Modulation of human keratinocyte responses to solar UV by plant polyphenols as a basis for chemoprevention of non-melanoma skin cancers.

Excessive exposure to solar UVA and UVB radiation is widely considered to cause skin cancers such as squamous cell carcinoma and basalioma. Direct UVB damage to skin cell DNA as well as UV-induced chronic skin inflammation, accelerated keratinocyte proliferation, inhibited apoptosis, and immunosuppression seem to underlie the UV-induced carcinogenesis. Also, UVB induces cytochrome P450 subfamilies (CYP1A1 and CYP1B1) involved in metabolic activation of organic pro-carcinogens and their conversion to ultimate carcinogens. Here, the effects of several glycosylated and non-glycosylated plant polyphenols (verbascoside, resveratrol, polydatin, rutin, and quercetin) on the inflammatory, apoptotic, metabolic, and proliferative responses of cultured human epidermal keratinocytes (HEK) to non-cytotoxic doses of solar-simulated UVA+UVB and chemical mediators of UV signalling in HEK, 6-formylindolo[3,2-b]carbazole and squalene isolated from photo-oxidized skin surface lipids (SSL), were evaluated. We showed that the stilbenes and quercetin being exposed to UV were photo-destroyed within a short period of time, while verbascoside and rutin were photo-stable. When SSL were exposed to UV, the stilbenes and quercetin remarkably accelerated photo-oxidation of alpha-tocopherol, squalene, and cholesterol fractions, whilst verbascoside protected them. Verbascoside invariably inhibited molecular pathways in HEK leading to inflammatory cytokine expression (NFkappaB and EGFR/ERK phosphorylation), and cell proliferation (EGFR nuclear translocation), and displayed a stimulus-specific effect on the metabolic axis aryl hydrocarbon receptor (AhR)-CYP1A1/CYP1B1. By contrast, the stilbenes inhibited UV-connected inflammatory cytokines excluding IL-8, but they prevalently stimulated NFkappaB, EGFR nuclear translocation and the AhR-CYP pathway. We conclude that, among the PPs investigated, verbascoside does interfere with multiple UV-sensitive signalling in HEK in a way that it could have a major impact on skin cancer chemoprevention.

alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.

BACKGROUND AND PURPOSE: The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. EXPERIMENTAL APPROACH: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. KEY RESULTS: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. CONCLUSIONS AND IMPLICATIONS: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Metal complexes of dietary flavonoids: evaluation of radical scavenger properties and protective activity against oxidative stress in vivo.

Interaction of certain flavonoids with transition metals increases their water solubility and leads to the formation of flavonoid-metal complexes, which may act as superoxide dismutase mimics with high scavenger potencies toward superoxide. Effect of serum albumin on stability of flavonoid-metal complexes was studied and complex of rutin with iron (II) was found to be the most stable. The ability of flavonoid metal complexes to catalyze homolytic cleavage of hydrogen peroxide was also studied and rutin iron (II) complex was found to be relatively poor Fenton catalyst. The potential therapeutic benefits of this new antioxidant agent were studied using experimental model of pathological states associated with oxidative stress in vivo. Acute hepatic injury in MT-I/MT-II null transgenic mice induced by injections of thioacetamide was used for this purpose. It was found that pretreatment with rutin- iron complex protected against thioacetamide induced hepatotoxicity as observed by a significant reduction in the elevated levels of serum enzymes and partial normalization of GSH/GSSG ratio, glutathione peroxidase II and glutathione reductase activity in mice liver. The results demonstrate that flavonoid-metal complexes possess effective free radical scavenger ability and have potent therapeutic benefits for the treatment of oxidative stress-related diseases and dysfunction.

Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron.

Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1-2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase.

