The cytotoxic and antiproliferative properties of ruthenium nitrosyl complexes and their modulation effect on cytochrome P450 in the HepG2 cell line.

Ruthenium nitrosyl complexes are actively investigated as antitumor agents. Evaluation of potential interactions between cytochromes P450 (CYPs) with new compounds is carried out regularly during early drug development. In this study we have investigated the cytotoxic and antiproliferative activities of ruthenium nitrosyl complexes with methyl/ethyl esters of nicotinic and isonicotinic acids and γ-picoline against 2D and 3D cultures of human hepatocellular carcinoma HepG2 and non-cancer human lung fibroblasts MRC-5, assessed their photoinduced activity at λrad = 445 nm, and also evaluated their modulating effect on CYP3A4, CYP2C9, and CYP2C19. The study of cytotoxic and antiproliferative activities against 2D and 3D cell models was performed using phenotypic-based high content screening (HCS). The expression of CYP3A4, CYP2C9, and CYP2C19 mRNAs and CYP3A4 protein was examined using target-based HCS. The results of CYP3A4 mRNA expression were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ruthenium nitrosyl complexes exhibited a dose-dependent cytotoxic effect against HepG2 and MRC-5 cells. The cytotoxic activity of complexes with ethyl isonicotinate (1) and nicotinate (3, 4) was significantly lower for MRC-5 than for HepG2, for a complex with methyl isonicotinate (2) it was higher for MRC-5 than for HepG2, for a complex with γ-picoline (5) it was comparable for both lines. The antiproliferative effect of complexes 2 and 5 was one order of magnitude higher for MRC-5; for complexes 1, 3, and 4 it was comparable for both lines. The cytotoxic activity of all compounds for 3D HepG2 was lower than for 2D HepG2, with the exception of 4. Photoactivation affected the activity of complex 1 only. Its cytotoxic activity decreased, while the antiproliferative activity increased. The ruthenium nitrosyl complexes 1-4 acted as inducers of CYP3A4 and CYP2C19, while the complex with γ-picoline (5) induced of CYP3A4. Among the studied ruthenium nitrosyl complexes, the most promising potential antitumor compound is the ruthenium compound with methyl nicotinate (4).

Hepatotropic activity of a betulonic acid based compound.

Using the model of cyclophosphamide (CP)-induced immunosuppression in C57BL/6 mice, the hepatotropic effects of a conjugate of betulonic acid with 9-(4-methylpiperazin-1-ylmethyl)-2-(1,2,3-triazolyl) oreozelone (BABC) have been studied. In the liver of treated animals the expression of genes for cytochromes (CYP 1A1, CYP 1A2, CYP 3A44, CYP 2B10, CYP 2C29, CYP 17A1), PPARA, and cytokines (TNF-α, IL-1ß, IL-12α, IL-10) and the relative levels of NF-κB p65, GST-π, and NAT-1 proteins were determined. On day six after administration of the compound and CP to animals a significant (3.2-fold) increase in the expression of the CYP 2B10 as compared to the control group was observed. Treatment of mice with the compound and CP also caused a 2.4-fold increase in the mRNA level of the pro-inflammatory TNF-α gene as compared to the group of animals receiving CP. Administration of the studied compound to intact animals was accompanied by a 2.5-fold increase in the IL-1ß expression and a 1.8-fold decrease in the IL-10 expression as compared to the control group. An increase in the expression of pro-inflammatory cytokine genes in the liver of animals treated with the compound was accompanied by an increase in the content of NF-κB p65 (by 1.6 times), as well as an increase in the relative amount of NAT-1 protein (by 2.7 times) as compared to control animals.

The immunomodulatory activity of the betulonic acid based compound.

