Effect of Inducers and Inhibitors of the Keap1/Nrf2/ARE System on the Viability and Functional Activity of Model Neuronal-Like and Glial Cells.

On mouse neuroblastoma (Neuro-2a) and human glioblastoma (U-87 MG) cell lines, we studied the effect of inducers and inhibitors of redox-sensitive signaling system of the antioxidant-responsive element Keap1/Nrf2/ARE on the main processes that determine nerve cell viability and vital activity (proliferative activity, apoptosis, autophagy, and activation of the Keap1/Nrf2/ARE system). Inhibitors of the Keap1/Nrf2/ARE system stimulate apoptosis more pronouncedly than inducers, have a weaker effect on autophagy, and do not change the nuclear to cytoplasmic Nrf2 ratio. In general, the revealed effects testify in favor of the potential effectiveness of stimulating the Keap1/Nrf2/ARE system for the prevention and adjuvant therapy of neurodegenerative diseases.

Protective Effect of a New Monophenolic Antioxidant TS-13 in a Mouse Model of Parkinson's Disease.

The development of means of the prevention and treatment of age-related neurodegenerative diseases, as well as geroprotectors, among other things, is based on the inflammatory and free radical theories of aging. In this context, we studied the effect of sodium monophenol 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13) on the behavioral and locomotor activity of C57BL/6 mice in modeling Parkinson's disease by MPTP neurotoxin injection. TS-13 administration significantly improved orientation and exploratory activity and emotional response of the animals in the open field test, but did not affect the increase in anxiety caused by MPTP injection. Long-term (6 months) TS-13 administration did not suppress spontaneous motor activity in BALB/c mice and slightly increased their exploratory activity.

[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.

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.

Effect of 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate sodium on expression of GSTP1 and NQO1 genes and protein transcription factors in BALB/c mouse liver.

The study examined dynamics of the effect of novel phenol antioxidant preparation 3-(3'-tertbutyl- 4'-hydroxyphenyl)propyl thiosulfonate sodium (TS-13) on expression of antioxidant protection enzymes genes GSTP1 and NQO1 and on the content of protein transcription factors NF-κB and ATF-2 in mouse liver. Expression of GSTP1 gene decreased significantly on days 4 and 7 after per os administration of TS-13 (100 mg/kg), but increased on post-administration day 14. On days 7 and 14 post-administration, expression of NQO1 gene was significantly increased. On day 7, the hepatic content of the phosphorylated form of ATF-2 and two subunits of nuclear factor NF-κB (p50, p65) decreased significantly.

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.

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.

[Evaluation of cytotocicity and efficiency of antioxidant protection of hydrophilic derivatives of 2,4,6-trialkylphenols in Escherichia coli cells].

The effects of five new derivatives of 2,6-dialkyl-4-propylphenole containing in para-radical different ionogenic groups (-SO3Na, -S-SO3Na, -S-(NH2)2Cl) in the presence and in the absence of hydrogen peroxide on the survival of E. coli AB1157 cells and its isogenic strain defective in repair enzyme genes were studied. Cell survival treated with hydrogen peroxide was remarkably increased in the presence of (3-(3,5-dimethyl-4-hydroxyphenyl)propyl)-1-sulphonate of sodium (Cl). Replacement of methyl ortho-radicals in the structure of Cl for tret-buthyl or cyclohexyl groups led to a decrease of the compounds ability to protect the cells from exogenic hydrogen peroxide. Between derivatives of 2,6-di-tret-buthylphenol the compound with thiosulphonate group demonstrated properties comparable with those for its sulphonate analog, then a chloride of isotiurone at concentration 3 mM completely suppressed the growth of cells in presence and in the absence of H2O2. Compound Cl may be considered as most perspective for detail analysis as antioxidant.

[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.

[Hepatoprotective effect of thiophane in rats with experimental carbon tetrachloride-induced hepatitis].

A hepatoprotective effect of thiophan was studied on the model of carbon tetrachloride-induced hepatitis in rats. Therapeutic administration of thiophan repairs the antitoxic function of liver, normalizes cytolysis marker activity, and improves the synthetic function of liver and the carbohydrate and lipid metabolism. The hepatoprotective activity of thiophan is similar to effect of silimarin.

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.

[Hemorheological effects of thiophane on tetrachloromethane induced hepatic damage].

Carbon tetrachloride-induced hepatitis in rats is accompanied by blood hyperviscosity syndrome development. A course intragastric administration of thiophane under these conditions prevents the increase in whole blood viscosity by normalizing the microrheological indices (deformability and aggregation of erythrocytes), which is manifested by increasing oxygen availability for tissues.

[The dependence of cytotoxicity and antioxidant activity of ammonii derivatives of alkylphenols upon percularities of their structure].

Effect of seven structurally similar N, N-dimethyl-(4-hydroxyaryl)alkylammonium chlorides in the presence and in the absence of hydrogen peroxide on the survival of E. coli cells AB1157 and its isogenic strain BH910 defective in genes of repair enzymes has been analyzed. Among the studied compounds only chloride of N,N-dimethyl-(3,5-dimethyl-4-hydroxybenzyl)ammonium (C1) has no cytotoxic properties and increases the survive of the cells of both strains in the presence of H2O2 better than trolox (water soluble analog of alpha-tocopherol). C1 analogs: 3-methyl-(5-di(tert-butyl)-4-hydroxybenzyl) and 3-(3,5-di(tert-butyl)-4-hydroxyphenyl)propyl)amines derivatives effectively protected from H2O2 only mutant cells BH910. Among the structural analogs of C1 cytotoxicity increases at substitution of methyl groups in aromatic cycle by tert-butyl and cyclohexyl groups. Only C1 among the seven new compounds is the most promising antioxidant for the subsequent more detailed analysis.

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.

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.

[Antioxidative activity of thiophane [bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl)sulfide].

The antioxidant activity, mutagenicity, and genotoxicity of bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl)sulfide (thiophane) were studied using bacterial tests. The results of both an Ames test and SOS chromotest, as well as those studying the survival of E. coli cells deficient in enzymes responsible for the repair of DNA oxidative damage, testify to the fact that thiophane is not mutagenic and genotoxic, and it protects Salmonella typhimurium cells better than the well-known antioxidant trolox.