfamily. A variety of methods and dosages to effectively use GHK to reset genes to a healthier state are also discussed.
The main purpose of current study is to analyze the structural behaviour of a skin protective tripeptide Gly-His-Lys (GHK) with anti-oxidant and anti-cancer properties, design and characterize its nanoformulation by experimental and spectroscopic techniques to ensure its stability and enhance bioavailability and biocompatibility. GHK is extensively used as a cosmetic products for the therapy of skin and hair deformation. In this study the calculations on GHK were performed at DFT/B3LYP level of theory with the 6–311++G (d,p) basis set to obtain optimized geometry, HOMO-LUMO energy gap, MEP analysis and vibrational wavenumbers. The spectroscopic investigation of GHK tripeptide was performed experimentally through optical spectroscopic techniques (such as FT-IR, FT-IR-ATR and Micro-RAMAN) and compared with theoretical wavenumbers. The Potantial Energy Distributions (PED) of the normal modes of vibrations were also carried out with the help of GAR2PED program. By using molecular dynamics simulation, the stability of the GHK in water and methanol mediums have been simulated. To reveal the mechanism of interaction between GHK tripeptide and Fibroblast Growth Factor, the hydrogen bonding interactions are also investigated by Molecular docking calculations. Besides, GHK-loaded Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles (NPs) were synthesized with double emission (water/oil/water) method, and characterized with Zeta Sizer, UV–Vis Spectrometry, Transmission Emission Microscope and FT-IR Spectrometry. Due to the encapsulation, the shifts in the wavenumbers occurring at the characteristic peaks of GHK in histidine, peptide and carboxyl groups were also examined. The encapsulation and loading efficiencies were determined as 94% and 4%, also the in vitro release profile was performed. Peptide loaded PLGA NPs which have a spherical morphology were visualized by TEM. In vitro cell culture studies of both peptide-loaded PLGA NPs and GHK tripeptide were studied and non-toxic effect on L929 cells were found. This study is a pioneer in the development and design of products with nano-drug formulations with better effectiveness and stability, especially in cosmetic products.
The copper-binding tripeptide GHK (glycyl-l-histidyl-l-lysine) is a naturally occurring plasma peptide widely used in skin care products. It is especially popular in antiaging cosmetic formulations due to its various and well-established positive biological effects on aging skin. It has been established that GHK-Cu improves wound healing and tissue regeneration and stimulates collagen and decorin production. GHK-Cu also supports angiogenesis and nerve outgrowth, improves the biological condition of aging skin and hair and possesses DNA repair, antioxidant, and anti-inflammatory effects. In addition, it increases cellular stemness and secretion of trophic factors by mesenchymal stem cells. GHK’s antioxidant actions have been demonstrated in vitro and in animal studies. They include blocking the formation of reactive oxygen and carbonyl species, detoxifying toxic products of lipid peroxidation such as acrolein, protecting keratinocytes from lethal UVB radiation, and blocking hepatic damage by dichloromethane radicals. In recent studies, GHK has also been found to switch cellular gene expression from a diseased state to a healthier state for certain cancers and for chronic obstructive pulmonary disease (COPD). The human gene expression actions provide a unique view of the complex and intricate gene actions underlying visible changes in human skin. This chapter reviews biological and gene data related to the positive antiaging effects of GHK on human skin.
The copper-binding tripeptide GHK (glycyl-l-histidyl-l-lysine) is a naturally occurring plasma peptide widely used in skin care products. It is especially popular in antiaging cosmetic formulations due to its various and well-established positive biological effects on aging skin. It has been established that GHK-Cu improves wound healing and tissue regeneration and stimulates collagen and decorin production. GHK-Cu also supports angiogenesis and nerve outgrowth, improves the biological condition of aging skin and hair and possesses DNA repair, antioxidant, and anti-inflammatory effects. In addition, it increases cellular stemness and secretion of trophic factors by mesenchymal stem cells. GHK’s antioxidant actions have been demonstrated in vitro and in animal studies. They include blocking the formation of reactive oxygen and carbonyl species, detoxifying toxic products of lipid peroxidation such as acrolein, protecting keratinocytes from lethal UVB radiation, and blocking hepatic damage by dichloromethane radicals. In recent studies, GHK has also been found to switch cellular gene expression from a diseased state to a healthier state for certain cancers and for chronic obstructive pulmonary disease (COPD). The human gene expression actions provide a unique view of the complex and intricate gene actions underlying visible changes in human skin. This chapter reviews biological and gene data related to the positive antiaging effects of GHK on human skin.
Human aging is marked by increased inflammation, a decline in tissue repair, and a deterioration of organ function. Externally, this diminution leads to wrinkles, photodamage, looser skin, bone loss, and other signs of aging. Growing old is also accompanied by a lessened resistance to infections. The human peptide GHK (glycyl-l-histidyl-l-lysine), as its copper 2+ complex (GHK-Cu), acts as an anti-aging activator for the late stages of tissue repair while also suppressing inflammatory cytokines. This unique molecule also increases resistance to infections. Results of an analysis of its actions on wound repair point to the existence of a biochemical copper switch that separates inflammatory and anti-inflammatory conditions. GHK may provide the key to improving organ rebuilding and maintenance. Furthermore, GHK-Cu’s actions on stem cells suggest that it might be used systemically to drive the progression of dormant stem cells within organs into the types of differentiated cells needed for organ rebuilding and maintenance. It is also possible that it could increase the therapeutic success of externally administered stem cells.