The immunomodulatory activity of a betulonic acid-based compound with furocoumarin (BABCF; 2-azido, 9-N-methylpiperazinomethyl oreozelone) has been investigated. Male C57BL/6 mice (aged 3 months) treated with the cytostatic agent cyclophosphamide (CP) and intact individuals served as experimental models. The expression of genes was studied in bone marrow (IL-12, IL-10, IL-1ß, TNF-α, TGF-ß, M-CSF, GM-CSF) or in the suspension of peritoneal cells (IL-12, IL-10; as the injection site). The surface markers of T-lymphocytes (CD3, CD4, and CD8) in fractions of venous blood mononuclear cells (MNCs) were determined by means of flow cytometry using antibodies. Histological and morphometric studies were performed to assess the impact of CP and BABCF on the thymus. BABCF caused a pronounced (about 3-fold) increase in relative expression of the GM-KSF gene. BABCF caused a local increase in the expression of IL-12 in the peritoneal cavity cells and restored the relative content of T-lymphocytes in the blood of CP-treated mice treated affecting mainly CD3⁺CD4⁺ lymphocytes. This substance reduced the tissue density of the thymic cortex and thymic medulla in CP-treated mice. Thus, results of this study suggest that BABCF exhibits a stimulating effect on the cellular link of immunity and promotes maintenance of the number of T-lymphocytes in the blood due to their migration from the central organs of the immune system.

Personalized Approach to Assessing mRNA Expression of Histidine-Rich Glycoprotein and Immunohistochemical Markers in Diseases of the Breast.

Biopsy material of patients with malignant and benign breast diseases was examined. HRG mRNA expression was detected in 70% of cases in biopsy material obtained from patients with nonspecific invasive carcinoma and in 66.7% of cases in biopsy material of patients with benign breast diseases. Immunohistochemical analysis revealed expression of collagen II, the beta-1 integrin, and E-cadherin-markers of epithelial-mesenchymal transition. The use of RT-qPCR combined with immunohistochemical study made it possible to identify atypical cells, which can be regarded as precancerous changes, in individual patients.

Nitrate denitrification with nitrite or nitrous oxide as intermediate products: Stoichiometry, kinetics and dynamics of stable isotope signatures.

A kinetic analysis of nitrate denitrification by a single or two species of denitrifying bacteria with glucose or ethanol as a carbon source and nitrite or nitrous oxide as intermediate products was performed using experimental data published earlier (Menyailo and Hungate, 2006; Vidal-Gavilan et al., 2013). Modified Monod kinetics was used in the dynamic biological model. The special equations were added to the common dynamic biological model to describe how isotopic fractionation between N species changes. In contrast to the generally assumed first-order kinetics, in this paper, the traditional Rayleigh equation describing stable nitrogen and oxygen isotope fractionation in nitrate was derived from the dynamic isotopic equations for any type of kinetics. In accordance with the model, in Vidal-Gavilan's experiments, the maximum specific rate of nitrate reduction was proved to be less for ethanol compared to glucose. Conversely, the maximum specific rate of nitrite reduction was proved to be much less for glucose compared to ethanol. Thus, the intermediate nitrite concentration was negligible for the ethanol experiment, while it was significant for the glucose experiment. In Menyailo's and Hungate's experiments, the low value of maximum specific rate of nitrous oxide reduction gives high intermediate value of nitrous oxide concentration. The model showed that the dynamics of nitrogen and oxygen isotope signatures are responding to the biological dynamics. Two microbial species instead of single denitrifying bacteria are proved to be more adequate to describe the total process of nitrate denitrification to dinitrogen.

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.

Effects of simvaglyzin and atorvaglyzin on the expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase in rat liver.

Activity and levels of protein and mRNA of 3-hydroxy-3-methyl-glutaryl-CoA reductase were estimated in rat liver after the administration of atorvastatin and simvastatin and their complexes with glycyrrhizic acid (atorvaglyzin and simvaglyzin). The amount of 3-hydroxy-3-methyl-glutaryl-CoA reductase protein in rats decreased by 13 and 25% (p<0.05) 24 h after treatment with atorvaglyzin and simvaglyzin, respectively. Activity of this enzyme decreased by 46% in rats treated with atorvaglyzin. The amount of messenger RNA in these groups significantly increased as compared to control group (untreated animals).

