[Oxidative stress in aging.]

The free-radical theory of aging, advanced more than 50 years ago by D.Harman, remains popular today. The review analyzes age-related changes in the main endogenous mechanisms of reactive oxygen species (ROS) production and antioxidant defense mechanisms. With age, ROS generation by mitochondria, peroxisomes, and NAD(P)H oxidases is enhanced, while the transcriptional activity of the important system Keap1/Nrf2/ARE maintaining redox balance decreases. In old animals, autophagy activity is also low, which removes damaged organelles and aggregated structures from cells. The age-related shift of the redox balance towards oxidative stress can cause the development of age-associated neurodegenerative, autoimmune and inflammatory pathologies.

Synthetic Phenolic Antioxidant TS-13 Suppresses the Growth of Lewis Lung Carcinoma and Potentiates Oncolytic Effect of Doxorubicin.

ROS are important intracellular messengers; their ambiguous role in malignant processes was demonstrated in many studies. The effects of a synthetic phenolic antioxidant sodium 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate sodium (TS-13) on the tumor growth and oncolytic properties of doxorubicin were studied in the experimental model of Lewis lung carcinoma in mice. In mice receiving TS-13 with drinking water (100 mg/kg), suppression of tumor growth by 32.3% was observed on day 21 after inoculation of Lewis lung carcinoma cells. Two-fold intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg were followed by inhibition of tumor growth by 49.5%. Combined treatment with TS-13 and doxorubicin suppressed the tumor growth by 55.4%. In contrast to doxorubicin, TS-13 inhibited NO generation by peritoneal macrophages. The results show the prospect of studying TS-13 in the context of overcoming drug-resistance of tumors.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

Mazes of Nrf2 Regulation.

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

Plant Phenols and Autophagy.

Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular, neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redox-sensitive signaling system and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are presented.

[Phenolic antioxidant TS-13 regulating ARE-dependent genes induces tumor cell death by mitochondria-dependent pathway].

Effects of water-soluble phenolic antioxidant sodium 3-(3'-tret-butyl-4'-hydroxyphenyl)-propyl thiosulfonate (TS-13), potassium 3,5-dimethyl-4-hydroxybenzyl thioetanoate (BEP-11-K) and potassium 3-(3',5'-ditretbutyl-4'-hydroxyphenyl)-propionate (potassium phenosan) on tumor cells proliferative activity and the role of redox-dependent and calcium-dependent signaling mechanisms in realization of tumor cell response to the antioxidant action were studied. Potassium phenosan and BEP-11-K were found to stimulate proliferation and ARE-inducing phenolic antioxidant TS-13 was found to inhibit tumor cell growth in culture. The tumor cell growth rate depended on the rate of intracellular reactive oxygen species production and was decreased by apocynin (a NADPH-oxidase inhibitor) and antimycin A (an ubiquinol-cytochrome c oxidoreductase inhibitor). TS-13 action on tumor cells was accompanied by a transient increase in intracellular reactive oxygen species production and the intracellular calcium concentration, whereas cell incubation with potassium phenosan and BEP-11-K did not influence the reactive oxygen species level and intracellular calcium ions. Cyclosporine A blocked the inhibitory effect of TS-13. Thus, it can be reasonably speculated that phenolic antioxidant TS-13 starts mitochondria-dependent apoptosis in tumor cells by the opening of permeability transition pores.

Protective effect of ARE-inducing phenol antioxidant TS-13 in chronic inflammation.

The protective effect of partially substituted monophenol TS-13 inducing the Nrf2/Keap1/ARE signaling system was studied on the model of chronic inflammation in vivo. It was found that during simulation of inflammation in an air pouch lined with synovial-like membrane, TS-13 did not affect the exudate volume, protein content, and cell count, but significantly reduced the intensity of oxidative metabolism in leukocytes of the exudate. In rheumatoid polyarthritis induced by heterologous collagen, TS-13 reduced the severity of clinical signs of inflammation only at the early stages, but inhibited H2O2 generation by monocytes and, partially, by blood neutrophils. These results suggest that the phlogolytic effect of the redox sensitive Nrf2/Keap1/ARE signaling system is less pronounced in chronic immune-mediated inflammatory processes than in acute inflammation.

Anti-inflammatory activity of TS-13, ARE-inducing phenol antioxidant.

The protective effect of water-soluble TS-13 monophenol inducing the antioxidant-responsive element (ARE) system was studied on the models of acute inflammation. Intragastric administration of TS-13 to rats significantly reduced the severity of acute aseptic inflammation induced by intravenous injection of zymosan particles: granulocyte blood count and volume density of infiltrates in the liver decreased on day 3, spontaneous production of activated oxygen metabolites and respiratory burst in blood granulocytes decreased on days 2 and 3. A single dose of TS-13 improved survival of mice with endotoxin shock induced by intraperitoneal injection of E. coli LPS. These results confirmed high anti-inflammatory activity of TS-13.