The surface activity of chroman-6 (CR-6) and chroman-6 palmitoyl ester (PCR-6) and the interactions with lipid membranes, using 1,2-dipamitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) monolayers, were determined. 8-Anilino-1-naphthalenesulfonic acid titration indicates that none of these molecules was able to form aggregates in aqueous media. The presence of a palmitoyl chain in PCR-6 increases strongly the surface activity of the parent compound (CR-6), rendering a molecule to form stable monomolecular layers. The interaction of both compounds with DPPC and DOPC, measured at constant area or in a compression isotherm model, follows the same trend. Miscibility studies performed with DPPC/CR-6 or PCR-6 indicate that the energies involved are small. The presence of CR-6 and PCR-6 has a soft influence on the compressibility of the Langmuir mixed films. Differential scanning calorimetry studies indicate that CR-6 and PCR-6 modify the temperature and cooperativity of the transition from gel to liquid crystal process in DPPC vesicles.
Singlet-oxygen is a non-radical reactive oxygen species believed to play a major role in many photooxidation processes in connection with diverse photo-biological processes such as skin ageing or photocarcinogenesis. Dimethylmethoxy chromanol (3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran) is a potent antioxidant used in cosmetic and pharmaceutical formulations. We have assessed the singlet oxygen quenching ability of dimethylmethoxy chromanol, by monitoring the near-IR phosphorescence of singlet-oxygen in solution and in ex vivo porcine skin samples. Dimethylmethoxy chromanol quenches singlet oxygen with a rate constant of (1.3 ± 0.1) × 108 M−1 s−1 in solution. Consistent with this, a clear reduction in the singlet oxygen lifetime and emission intensity was observed when ex vivo porcine skin samples were treated with dimethylmethoxy chromanol.
L'oxygène singulet est une espèce non-radicalaires réactive de l'oxygène réputée jouer un rôle majeur dans des nombreux processus de photo-oxydation dans le cadre de divers processus photo-biologiques comme le vieillissement de la peau ou la photocarcinogénèse. Le Dimethylmethoxy chromanol (3,4-dihydro-6-hydroxy-2,2-diméthyl-7-méthoxy-1 (2H)-benzopyran) est un antioxydant puissant utilisé dans des formulations pharmaceutiques et cosmétique. Nous avons évalué la capacité de quenching de l'oxygène singulet par le chromanol dimethylmethoxy, en suivant la phosphorescence dans l'infrarouge proche de l'oxygène singulet en solution et dans des échantillons de peau de porc ex-vivo. Le Dimethylmethoxychromanol épuise l'oxygène singulet avec une vitesse constante de (1,3 ± 0,1) × 108M−1s−1en solution. Conformément à cela, une nette diminution de la durée de vie et de l'intensité des émissions de l'oxygène singulet a été observée lorsque des échantillons de peau de porc ex-vivo ont été traités avec le chromanol dimethylmethoxy.
Tissue remodeling follows the initial phase of wound healing and stops inflammatory and scar-forming processes, then restores the normal tissue morphology. The human peptide Gly-(L-His)-(L-Lys) or GHK, has a copper 2+ (Cu(2+)) affinity similar to the copper transport site on albumin and forms GHK-Cu, a complex with Cu(2+). These two molecules activate a plethora of remodeling related processes: (1) chemoattraction of repair cells such as macrophages, mast cells, capillary cells; (2) anti-inflammatory actions (suppression of free radicals, thromboxane formation, release of oxidizing iron, transforming growth factor beta-1, tumor necrosis factor alpha and protein glycation while increasing superoxide dismutase, vessel vasodilation, blocking ultraviolet damage to skin keratinocytes and improving fibroblast recovery after X-ray treatments); (3) increases protein synthesis of collagen, elastin, metalloproteinases, anti-proteases, vascular endothelial growth factor, fibroblast growth factor 2, nerve growth factor, neutrotropins 3 and 4, and erythropoietin; (4) increases the proliferation of fibroblasts and keratinocytes; nerve outgrowth, angiogenesis, and hair follicle size. GHK-Cu stimulates wound healing in numerous models and in humans. Controlled studies on aged skin demonstrated that it tightens skin, improves elasticity and firmness, reduces fine lines, wrinkles, photodamage and hyperpigmentation. GHK-Cu also improves hair transplant success, protects hepatic tissue from tetrachloromethane poisoning, blocks stomach ulcer development, and heals intestinal ulcers and bone tissue. These results are beginning to define the complex biochemical processes that regulate tissue remodeling.
Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.
Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.
Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of .NO and O2.- during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how gamma-tocopherol (gammaT), the principal form of vitamin E in the United States diet, and alpha-tocopherol (alphaT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the .NO and O2.- generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by gammaT than alphaT. More importantly, nitration of gammaT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of approximately 50% and approximately 75%, respectively, was not affected by the presence of alphaT. These results suggest that despite alphaT's action as an antioxidant gammaT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that gammaT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in alphaT. Because large doses of dietary alphaT displace gammaT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily alphaT should be reconsidered.