Cytochrome P450 4F2 polymorphism in patients with liver cirrhosis.

1297C/T polymorphism of CYP4F2 gene was studied in 108 patients with chronic liver diseases. No significant correlation with predisposition to rapid liver cirrhosis was revealed without consideration for cirrhosis etiology (OR=0.93, 95% CI=0.28-2.99, p=0.885). In patients with viral cirrhosis, a tendency to association of 1297T allele genotypes with rapid cirrhosis development was found (OR=6.0, 95% CI=0.28-382.64, p=0.222). At the same time, CYP4F2 1297T allele was associated with mild (Child-Pugh class A-B) cirrhosis (OR=2.9, p=0.044).

Non-linear dynamics of carbon and hydrogen isotopic signatures based on a biological kinetic model of nitrite-dependent methane oxidation by "Candidatus Methylomirabilis oxyfera".

System dynamics of nitrite-dependent anaerobic methane oxidation (N-DAMO) in a "Candidatus Methylomirabilis oxyfera" culture are described using a mathematical model based on chemical kinetics, microbial growth dynamics and equations for (13)C and (2)H isotopic fractionation. Experimental data for the N-DAMO model were taken from Rasigraf et al. (2012), who studied N-DAMO in a batch culture of "Ca. M. oxyfera" started at two different conditions with varying methane, nitrite and biomass concentrations. In the model, instead of using concentrations of each isotopologue ((12)C and (13)C, (1)H and (2)H), total concentrations and respective isotope ratios were considered as variables. The empirical Monod equations, which included methane and nitrite as two rate-limiting substrates, a threshold methane concentration CH 4min below which there was no biomass growth, and the same kinetic coefficients for the separate batch experiments, fitted the experimental data much better than apparent first-order kinetics that required rather different kinetic coefficients for the two experiments. Non-linear dynamics of (13)C and (2)H isotopic signatures were obtained based on the N-DAMO model. It was shown that rate limitation by methane or nitrite concentrations significantly affected the dynamics of carbon and hydrogen isotopic signatures. Fractionation rate increased at higher initial biomass concentration. The non-linear N-DAMO model satisfactorily described experimental data presented in the two-dimensional plot of hydrogen versus carbon stable isotopic signatures.

Estimating changes of isotopic fractionation based on chemical kinetics and microbial dynamics during anaerobic methane oxidation: apparent zero- and first-order kinetics at high and low initial methane concentrations.

Changes in natural isotopic composition may be used to reveal metabolic pathways of substrate transformation by microbial communities (Vavilin in Ecol Model 240:84-92, 2012b). Anaerobic oxidation of methane (AOM) by sulfate has been described using a mathematical model based on chemical kinetics, microbial dynamics and equations for (13)C isotope accumulation in products as well as their redistribution between substrate and products. Experimental data for two batch cultures that originated from microbial mats covering methane seep chimneys in the Black Sea, previously obtained by Seifert et al. (Org Geochem 37:1411-1419, 2006) and Holler et al. (Env Microbiol Reports 1(5):370-376, 2009), were used to model AOM. During long-time incubation, changes of isotope signatures in CH(4) showed that in the Seifert et al. batch tests (low methane concentration), in contrast to the Holler et al. batch tests (high methane concentration), methane production occurred along with methane oxidation. In accordance with the model, apparent zero and first-order kinetics of methane oxidation were valid for the Holler et al. and Seifert et al. batch tests, respectively. The observed change of [Formula: see text] was explained by microbial kinetics reflecting that the rate is lower for heavy substrate microbial utilization when compared to light substrate microbial utilization. The model showed that small amounts of methanogenesis will change the carbon isotopic composition of methane because biogenic methane has a distinct isotopic composition and due to the large difference between the maximum specific rates of methane oxidation and production. The estimated biomass doubling time of methane-oxidizers for high and low methane concentration was 408/126 days and 4640/1160 days, respectively, depending on the value of the half-saturation constant K ( S ) (5 and 20 mM).