Radical scavenging activities of flavonoids rutin, taxifolin, (-)-epicatechin, luteolin, and their complexes with transition metal (Fe2+, Fe3+, and Cu2+) towards superoxide were determined using illumination of riboflavin as source and NBT as detector of O*2-. The scavenger potencies of flavonoid metal complexes were significantly higher than those of the parent flavonoids. To elucidate the mechanism of this phenomenon, the rates of superoxide-dependent oxidation of flavonoids and their metal complexes in photochemical system with riboflavin were examined. It was found for the first time that flavonoids bound to metal ions were much less subjected to oxidation compared with those of free compounds. The findings directly demonstrate superoxide scavenging activity of metal ions in complexes with flavonoids and support earlier suggestions that flavonoid metal complexes may exhibit superoxide dismuting activity.

Comparative study of antioxidant properties and cytoprotective activity of flavonoids.

Antioxidant properties and cytoprotective activity of flavonoids (rutin, dihydroquercetin, quercetin, epigallocatechin gallate (EGCG), epicatechin gallate (ECG)) were studied. All these compounds inhibited both NADPH- and CCl4-dependent microsomal lipid peroxidation, and the catechins were the most effective antioxidants. The I(50) values calculated for these compounds by regression analysis were close to the I(50) value of the standard synthetic antioxidant ionol (2,6-di-tert-butyl-4-methylphenol). The antiradical activity of flavonoids to O2-* was studied in a model photochemical system. Rate constants of the second order reaction obtained by competitive kinetics suggested flavonoids to be more effective scavengers of oxygen anion-radicals than ascorbic acid. By competitive replacement all flavonoids studied were shown to be chelating agents capable of producing stable complexes with transition metal ions (Fe2+, Fe3+, Cu2+). The flavonoids protected macrophages from asbestos-induced damage, and the protective effect increased in the following series: rutin < dihydroquercetin < quercetin < ECG < EGCG. The cytoprotective effect of flavonoids was in strong positive correlation with their antiradical activity to O2-*.

Protective effect of copper-rutin complex in animals with experimental epilepsy.

Copper-rutin complex (2 mg/kg) completely eliminated epileptiform potentials induced by a combination of chlorpromazine and microwave radiation 1-2 min postinjection and suppressed convulsive activity provoked by application of penicillin to the sensorimotor cortex.

Influence of metal ions on flavonoid protection against asbestos-induced cell injury.

Influence of metal ions (Fe2+, Fe3+, Cu2+, Zn2+) on the protective effect of rutin, dihydroquercetin, and green tea epicatechins against in vitro asbestos-induced cell injury was studied. Metals have been found to increase the capacity of rutin and dihydroquercetin to protect peritoneal macrophages against chrysotile asbestos-induced injury. The data presented here show that this effect is due to the formation of flavonoid metal complexes, which turned out to be more effective radical scavengers than uncomplexed flavonoids. At the same time epicatechins and their metal complexes have similar antiradical properties and protective capacities against the asbestos induced injury of macrophages. Metal complexes of all flavonoids were found to be considerably more potent than parent flavonoids in protecting red blood cells against asbestos-induced injury. It was also found that the metal complexes of all flavonoids were absorbed by chrysotile asbestos fibers considerably better than uncomplexed compounds and probably for this reason flavonoid metal complexes have better protective properties against asbestos induced hemolysis. Thus, the results of the present study show that flavonoid metal complexes may be effective therapy for the inflammatory response associated with the inhalation of asbestos fiber. The advantage of their application could be the strong increase in ROS scavenging by flavonoids and finally a better cell protection under the conditions of cellular oxidative stress.

Protective effects of green tea catechins against asbestos-induced cell injury.

Green tea extract was found to provide a strong protective effect against asbestos-induced injury of peritoneal macrophages and red blood cells in vitro. The main polyphenolic constituents of green tea extract, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG), were also efficient in preventing injury of cells following exposure to asbestos fibers. The protective efficacies of EGCG and ECG expressed as IC50 values were, respectively, 10 microM and 12 microM if peritoneal macrophages were injured by chrysotile and 4 microM and 5 microM in the case of crocidolite-induced cell injury. Antiradical and chelating properties of ECG and EGCG were evaluated and it was concluded that the protective effect of catechins against asbestos-induced injury may be related to both scavenger properties towards to superoxide anion and the ability to chelate iron ions.