Lipoprotein peroxidation, especially the modification of apolipoprotein B-100, has been implicated to play an important role in the pathogenesis of atherosclerosis. However, there have been few detailed insights into the chemical mechanism of derivatization of apolipoproteins during oxidation. In the present study, we provide evidence that the formation of the toxic pollutant acrolein (CH2=CH-CHO) and its conjugate with lysine residues is involved in the oxidative modification of human low density lipoprotein (LDL). Upon incubation with LDL, acrolein preferentially reacted with lysine residues. To determine the structure of acrolein-lysine adduct in protein, the reaction of acrolein with a lysine derivative was carried out. Employing Nalpha-acetyllysine, we detected a single product, which was identified to be a novel acrolein-lysine adduct, Nalpha-acetyl-Nepsilon-(3-formyl-3,4-dehydropiperidino )lysine. The acid hydrolysis of the adduct led to the derivative that was detectable with amino acid analysis. It was revealed that, upon in vitro incubation of LDL with acrolein, the lysine residues that had disappeared were partially recovered by Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine. In addition, we found that the same derivative was detected in the oxidatively modified LDL with Cu2+ and that the adduct formation was correlated with LDL peroxidation assessed by the consumption of alpha-tocopherol and cholesteryl ester and the concomitant formation of cholesteryl ester hydroperoxide. Enzyme-linked immunosorbent assay that measures free acrolein revealed that a considerable amount of acrolein was released from the Cu2+-oxidized LDL. Furthermore, metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, indicating that polyunsaturated fatty acids including arachidonate represent potential sources of acrolein generated during the peroxidation of LDL. These results indicate that acrolein is not just a pollutant but also a lipid peroxidation product that could be ubiquitously generated in biological systems.
In the present study, we studied the signal transduction mechanism that is involved in the expression of c-Jun protein evident after exposure of rat liver epithelial RL34 cells to the major end product of oxidized fatty acid metabolism, 4-hydroxy-2-nonenal (HNE). HNE treatment of the cells resulted in depletion of intracellular glutathione (GSH) and in the formation of protein-bound HNE in plasma membrane. In addition, HNE strongly induced intracellular peroxide production, suggesting that HNE exerted oxidative stress on the cells. Potent expression of c-Jun occurred within 30 min of HNE treatment, which was accompanied by a time-dependent increase in activator protein-1 (AP-1) DNA binding activity. We found that HNE caused an immediate increase in tyrosine phosphorylation in RL34 cells. In addition, HNE strongly induced phosphorylation of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases and also moderately induced phosphorylation of extracellular signal-regulated kinases. The phosphorylation of JNK was accompanied by a rapid and transient increase in JNK and p38 activities, whereas changes in the activity of extracellular signal-regulated kinase were scarcely observed. GSH depletion by L-buthionine-S, R-sulfoximine, a specific inhibitor of GSH biosynthesis, only slightly enhanced peroxide production and JNK activation, suggesting that HNE exerted these effects independent of GSH depletion. This and the findings that (i) HNE strongly induced intracellular peroxide production, (ii) HNE-induced JNK activation was inhibited by pretreatment of the cells with a thiol antioxidant, N-acetylcysteine, and (iii) H2O2 significantly activated JNK support the hypothesis that pro-oxidants play a crucial role in the HNE-induced activation of stress signaling pathways. In addition, we found that, among the inhibitors of tyrosine kinases, cyclooxygenase, and Ca2+ influx, only quercetin exerted a significant inhibitory effect on HNE-induced JNK activation. In light of the JNK-dependent induction of c-jun transcription and the AP-1-induced transcription of xenobiotic-metabolizing enzymes, these data may show a potential critical role for JNK in the induction of a cellular defense program against toxic products generated from lipid peroxidation.
The use of genetically altered laboratory animals offers a direct means to evaluate these alternative hypotheses in living systems and to probe aspects of the pathology of oxidative stress that are not readily amenable to other types of investigation. A particularly compelling illustration of the power of the approach is the study reported by Heinecke and associates in this issue of the JCI (13). They found that nitrotyrosine levels in peritoneal fluids from MPO-deficient mice infected with Klebsiella pneumoniae are markedly reduced relative to those from wild-type mice — this despite the induction of iNOS in both strains and a comparable accumulation of NO2–. By simultaneously measuring chlorotyrosine and nitrotyrosine in these fluids, the authors showed that active MPO is present in the peritoneum of the infected wild-type mice but not in the MPO knockout mice; specifically, the yields of the two tyrosine derivatives were comparable in the wild-type mice, but chlorotyrosine was completely lacking in the MPO knockouts. Since the tyrosine-nitrating and -chlorinating activities of MPO should be roughly comparable under the prevailing in vivo conditions, it follows that the large increase in nitrotyrosine yields measured in normal mice most likely arose by MPO catalysis, the unattractive alternative being that MPO somehow enabled expression of another as-yet unidentified nitration mechanism. Very similar conclusions have been reached by Hazen and collaborators, who compared nitrotyrosine accumulation in several acute inflammatory models that made use of MPO knockout and eosinophil peroxidase knockout mice (14).