Keap1/Nrf2/ARE redox-sensitive signaling system as a pharmacological target.

The redox-sensitive signaling system Keap1/Nrf2/ARE plays a key role in maintenance of cellular homeostasis under stress, inflammatory, carcinogenic, and proapoptotic conditions, which allows us to consider it as a pharmacological target. Here we review the basic regulatory mechanisms of the Keap1/Nrf2/ARE system, key targets for pharmacological intervention, and interconnection of this system with other redox-sensitive transcriptional factors. We also discuss the range of currently available pharmaceuticals. Finally, we promote "indirect" antioxidants as a promising strategy for prevention and treatment of wide range of diseases associated with oxidative stress.

Changes in activity of free radical oxidation processes in the early stages of BCG granulomatosis.

We studied the intensity of free radical oxidation in the liver and activity of oxidative metabolism in mouse peritoneal exudate phagocytes at the early stages of chronic generalized BCG-induced granulomatosis (days 3 and 30 after a single intraperitoneal or intravenous administration of 0.5 mg of BCG vaccine). It was found that both methods of injection did not change the intensity of free radical lipid peroxidation in the liver in comparison with the control, but activity of free radical oxidation mediated by production of hydrogen peroxide was increased in the liver and peritoneal exudate at the stages of mature granuloma formation (day 30). At the same time, intraperitoneal injection contributed to more pronounced activation of lipid peroxidation and synthesis of hydrogen peroxide in the liver.

ARE-inducing phenol antioxidant TC-13 improves survival of Drosophila melanogaster in oxidative stress.

The effects of hydrophilic synthetic antioxidant TC-13 sodium (3'-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate on survival of various strains of Drosophila melanogaster were studied under conditions of oxidative stress induced with H(2)O(2)and paraquat. In a concentration of 1%, TC-13 significantly improved survival of Canton S males treated with H(2)O(2)and in a concentration of 0.2% it improved survival of H(2)O(2)-stressed Oregon R females. The protective effect of the antioxidant under conditions of paraquat-induced stress was observed in Canton S females and Oregon R flies of both genders. Addition of T-13 to diets led to prolongation of the maximum lifespan of insects in the majority of the experiment variants. A relationship between the protective effects of TC-13 and the genotype, gender, and environmental conditions was detected.

Mechanism of the Nrf2/Keap1/ARE signaling system.

Nrf2 regulates expression of genes containing antioxidant-respons(iv)e element (ARE) in their promoters and plays a pivotal role among all redox-sensitive transcription factors. Nrf2 is constitutively controlled by repressor protein Keap1, which acts as a molecular sensor of disturbances in cellular homeostasis. These molecular patterns are in close interconnection and function as parts of the integrated redox-sensitive signaling system Nrf2/Keap1/ARE. Depending on cellular redox balance, activity of this signaling system changes at the levels of transcription, translation, posttranslational modification, nuclear translocation of transcription factor, and its binding to ARE-driven gene promoters. This review summarizes current conceptions of Nrf2/Keap1/ARE induction and inactivation.

[Effect of antioxidant responsive element inducing phenol on D. melanogaster life span].

The effect of water-soluble synthetic antioxidant TS-13 (sodium 3-(3'-tert-butyl-4'-hydroxyphenyl) propyl thiosulfonate) on life span of different lines of Drosophila melanogaster under normal conditions and survival under oxidative stress induced by hydrogen peroxide and paraquat has been investigated. Introduction to the diet of 1% TS-13 prolonged the life span of males and females of D. melanogaster long-living line Canton S, had no effect on short-living Oregon R life span and reduced the life span of male D. melanogaster line IgI(558)OR/Cy, heterozygous on tumor suppressor recessive lethal mutation. When flies were exposed to hydrogen perexide, TS-13 significantly enhanced Canton Smale and Oregon R female survival. Under the influence of paraquat antioxidant protected Canton S female and Oregon R flies of both sexes. Despite the fact that the anti-aging and protective properties of synthetic phenol antioxidant TS-13 depend essentially on the genotype and gender, in the extreme conditions of oxidative stress its positive effect pronounced.

Effect of phenol inducing the antioxidant responsive element on Drosophila melanogaster lifespan.

The effect of hydrophilic synthetic antioxidant TC-13 (3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate sodium) inducing the antioxidant-responsive element on the lifespan of Drosophila melanogaster was studied. Addition of 1% TC-13 to diets prolonged the lifespan of long-lived D. melanogaster Canton S strain females and males, but not of short-lived Oregon R insects and reduced the mean lifespan of D. melanogaster males of the lgl558OR/Cy strain containing a recessive lethal mutation of tumor suppressor in the heterozygotic state. The geroprotective effects of TC-13 synthetic phenol antioxidant depended on D. melanogaster genotype and gender.