Antiradical and chelating effects in flavonoid protection against silica-induced cell injury.

Quercetin, dihydroquercetin, and rutin are capable of scavenging superoxide anion (rate constants of the reaction with superoxide at pH 10 were 1.7 x 10(5), 1.5 x 10(5), and 0.5 x 10(5) M-1 s-1, respectively). At the same time rutin and quercetin but not dihydroquercetin are iron ion chelators. These substances were used to elucidate the role of radical scavenging and iron chelating in flavonoid protection against asbestos-induced oxidative cellular injury. Exposure of rat peritoneal macrophages to chrysotile asbestos fibers resulted in "frustrated" phagocytosis, cell injury, and a LDH release. Quercetin, dihydroquercetin, and rutin were effective in protecting the phagocytic cells against injury caused by asbestos. Moreover, these flavonoids exhibited cellular protection in the same order of effectiveness as that observed for the quenching of superoxide: quercetin > dihydroquercetin > rutin. Exposure of human red blood cells to asbestos fibers also caused progressive cell injury and lysis. Quercetin and rutin protected the red cells (quercetin > rutin), whereas dihydroquercetin was ineffective in preventing asbestos-induced hemolysis. The protective ability of quercetin and rutin may be related to their iron-chelating activity. Due to this these flavonoids can be located on asbestos surface in sites of initiation of free radical reactions and their antiradical moieties can scavenge reactive oxygen species immediately after the appearance. Thus, both antiradical and chelating effects appear to be involved in the flavonoid protection against silica-induced cell injury.

Protective effect of natural flavonoids on rat peritoneal macrophages injury caused by asbestos fibers.

Exposure of macrophages to asbestos fibers resulted in enhancement of the production of oxygen radicals, determined by a lucigenin enhanced chemiluminescence (LEC) assay, a formation of thiobarbituric acid reactive substances (TBARS), a LDH release into the incubation mixture, and a rapid lysis of the cells. Rutin (Rut) and quercetin (Qr) were effective in inhibiting LEC, TBARS formation, and reducing peritoneal macrophages injury caused by asbestos. The concentrations pre-treatment of antioxidants that were required to prevent the injury of peritoneal macrophages caused by asbestos by 50% (IC50) were 90 microM and 290 microM for Qr and Rut, respectively. Both flavonoids were found to be oxidized during exposure of peritoneal macrophages to asbestos and the oxidation was SOD sensitive. The efficacy of flavonoids as antioxidant agents as well as superoxide ion scavengers was also evaluated using appropriate model systems, and both quercetin and rutin were found to be effective in scavenging O2.-. These findings indicate that flavonoids are able to prevent the respiratory burst in rat peritoneal macrophages exposed to asbestos at the stage of activated oxygen species generation, mainly as superoxide scavengers. On the basis of this study it was concluded that natural flavonoids quercetin and rutin would be promising drug candidates for a prophylactic asbestos-induced disease.

[Status of the antioxidant protective system of rat liver exposed to carbon tetrachloride]. / Sostoianie antiokislitel'noi zashchitnoi sistemy pecheni krys pri vozdeistvii chetyrekhkhloristogo ugleroda.

The dynamics of enzyme activity of antioxidative protective system of the liver and the content of restored glutathione have been studied in rats poisoned by CCl4 injection. During the first hours followed the injection against the background of maximum accumulation of dienic conjugates and decrease of the restored glutathione level no significant changes in the enzyme activity of the antioxidative protective liver system were observed. At the same time 48 hours later the superoxide dismutase and catalase activity decreased by 38% and 36%, respectively, with relative stability of glutathione-dependent enzymes and a two-fold increase of the restored glutathione level. It is shown that a fall of activity of the cytoplasmic antioxidative liver enzymes is not a result of the immediate inactivating effect of free-radical reactions initiated by CCl4, but is, evidently, caused by the covalent binding of its radical metabolites with corresponding macromolecules.