Other studies using knockouts to examine the role of MPO in tissue damage have not been so readily interpretable. In an earlier JCI publication, Heinecke and associates reported that LDL receptor- and MPO-deficient double-knockout mice were more susceptible to atherosclerosis than were littermates whose neutrophils contained normal MPO activity (15). Similarly, Takizawa and coworkers have recently reported that ischemia/reperfusion–induced cerebral damage was greater in MPO knockout than in wild-type mice and was accompanied by increased levels of nitration of protein tyrosyls (16). In both studies, MPO was apparently not present at the nitration sites. Numerous potential explanations (summarized in ref. 15) can be imagined for the apparent protection by MPO, although the actual mechanisms remain to be determined. An even more provocative study has involved use of mice deficient in neutrophil granule proteases, but with apparently normal oxidative capacities (17). These protease-deficient mice were unusually susceptible to infection by Staphylococcus aureus and Candida albicans, which was reflected in inefficient killing of these organisms by their isolated neutrophils. The authors of this study suggest that proteases are the central agents of microbicidal action, the primary purpose of respiratory activation being to electrogenically increase the intraphagosomal ionicity, thereby electrostatically triggering release of the proteases from inhibitory complexes with acid proteoglycans. MPO in this model is assigned the role of protecting the proteases from inactivation by H2O2 via catalatic degradation to O2 and H2O2.
Reactive oxygen species (ROS) are associated not only with initiation, but also with promotion and progression in the multistage carcinogenesis model. In the present review, we will focus on the involvement of ROS in skin carcinogenesis, especially that induced by ultraviolet (UV) radiation. UV-specific DNA damage has been well studied thus far. However, recent reports have revealed the previously unknown participation of oxidative stress in UV-induced skin carcinogenesis. Indeed, in addition to transition-type mutations at dipyrimidine sites, G:C to T:A transversions, which may be induced by the presence of 8-oxoguanine during DNA replication, are frequently observed in the ras oncogene and p53 tumor suppressor gene in human skin cancers of sun-exposed areas and in UV-induced mouse skin cancers. Recent studies have shown that not only UV-B, but also UV-A is involved in UV-induced carcinogenesis. A wide variety of biological phenomena other than direct influence by UV, such as inflammatory and immunological responses and oxidative modifications of DNA and proteins, appear to play roles in UV-induced skin carcinogenesis. Furthermore, it has become clear that genetic diseases such as xeroderma pigmentosum show deficient repair of oxidatively modified DNA lesions. The involvement of ROS in skin carcinogeneisis caused by arsenic and chemical carcinogens will also be discussed.
Cardiovascular complications, characterized by endothelial dysfunction and accelerated atherosclerosis, are the leading cause of morbidity and mortality associated with diabetes. There is growing evidence that excess generation of highly reactive free radicals, largely due to hyperglycemia, causes oxidative stress, which further exacerbates the development and progression of diabetes and its complications. Overproduction and/or insufficient removal of these free radicals result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids. Despite overwhelming evidence on the damaging consequences of oxidative stress and its role in experimental diabetes, large scale clinical trials with classic antioxidants failed to demonstrate any benefit for diabetic patients. As our understanding of the mechanisms of free radical generation evolves, it is becoming clear that rather than merely scavenging reactive radicals, a more comprehensive approach aimed at preventing the generation of these reactive species as well as scavenging may prove more beneficial. Therefore, new strategies with classic as well as new antioxidants should be implemented in the treatment of diabetes.
The use of genetically altered laboratory animals offers a direct means to evaluate these alternative hypotheses in living systems and to probe aspects of the pathology of oxidative stress that are not readily amenable to other types of investigation. A particularly compelling illustration of the power of the approach is the study reported by Heinecke and associates in this issue of the JCI (13). They found that nitrotyrosine levels in peritoneal fluids from MPO-deficient mice infected with Klebsiella pneumoniae are markedly reduced relative to those from wild-type mice — this despite the induction of iNOS in both strains and a comparable accumulation of NO2–. By simultaneously measuring chlorotyrosine and nitrotyrosine in these fluids, the authors showed that active MPO is present in the peritoneum of the infected wild-type mice but not in the MPO knockout mice; specifically, the yields of the two tyrosine derivatives were comparable in the wild-type mice, but chlorotyrosine was completely lacking in the MPO knockouts. Since the tyrosine-nitrating and -chlorinating activities of MPO should be roughly comparable under the prevailing in vivo conditions, it follows that the large increase in nitrotyrosine yields measured in normal mice most likely arose by MPO catalysis, the unattractive alternative being that MPO somehow enabled expression of another as-yet unidentified nitration mechanism. Very similar conclusions have been reached by Hazen and collaborators, who compared nitrotyrosine accumulation in several acute inflammatory models that made use of MPO knockout and eosinophil peroxidase knockout mice (14).