Cytochrome P450 2D6 polymorphism is a molecular genetic marker of liver cirrhosis progression.

Patients with infectious viral or toxic cirrhosis of the liver participated in complex clinical pathomorphological and molecular-genetic study aimed at the search for markers of predisposition to accelerated liver fibrosis, in which the xenobiotic biotransformation system is involved. The results demonstrate association between CYP2D6 (1846G/A) genotype and rapid cirrhosis development and indicate the necessity of studying the mechanisms underlying this association.

Estimating evolution of δ13CH4 during methanization of cellulosic waste based on stoichiometric chemical reactions, microbial dynamics and stable carbon isotope fractionation.

A change in δ(13)CH(4) during mesophilic methanization of cellulosic waste (paper and cardboard) was described using a mathematical model based on stoichiometric chemical reactions, microbial dynamics and the equation for the (13)C isotope accumulation in products including isotope fractionation. In this study, experimental data, previously obtained by Qu et al. (2009), was used to model metabolic pathways of cellulose transformation. A significant change in δ(13)CH(4) occurred in time during cellulosic waste methanization which was in accordance with the model. It was explained by the change in input of acetoclastic and hydrogenotrophic methanogenesis as well as by fractionation of stable carbon isotopes (13)C and (12)C which was much higher for hydrogenotrophic methanogenesis when compared to acetoclastic methanogenesis.

Estimating evolution of δ(13)CH(4) during methanization of municipal solid waste based on chemical reactions, isotope accumulation in products and microbial ecology.

Natural isotopic composition in substrate may be used to reveal the metabolic pathways of substrate transformation by microbial community. In this paper, a change in δ(13)CH(4) during methanization of reconstituted municipal solid waste was described using a mathematical model based on stoichiometric chemical reactions, equation for the (13)C isotope accumulation in products at the low natural C(13)/C(12) ratio and microbial ecology. A set of experimental data used in the model was taken from Qu et al. (2009a). According to the model, during mesophilic municipal solid waste methanization initially hydrogenotrophic and further aceticlastic methanogenesis dominated. At the final stage hydrogenotrophic methanogenesis followed by acetate oxidation dominated again. In spite of rather high measured values of δ(13)C for CO(2) above -21‰, a sharp decrease in δ(13)CH(4) from -20‰ to -60‰ at the final stage was explained by a larger fractionation against (13)C during methanogenesis from H(2)/H(2)CO(3) due to a kinetic isotope effect when hydrogenotrophic methanogens preferentially take down light (12)C. The model also confirmed that in thermophilic conditions a comparatively stable value of δ(13)CH(4) about -60‰ measured earlier (Qu et al. 2009b) was due to a dominance of hydrogenotrophic methanogenesis during all methanization process of cardboard waste.

Relationship between CYP2E1 polymorphism and increase of ALT activity during therapy of patients with pulmonary tuberculosis.

Association of CYP2E1 polymorphism with ALT activity increase was studied in patients with pulmonary tuberculosis receiving therapy by intermittent and daily protocols. The greatest increment of ALT activity in the group receiving therapy by intermittent protocol was seen in the patients with CYP2E1*7632TA genotype. In patients with wild homozygotic 1C/1C (6/6) genotype, ALT activity significantly increased, but remained within the normal range (p=0.048). In the group on daily regimen, activity of ALT increased significantly in patients with all genotypes identified. A more pronounced elevation surpassing the median of the upper threshold of ALT norm was observed in patients with 7632TA genotype (p=0.0051) and in patients with 7632TA or -71GT or 1C/1D genotypes in combinations with wild type alleles by other detected polymorphisms (p=0.0277). Detection of the CYP2E1 gene 7632T

Effect of bovine serum albumin on mitochondrial respiration in the brain and liver of mice and rats.