[The mechanism of the antioxidant action of D-penicillamine]. / Mekhanizm antioksidantnogo deistviia D-penitsillamina.

The mechanism of the antioxidative effect of D-penicillamine was studied under the conditions of NADPH- and CCl4-dependent lipid peroxidation in microsomes of rat liver and adrenalin autooxidation reaction. The antioxidative action of the drug was shown to be due to both its chelating properties and direct anti-radical influence. The chelating effect was more pronounced and depended on the concentration of ferric ions in the medium.

4-(4-R-phenylamino)-5-methoxy-1,2-benzoquinones are new selective inhibitors of carbon tetrachloride-initiated free radical reactions in liver.

The action of several 1,2-benzoquinone derivatives on free radical processes initiated by carbon tetrachloride was studied. Among them a substance that effectively inhibited lipid peroxidation without substantial influence on covalent binding of radical products of metabolic cleavage of carbon tetrachloride as well a substance that equally inhibited both processes were found. It was shown that 1,2-benzoquinones can be useful tool for investigation of free radical mechanisms of carbon tetrachloride-initiated liver cell damage in vivo.

Damage of rat liver microsomal mixed function oxidase system by carbon tetrachloride. In vivo study with selective inhibitor of lipid peroxidation.

Pretreatment of rats with 4-[4-N-sodium-N-(5-ethyl-1-thia-3,4-diazol-2-yl)sulfophenylamin o]-5- methoxy-1,2-benzoquinone (Q) before carbon tetrachloride intoxication inhibited lipid peroxidation by 85% but did not prevent cytochrome P-450 destruction, decrease of hydroxylase activity, and loss of the capability to bioactivate carbon tetrachloride in rat liver microsomes. Also no influence of Q on 10-day lethality was found. We conclude that covalent binding of free radical products of metabolic cleavage to various cellular structures is apparently the main damage factor of carbon tetrachloride hepatotoxicity rather than lipid peroxidation.

[Antioxidative activity of antiarthritic preparations]. / Antiokislitel'naia aktivnost' protivoartritnykh preparatov.

Antioxidative activity of acetyl salicylic acid, indometacin, ibuprophen, orthophen, delagil, D-penicillamine and levamysol was studied under conditions of NADPH- and CCl4-dependent lipid peroxidation both in rat liver microsomes and in adrenaline autioxidation. All the drugs studied exhibited only slight antioxidative activity being dissolved in mater, and especially towards lipid peroxidation, as compared with quercitrol and ionol; their activity was noted at concentrations exceeding distinctly the therapeutic doses. At the same time, D-penicillamine inhibited lipid peroxidation not only by means of free radicals neutralization but also via binding of iron ions. This property of the drug may be responsible for its effects, to some extent, in treatment of joint inflammations.

[A simple and sensitive method of determination of superoxide dismutase activity based on the reaction of quercetin oxidation]. / Prostoi i chuvstvitel'nyi metod opredeleniia aktivnosti superoksid- dismutazy, osnovannyi na reaktsii okisleniia kvertsetina.

An indirect method for determination of superoxide dismutase (SOD) activity, based on inhibition of quercetin autooxidation, was developed. The optimal conditions of reaction ensuring high sensitivity of measurement (I50 = 1.5 ng/ml) were selected. NaN3, catalase and physiological concentrations of ascorbic acid did not interfere with the SOD estimation in blood and tissue homogenates.

Superoxide--driven oxidation of quercetin and a simple sensitive assay for determination of superoxide dismutase.

Oxidation of quercetin at pH 10 was shown to be a free radical chain reaction involving superoxide and hence inhibitable by superoxide dismutase (SOD) (EC 1.15.1.1). The degree of inhibition of quercetin oxidation was a function of SOD concentration, and fifty percent inhibition was produced by approximately 1.5 ng/ml of pure enzyme. This reaction proved to be a very useful tool for a rapid and highly sensitive measurement of SOD in crude tissue extracts and other biological samples.