Other studies using knockouts to examine the role of MPO in tissue damage have not been so readily interpretable. In an earlier JCI publication, Heinecke and associates reported that LDL receptor- and MPO-deficient double-knockout mice were more susceptible to atherosclerosis than were littermates whose neutrophils contained normal MPO activity (15). Similarly, Takizawa and coworkers have recently reported that ischemia/reperfusion–induced cerebral damage was greater in MPO knockout than in wild-type mice and was accompanied by increased levels of nitration of protein tyrosyls (16). In both studies, MPO was apparently not present at the nitration sites. Numerous potential explanations (summarized in ref. 15) can be imagined for the apparent protection by MPO, although the actual mechanisms remain to be determined. An even more provocative study has involved use of mice deficient in neutrophil granule proteases, but with apparently normal oxidative capacities (17). These protease-deficient mice were unusually susceptible to infection by Staphylococcus aureus and Candida albicans, which was reflected in inefficient killing of these organisms by their isolated neutrophils. The authors of this study suggest that proteases are the central agents of microbicidal action, the primary purpose of respiratory activation being to electrogenically increase the intraphagosomal ionicity, thereby electrostatically triggering release of the proteases from inhibitory complexes with acid proteoglycans. MPO in this model is assigned the role of protecting the proteases from inactivation by H2O2 via catalatic degradation to O2 and H2O2.
The browning of proteins via the Maillard reaction in vivo involves nonenzymatic autoxidation and/or enzyme-catalysed oxidation of carbohydrates, lipids and amino acids. Carbohydrates also contribute to non-oxidative browning of proteins by rearrangement and elimination pathways which generate deoxydicarbonyl compounds, such as deoxyglucosone and methylglyoxal. A common feature of both the oxidative and non-oxidative reactions is the formation of reactive carbonyl compounds, suggesting that carbonyl stress, as well as oxidative stress, is involved in chemical modifications of proteins during the Maillard reaction. Oxidative stress may enhance the damage produced by carbonyl stress, both by providing an additional source of carbonyls and by limiting the rate of their detoxification.
Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is
formed by the near diffusion-limited reaction of \cdot NO and
O2{\cdot} during activation of phagocytes. Chronic
inflammation induced by phagocytes is a major contributor to cancer and
other degenerative diseases. We examined how γ -tocopherol
(γ T), the principal form of vitamin E in the United States diet,
and α -tocopherol (α T), the major form in supplements,
protect against peroxynitrite-induced lipid oxidation. Lipid
hydroperoxide formation in liposomes (but not isolated low-density
lipoprotein) exposed to peroxynitrite or the \cdot NO and
O2{\cdot} generator SIN-1
(3-morpholinosydnonimine) was inhibited more effectively by γ T
than α T. More importantly, nitration of γ T at the
nucleophilic 5-position, which proceeded in both liposomes and human low
density lipoprotein at yields of ≈ 50% and ≈ 75%, respectively,
was not affected by the presence of α T. These results suggest
that despite α T's action as an antioxidant γ T is required
to effectively remove the peroxynitrite-derived nitrating species. We
postulate that γ T acts in vivo as a trap for membrane-soluble
electrophilic nitrogen oxides and other electrophilic mutagens, forming
stable carbon-centered adducts through the nucleophilic 5-position,
which is blocked in α T. Because large doses of dietary α T
displace γ T in plasma and other tissues, the current wisdom of
vitamin E supplementation with primarily α T should be
reconsidered.
The effects on rat liver microsomal lipid peroxidation elicited by 2,2-dimethylchromenes and chromans structurally related to precocenes was investigated in NADPH-dependent incubations by using the thiobarbituric acid reactive substances (TBARS) and oxygen uptake rate tests as evaluation methods. Precocene II (1) exhibited an unexpected inhibitory activity on lipid peroxidation (IC50 = 11.4-mu-M, TBARS production test). Among the compounds tested, those which had a hydroxyl group on the aromatic ring, particularly at the C-6 position (i.e., 6-hydroxy-7-methoxy-2,2-dimethyl-1-2H-benzopyran (2) and its 3,4-dihydro derivative 5), appeared to be the best inhibitors. In this context, the inhibitory effect elicited by the hydroxychroman 5 (IC50 = 0.3-mu-M, TBARS production test) was higher than that shown by 2,6-di-tert-butyl-4-methylphenol (BHT, 11). These results suggested that the lipid peroxidation inhibitory effect exhibited by precocene II and related derivatives could be due to a vitamin E-like free radical scavenger based mechanism. The identification of phenolic metabolites in the incubations performed with precocene II or its benzopyranyl analogues lacking aromatic hydroxyl substituents supported the above postulated mechanism.
The presence of stratum corneum carbonyls may serve as an intrinsic dosimeter for environmental oxidative damage to skin. To investigate the accumulation of carbonyls in human stratum corneum, skin was tape-stripped, then tapes were sequentially incubated with 2,4-dinitrophenyl hydrazine (DNPH), rat anti-DNP, mouse anti-rat IgG conjugated with alkaline phosphatase, p-nitrophenyl phosphate and absorbance (405 nm) measured and carbonyls estimated. Stratum corneum exposed in vitro to oxidants: hypochlorous acid (1, 10, 100 mM), ozone (0, 1, 5, 10 ppm for 2 h) or UV light (280-400 nm; 0, 4, 88, or 24 J/cm2) contained increased carbonyls. Furthermore, stratum corneum carbonyls were elevated in tanned compared with untanned sites: dorsal hand (0.43 +/- 0.06 nmol/cm2) vs. lower arm (0.32 +/- 0.04, mean +/- S.E.M., n = 11; P < 0.003) and lower back (0.26 +/- 0.02) vs. buttock (0.21 +/- 0.02; n = 6, P < 0.01) indicating in vivo oxidative damage.