We studied the effect of BSA (in the isolation medium) on the oxidation rate of succinate, glutamate, pyruvate, and α-ketoglutarate by mitochondria of the brain and liver from C57Bl/6g mice and Taconic Sprague Dawley rats. BSA had no effect on liver mitochondrial respiration, but increased oxidation of substrates (particularly of succinate) in brain mitochondria. Therefore, the major effect of BSA on brain mitochondria is manifested in activation of SDH. The improvement of mitochondrial properties in the brain after treatment with BSA is associated with antioxidant activity of this agent. Our results confirm the hypothesis that inhibition of SDH in brain mitochondria is not the artifact. This process serves as a mechanism protecting neurons from free oxygen radicals during succinate oxidation.

Similar evolution in delta 13CH4 and model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes.

Similar evolution was obtained for the stable carbon isotope signatures delta (13)CH(4) and the model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes. In batch incubations, the importance of aceticlastic and hydrogenotrophic methanogenesis changes in time. Initially, hydrogenotrophic methanogenesis dominated, but increasing population of Methanosarcina sp. enhances aceticlastic methanogenesis. Later, hydrogenotrophic methanogenesis intensified again. A mathematical model was developed to evaluate the relative contribution of hydrogenotrophic and aceticlastic pathways of methane generation during mesophilic batch anaerobic biodegradation of the French and the Chinese Municipal Solid Wastes (FMSW and CMSW). Taking into account molecular biology analysis reported earlier three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) were considered in the model. The total organic and inorganic carbon concentrations, methane production volume, methane and carbon dioxide partial pressures values were used for the model calibration and validation. Methane isotopic composition (delta (13)CH(4)) evolution during the incubations was used to independently validate the model results. The model demonstrated that only the putrescible solid waste was totally converted to methane.

Anaerobic biodegradation of cellulosic material: batch experiments and modelling based on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis.

Utilizing stable carbon isotope data to account for aceticlastic and non-aceticlastic pathways of methane generation, a model was created to describe laboratory batch anaerobic decomposition of cellulosic materials (office paper and cardboard). The total organic and inorganic carbon concentrations, methane production volume, and methane and CO(2) partial pressure values were used for the model calibration and validation. According to the fluorescent in situ hybridization observations, three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) as well as acetate-oxidizing syntrophs, were considered. It was shown that temporary inhibition of aceticlastic methanogens by non-ionized volatile fatty acids or acidic pH was responsible for two-step methane production from office paper at 35 degrees C where during the first and second steps methane was generated mostly from H(2)/H(2)CO(3) and acetate, respectively. Water saturated and unsaturated cases were tested. According to the model, at the intermediate moisture (150%), much lower methane production occurred because of full-time inhibition of aceticlastic methanogens. At the lowest moisture, methane production was very low because most likely hydrolysis was seriously inhibited. Simulations showed that during cardboard and office paper biodegradation at 55 degrees C, non-aceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens were the dominant methanogenic pathways.

[From a periodic chemical reaction to the dynamics of microbial communities].

The first articles published by Anatol Zhabotinsky have been analyzed. The mechanisms of the Belousov-Zhabotinsky and Bray-Liebhafsky oscillating chemical reactions were compared. It was shown that the traditional chemical kinetics, the new methods of molecular biology as well as isotopic composition analysis made it possible to consider new constraints concerning the degradation of organic matter and microbial dynamics. A mathematical model was developed to describe isotope accumulation in biomass and reaction products.

Study of cholesterol-lowering effect and safety of simvaglisin on rabbit model of hypercholesterolemia.

Using rabbit model of experimental hypercholesterolemia we showed that the hypocholesterolemic effect of simvaglisin, a complex preparation containing simvastatin and glycyrrhizic acid, in doses corresponding to 40, 66.5, and 100 mg/kg/day simvastatin is equal to the hypocholesterolemic effect of 200 mg/kg/day simvastatin alone. The total blood cholesterol decreased by 39, 36, 47, and 38% (p < 0.05), respectively, after 20-day course of the preparation. Myotoxicity of simvaglisin evaluated by serum creatine phosphokinase was lower than that of simvastatin. After 30-day treatment, this parameter was lower by 26, 24, and 29% (p < 0.05) than the corresponding parameter for simvastatin.