Synthetic water-soluble phenolic antioxidant regulates l-arginine metabolism in macrophages: a possible role of Nrf2/ARE.

Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150-1000 microM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1-100 microM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.

[Redox-dependent signaling system Nrf2/are in inflammation].

Transcription factor Nrf2 plays a key role in cell defense against oxidative stress, as well as in counteracting chemical and physical factors that induce apoptosis and carcinogenesis. The mechanism of Nrf2/ARE redox regulation and ARE-mediated gene expression are analyzed in the review. Special attention was paid to anti-inflammatory effects of Nrf2/ARE system, which significantly manifest in Nrf2 knockout animal.

[Variability of the exogenic antioxidant effect on survival: modeling in Drosophila lines with different lifespan and l(2)gl-tumor suppressor dosage].

Different exogenic antioxidants and geroprotectors are used to decrease age abnormalities and enhance the human life span. However, the antioxidant effect on lifespan is variable and requires detailed analysis. In the present report, we modeled in Drosophila the peculiar character of action of various doses of a new phenol antioxidant TC-13. We studied the TC-13 effect on aging of two Drosophila lines with genetically determined contrast lifespan dynamics. In addition, we tested the TC-13 antioxidant influence on the background of heterozygozity on the loss-of-function mutation of the l(2)gl tumor suppressor. The differing effect of TS-13 on LS, the character of which depends on the antioxidant dosage, genotype of line, and sex of Drosophila, was found. TS-13 in the concentration 0.2% did not affect the lifespan in all studied lines and decreased survival, whereas the antioxidant in a concentration of 1% positively affected the lifespan in both males and females of long-living lines. The antioxidant effect on animal lines with a smaller dose of tumor suppressor l(2)gl resulted in a decrease of the lifespan.

Structural and functional characteristics for the antiinflammatory effect of new water-soluble sulfur-containing phenol antioxidants.

Structurally related phenols containing the p-alkylthiosulfonate substituent and partially hindered by tert-butyl groups were synthesized for the search and development of new synthetic antioxidant, which would be effective in vivo in preventing free radical-induced pathological processes. Sodium 3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate had high antiinflammatory activity and produced a dose-dependent effect (IC(50)=36 mg/kg). Changes in the length of the carbohydrate chain in the p-alkyl substituent had no effect on in vitro antiradical activity of the test compounds. However, the decrease in the length of this chain by one methylene unit was accompanied by reduction of antiinflammatory activity of the end product. Lengthening of the chain did not modulate these properties.

Combination of methods for in vitro study of antioxidant properties of chemical compounds.

We proposed a combination of methods to study antioxidant properties of compounds, including evaluation of the capacity of the test preparations to inhibit peroxidation of unsaturated lipids (in model systems with oxidation of ethyl oleate; aqueous emulsion medium) and low-density lipoproteins (in the presence of transition metal ions), generation of superoxide anion radical (system of lucigenin, xanthine oxidase, and xanthine) and NO(*)/ONOO(-) (system of SIN-1 and lucigenin), and induction of respiratory burst in blood granulocytes (luminol-induced and lucigenin-induced reaction after zymosan stimulation). In vitro study showed that the antioxidant properties of synthetic water-soluble phenols depend strongly on masking of the phenol OH group and nature of the ionogenic fragment in the p-propyl substituent.

Antioxidant and antiinflammatory activity of new water-soluble sulfur-containing phenolic compounds.

We synthesized a series of structurally related water-soluble alkyl phenols - sodium 4-hydroxyphenyl propyl sulfonates and thiosulfonates with different number of tert-butyl groups at the ortho-position. In experimental systems of transient metal-induced ethyl oleate and low-density lipoprotein oxidation the antioxidant activity of the compounds increased when the tert-butyl group number at the ortho-position increased and when the sulfonate group was replaced with thiosulfonate. Compounds containing thiosulfonate group in para-propyl substituent also more effectively inhibited reactive oxygen metabolites generated in xanthine-xanthine oxidase system and during morpholinosydnonimine decomposition compared to sulfonate-containing analogs. Phenols with one tert-butyl group at the ortho-position have been shown to exhibit the highest antiinflammatory activity in the model of carrageenan-induced rat paw inflammation, as well as with regard to the expression of the glutathione S-transferase P1-1 gene in HepG2 human hepatoma cell line. Thus, it can be reasonably speculated that the antiinflammatory activity of sulfur-containing phenolic antioxidants in vivo is mediated by their effect on redox-sensitive transcription factors.