Cutaneous aging represents a complex situation in which at least two independent factors--innate aging and solar exposure--contribute to the development of degenerative changes in the dermis. The biochemical and ultrastructural evidence reviewed in this article indicates that reduced collagen deposition, as a result of diminished collagen biosynthesis and reduced proliferative capacity of the fibroblasts, could explain the development of dermal atrophy and would relate to poor wound healing in the elderly. At the same time, perturbations in the supramolecular organization of the elastic fiber network lead to alterations in the mechanical properties of the skin, as manifested by loose and sagging skin with reduced resilience and elasticity.
Cutaneous aging represents a complex situation in which at least two independent factors--innate aging and solar exposure--contribute to the development of degenerative changes in the dermis. The biochemical and ultrastructural evidence reviewed in this article indicates that reduced collagen deposition, as a result of diminished collagen biosynthesis and reduced proliferative capacity of the fibroblasts, could explain the development of dermal atrophy and would relate to poor wound healing in the elderly. At the same time, perturbations in the supramolecular organization of the elastic fiber network lead to alterations in the mechanical properties of the skin, as manifested by loose and sagging skin with reduced resilience and elasticity.
Peroxynitrite, formed by combination of superoxide radical with nitric oxide, is a reactive tissue-damaging species apparently involved in the pathology of several human diseases. Peroxynitrite nitrates tyrosine residues and inactivates alpha 1-antiproteinase. We show that both lipoic acid and dihydrolipoic acid efficiently protect against damage by peroxynitrite. By contrast, other disulphides tested did not. The biological antioxidant effects of lipoate/dihydrolipoate may involve scavenging of reactive nitrogen species as well as reactive oxygen species.
Local intra-arterial infusion of high doses of nitric oxide (NO) donors, such as S-nitroso-N-acetyl-penicillamine or nitroprusside cause extensive gastric mucosal damage. The involvement of lipid peroxidation of mucosal tissue in the mechanism of such gastric damage has been investigated in the pentobarbitone-anaesthetised rat. Local intra-arterial infusion of nitroprusside (40 micrograms.kg-1.min-1) or S-nitroso-N-acetyl-penicillamine (40 micrograms.kg-1.min-1) induced macroscopically apparent injury and provoked a dose-dependent peroxidation of lipid in gastric tissue. By contrast, endothelin-1 infusion provoked mucosal injury of the mucosa, yet did not produce lipid peroxidation. Local co-infusion of superoxide dismutase (2000-4000 IU.kg-1) reduced both the lipid peroxidation and the mucosal damage provoked by S-nitroso-N-acetyl-penicillamine and nitroprusside. These findings indicate that lipid peroxidation accompanies the mucosal tissue damage induced by NO donors, while the action of superoxide dismutase implicates the involvement of peroxynitrite, formed from superoxide and NO, in this process.
There is increasing evidence that aldehydes generated endogenously during lipid peroxidation contribute to the pathophysiologic effects associated with oxidative stress in cells and tissues. A number of reactive lipid aldehydes, such as 4-hydroxy-2-alkenals and malondialdehyde, have been implicated as causative agents in cytotoxic processes initiated by the exposure of biologic systems to oxidizing agents. Recently, acrolein (CH2 = CH-CHO), a ubiquitous pollutant in the environment, was identified as a product of lipid peroxidation reactions. The basis for this finding is an experimental approach that provides a measure of acrolein bound to lysine residues of protein. The identification of acrolein as an endogenous lipid-derived product suggests an examination of the possible role of this aldehyde as a mediator of oxidative damage in a variety of human diseases.
Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.
In this work we describe the development of specific markers for determination of both the membrane and intracellular damage induced by free radicals generated by UVB radiation (5-150 mJ/cm2) in cultured keratinocytes. This using simple, specific and sensitive fluorescent probes: cis-parinaric acid (PNA) to monitor membrane lipid peroxidation and 2',7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) to evaluate the intracellular redox status, in parallel to the fluorimetric determination of the main intracellular antioxidant glutathione. To validate the methodologies, the changes in the intracellular oxidative status following exposure to low doses UVB were measured in both control and N-acetylcysteine-protected cells, in parallel with morphological analyses. UVB induces an early reduction of GSH inside the cell correlated with an increase in the intracellular peroxide content. The effects were time- and dose-dependent. In addition, using a sensitive fluorescent method, we quantitated the release of proteases, a family of proteolytic enzymes greatly involved in the onset/perpetuation of the free radical-induced skin damage from keratinocytes exposed to suberythemal UVB doses (5-15 mJ/cm2). The use of these fluorescent probes provides a reliable tool to detect the early signs of damage in keratinocyte cultures (when the apoptotic phenomenon has not yet been triggered) useful for future screening of protective molecules.
The inhibitory effects of endogenous and synthetic compounds on the nitration and oxidation of L-tyrosine by peroxynitrite were examined. Nitration and oxidation activities of L-tyrosine by peroxynitrite were estimated by monitoring the formation of 3-nitrotyrosine and dityrosine with a high-performance liquid chromatography-ultraviolet (HPLC-UV)-fluorescence detector system. Glutathione and synthetic compounds ((2S,3R,4S)-N-ethylmercapto-3,4-dihydroxy-2-hydroxymethylpyrrolidine, L-N-dithiocarboxyproline) inhibited both the nitration and the oxidation reactions of L-tyrosine effectively. On the other hand, 5-methoxytryptamine and lipoic acid inhibited only the nitration reaction of L-tyrosine, and instead increased the oxidation reaction. It was assumed that 5-methoxytryptamine and lipoic acid reacted only with the nitrating species of peroxynitrite. This is the first report of a selective inhibitor for the nitrating reaction of peroxynitrite.
Clues as to why long-lived species live so much longer than short-lived species may reside in the amount of reactive oxygen species (ROS) produced and their effect on damaging cell components (especially proteins) and alterations of crucial cellular processes. Rigorous evaluation of these concepts required critical comparisons of oxidative damage markers and/or parameters with assess difference in ROS flux and the critical age-modifying processes they influence. The limited experimental comparative results available implicate that ROS production per unit weight of total oxygen consumed is much less in the longer-lived species than in shorter-lived species. Mitochondria are the major site of ROS production. They are also the functional nexus for intracellular signaling thus modulating stress and growth factor mediated cellular survival, proliferation and apoptotic processes. Mitochondrial DNA mutations, perhaps caused by ROS, increase with age. Mutant mitochondria possess comparative replicative advantage, which leads to age-specific intracellular swarms. General inflammatory stress tends to increase with age. Disruption in coordinated cell-to-cell signaling triggered by alterations in intracellular signaling may be the basis of the age-related increases in tissue inflammation, which may explain some of the differences between long-lived species and short-lived species.
Acrolein and 4-hydroxy-2-nonenal (HNE) are both byproducts of a lipid peroxidation reaction. Actinic elastosis in photodamaged skin of aged individuals is characterized by the accumulation of fragmented elastic fibers in the sun-exposed areas. To study whether a lipid peroxidation reaction is involved in the accumulation of altered elastic fibers in actinic elastosis, skin specimens obtained from sun-damaged areas were immunohistochemically examined using the antibodies against acrolein and HNE. Both antibodies were found to react with the accumulations of elastic material. Double immunofluorescence labeling demonstrated that acrolein/elastin and HNE/elastin were colocalized in the actinic elastosis. Western blot analysis showed that the polypeptide with a molecular weight of 62 kDa reacted with anti-acrolein, anti-HNE and anti-elastin antibodies. The results suggest that acrolein and HNE may be associated with actinic elastosis.
Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.
Recent discoveries in the biology of nitrosating agents, such as S-nitrosoglutathione (GSNO), have revealed that several proteins are particularly susceptible to S-nitrosation at specific cysteine residues. In searching for consensus nitrosation sites, it has been suggested that there are both hydrophobic and acid-base motifs in target proteins adjacent to the site of their modification (56). GSNO, a commonly used exogenous nitrosating agent, has been detected in vivo at micromolar concentrations in brain tissue and can directly transfer NO equivalents to target protein thiols (8). Additionally, other mechanisms of S-nitrosation of nucleophilic cysteines (e.g., by N2O3) are plausible (8). S-nitrosothiols in proteins can react with free sulfhydryl groups to create disulfide bonds and can be reduced by various reductants (8). Although the chemical pathways through which nitric oxide forms S-nitrosocysteine are complex, experiments using induced expression of NO synthase isoforms and neuronal NO synthase knockout cells have established that NO production increases levels of overall protein S-nitrosation (57, 58). S-nitrosation of proteins is potentially a key method that cells use to mediate inflammatory responses and other NO-regulated processes.
Proteome-wide screening for modified proteins promises to be an exciting area of research in the immediate future. High-throughput methods have recently been developed for examining global protein S-nitrosation, glutathionylation, and tyrosine nitration (57, 93, 94). Although most current methods use the power of mass spectrometry for identification of proteins, the approaches taken to enrich for and isolate modified proteins differ. For example, an elegant approach to the isolation of S-nitrosated proteins was recently reported (57). Free thiol groups were reacted with methyl methanethiosulfonate, and then S-NO groups were reduced to free thiols by treatment with ascorbate. Subsequently, using streptavidin agarose beads, the newly released protein thiols were converted to biotin conjugates to enrich for proteins. Peptides from isolated proteins were identified using matrix-assisted laser desorption ionization mass spectrometry. Approximately 15–20 proteins were identified as targets of nitrosating agents. The methodology was validated by confirmation that many targets of exogenous nitrosating agents are in vivo targets, as revealed by the lack of labeling in brain lysates from mice deficient in neuronal NO synthase.
The notion that oxidative stress contributes to the pathogenesis of vascular disease was originally driven by observations that low-density lipoprotein (LDL) modification is a prominent feature of atherosclerosis. More recently, it has become clear that the relation between oxidative stress and vascular disease goes beyond LDL oxidation and involves cellular production of reactive oxygen species (ROS). Considerable data now indicate that ROS represent an important means of cellular signaling, although the precise mechanisms whereby ROS accomplish this function remain unclear. Emerging data point to protein thiol groups as important targets for post-translational protein modification by ROS. In this review, the data linking ROS to cell signaling is discussed and the notion that ROS mediate a vascular "injury" response is proposed.
The anatomy and functions of the blood and lymph vessels of human skin are described. Variation in these due to site, ageing and events during life consequent to exposure to a threatening environment are emphasised. Gradual atrophy and greater heterogeneity are features of ageing. Responses to injury and repair are complex and the interaction of mechanical signals distorting skin cells with numerous chemical signals are referred to. The lymphatics are part of an immunosurveillance system to monitor skin barrier penetration. The review attempts to draw attention to key recent advances in our understanding of the cytokine and growth factor production of the skin in the context of previous mainly physiological reviews especially influenced by 50 years of clinical practice as a dermatologist with an eye on both the skin and the fields of microcirculation and lymphology.
Multiple lines of evidence demonstrate that oxidative stress is an early event in Alzheimer's disease (AD), occurring prior to cytopathology, and therefore may play a key pathogenic role in the disease. Indeed, that oxidative mechanisms are involved in the cell loss and other neuropathology associated with AD is evidenced by the large number of metabolic signs of oxidative stress as well as by markers of oxidative damage. However, what is intriguing is that oxidative damage decreases with disease progression, such that levels of markers of rapidly formed oxidative damage, which are initially elevated, decrease as the disease progresses to advanced AD. This finding, along with the compensatory upregulation of antioxidant enzymes found in vulnerable neurons in AD, indicates that reactive oxygen species (ROS) not only cause damage to cellular structures but also provoke cellular responses. Mammalian cells respond to extracellular stimuli by transmitting intracellular instructions by signal transduction cascades to coordinate appropriate responses. Therefore, not surprisingly stress-activated protein kinase (SAPK) pathways, pathways that are activated by oxidative stress, are extensively activated during AD. In this paper, we review the evidence of oxidative stress and compensatory responses that occur in AD with a particular focus on the roles and mechanism of activation of SAPK pathways.
The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although 1H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.
Skin aging appears to be the result of both scheduled and continuous "wear and tear" processes that damage cellular DNA and proteins. Two types of aging, chronological skin aging and photoaging, have distinct clinical and histological features. Chronological skin aging is a universal and inevitable process characterized primarily by physiologic alterations in skin function. In this case, keratinocytes are unable to properly terminally differentiate to form a functional stratum corneum, and the rate of formation of neutral lipids that contribute to the barrier function slows, causing dry, pale skin with fine wrinkles. In contrast, photoaging results from the UVR of sunlight and the damage thus becomes apparent in sun-exposed skin. Characteristics of this aging type are dry and sallow skin displaying fine wrinkles as well as deep furrows, resulting from the disorganization of epidermal and dermal components associated with elastosis and heliodermatitis. Understanding of the functions of the skin and the basic principles of moisturizer use and application is important for the prevention of skin aging. Successful treatment of dry skin with appropriate skin care products gives the impression of eternal youth.
Synopsis
Fighting skin ageing is one of the major targets of cosmetology research. However, traditional approaches to skin ageing using stimulation of basal keratinocyte proliferation and fibroblastic neosynthesis appear today to be incomplete, particularly considering changes occurring at the dermal–epidermal junction (DEJ) during the course of ageing. Unfortunately, the lack of in vitro model limits the exploration process of the phenomena of DEJ ageing, and particularly the evaluation of the changes of key components, that are laminin‐5, types IV and VII collagens. The aim of this work was to provide an in vitro model of reconstructed skin, base for new dosage and identification methods for qualitative and quantitative analysis of the key components of DEJ. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and quantitative RT‐PCR were successfully applied to this model to analyse mRNA of laminin‐5, types IV and VII collagens and their variation in ‘young’ and ‘mature’ reconstructed skin model. Finally, this model was used to test the activity of ingredients for cosmetic application, in order to modulate the expression of the major components of DEJ. To conclude, we demonstrated that this in vitro model of reconstructed young and mature skin provides a useful tool to get into the biology of the DEJ, key structure of the skin, and specifically into its dynamic changes during the ageing process.
The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of (.)NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO(-)) and nitrogen dioxide ((.)NO2), formed as secondary products of (.)NO metabolism in the presence of oxidants including superoxide radicals (O2(.-)), hydrogen peroxide (H2O2), and transition metal centers. The precise interplay between (.)NO and oxidants and the identification of the proximal intermediate(s) responsible for nitration in vivo have been under controversy. Despite the capacity of peroxynitrite to mediate tyrosine nitration in vitro, its role on nitration in vivo has been questioned, and alternative pathways, including the nitrite/H2O2/hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitration in vivo, and (ii) most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (CO3(.-)) and/or oxo-metal complexes oxidizing tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with (.)NO2 to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of (.)NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.
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